Patent Grant
11884940
The contents of the text file submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (filename: PRVI_019_03US_SeqList_ST25.txt, date recorded: Dec. 2, 2021, file size 325 kilobytes).
This application relates to recombinant microorganisms useful in the biosynthesis of biomass or one or more lipid from one or more fatty acid and one or more simple carbon co-substrate. The one or more lipid can be unsaturated C6-C24 fatty acids, fatty alcohols, aldehydes, and acetates which may be useful as final products or precursors to insect pheromones, fragrances, flavors, and polymer intermediates. The application further relates to methods to improve biomass or lipid production in a microorganism from one or more fatty acid and one or more simple carbon co-substrate. The application also relates to methods of producing one or more lipid using the recombinant microorganisms, as well as compositions comprising the recombinant microorganisms and/or optionally one or more of the product lipid.
Derivatives of microbial lipids can be harnessed as precursors of fuels, and as chemicals used in detergent formulation, fragrances, and insect control agents. By applying metabolic engineering strategies to increase lipid content in some microbes, several microbial oleochemicals have been produced at commercial scale. A pathway to produce lipids from simple carbon sources such as glucose, fructose, and glycerol is referred to as a de novo pathway.
De novo lipid biosynthetic pathways rely on several key enzymes, such as fatty acid synthase. De novo pathways yield a broad range of fatty acid moieties with differing chain lengths and unsaturation. Engineering microbial de novo pathways for the purpose of enriching certain lipid species is challenging. De novo lipid pathways also require reducing equivalents. Biosynthetic pathways, such as an insect fatty alcohol pathway to generate pheromone precursors, require NADPH and NADH. Therefore, an improvement in reducing equivalent pool is needed to achieve high level lipid production.
Furthermore, in the presence of multiple substrates, metabolic regulations prevent the full utilization of biosynthetic pathways for the formation of lipids and biomass. The present disclosure addresses these challenges with the development of microorganisms modified to improve production of valuable products such as lipids from multiple substrates while maintaining or increasing biomass of the microorganism. Products produced by these modified microorganisms can include a wide-range of unsaturated C6-C24 fatty acids, fatty alcohols, aldehydes, and acetates including insect pheromones.
The present application relates to microorganisms modified to improve production of valuable products such as lipids from one or more fatty acids and one or more simple carbon co-substrates while maintaining or increasing biomass of the microorganism. The recombinant microorganisms described herein may be used for the production of at least one compound, such as an insect pheromone, a fragrance, or a flavoring agent. In some embodiments, at least one compound comprise unsaturated C6-C24 fatty acids, fatty alcohols, aldehydes, and acetates.
In one aspect, the application relates to a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acids and one or more simple carbon co-substrates, wherein the recombinant microorganism comprises one or more modifications associated with: tricarboxylic acid cycle; lipid synthesis; reducing equivalent availability; metabolic intermediates availability; and/or increased product purity, wherein the recombinant microorganism has improved production of biomass or improved production of one or more lipids compared to a microorganism without the same modifications.
In some embodiments, the one or more modifications associated with comprising tricarboxylic acid cycle comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an AMP-insensitive isocitrate dehydrogenase (IDH) variant in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the at least one nucleic acid molecule is from Yarrowia lipolytica and comprises isoleucine to alanine substitutions at amino acid positions 279 and 280 of XP_503571.2 (SEQ ID NO: 30). In some embodiments, the one or more modifications associated with tricarboxylic acid cycle results in extended activation of the tricarboxylic acid cycle.
In some embodiments, the one or more modifications associated with tricarboxylic acid cycle or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a pyruvate transporter in the recombinant microorganism. In other embodiments, the one or more metabolic intermediates availability comprises mitochondrial pyruvate availability. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to pyruvate transporter from Saccharomyces cerevisiae, Hanseniaspora osmophila, Yarrowia lipolytica, or Talaromyces marneffei PM1. In further embodiments, the pyruvate transporter is selected from Saccharomyces cerevisiae mpc1, Saccharomyces cerevisiae mpc3 (NP_011759.1—SEQ ID NO: 31), Hanseniaspora osmophila mpc3 (OEJ86292.1), Yarrowia lipolytica mpc, and Talaromyces marneffei PM1 mpc3 (KFX48982.1), or homolog thereof. In yet a further embodiment, the recombinant microorganism is Saccharomyces cerevisiae comprising a deletion, disruption, or loss of function mutation in a gene encoding an mpc2 pyruvate transporter. In some embodiments, the recombinant microorganism is Yarrowia lipolytica.
In some embodiments, the one or more modifications associated with lipid synthesis comprises alleviation of acetyl-CoA carboxylase (ACC) inhibition. In certain embodiments, alleviation of ACC inhibition comprises the replacement of the endogenous ACC, or overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a feedback-insensitive ACC variant in the recombinant microorganism. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to ACC from Mus musculus, Rattus norvegicus, or Homo sapiens.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an NADP/NAD-dependent isocitrate dehydrogenase (IDH) in the cytosol of the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the IDH is selected from Escherichia coli Idh (WP_000444484.1—SEQ ID NO: 21), Mycobacterium smegmatis Icd2 (WP_011727802.1—SEQ ID NO: 23), Acidithiobacillus thiooxidans Idh (PDB: 2D4V_A—SEQ ID NO: 22), and Yarrowia lipolytica Idh1 (XP_503571.2—SEQ ID NO: 20 for mutant, or SEQ ID NO: 30 for wild type), or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability further comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an aconitase in the cytosol of the recombinant microorganism. In certain embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding the IDH and the at least one endogenous and/or exogenous nucleic acid molecule encoding the aconitase lack a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the one or more modifications associated with reducing equivalent availability or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous or exogenous nucleic acid encoding a citrate transporter in the recombinant microorganism. In certain embodiments, the one or more intermediate comprises cytosolic citrate/isocitrate. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to a citrate transporter from Yarrowia lipolytica, Saccharomyces cerevisiae, Rattus norvegicus, Caenorhabditis elegans, or Caliqus clemensi. In yet further embodiments, the citrate transporter is selected from Yarrowia lipolytica YALI0F26323p, Saccharomyces cerevisiae AAC48984.1, Rattus norvegicus AAA18899.1, Caenorhabditis elegans P34519.1, and Caliqus clemensi ACO14982.1, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one exogenous nucleic acid molecule encoding a decarboxylating malic enzyme in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to a decarboxylating malic enzyme from Arabidopsis thaliana, Amaranthus hypochondriacus, Rhizobium meliloti, Solanum tuberosum, Homo sapiens, or Escherichia coli. In further embodiments, the decarboxylating malic enzyme is selected from Arabidopsis thaliana Q9SIU0, Amaranthus hypochondriacus P37224, Rhizobium meliloti 030807, Solanum tuberosum P37221, Homo sapiens Q16798, and Escherichia coli P26616, or homolog thereof. In yet a further embodiment, the decarboxylating malic enzyme lacks a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the one or more modifications associated with one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an ATP-citrate lyase in the recombinant microorganism. In certain embodiments, the one or more intermediates availability comprises cytosolic oxaloacetate availability. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to an ATP-citrate lyase from Saccharomyces cerevisiae, Yarrowia lipolytica, Mus musculus, and Aspergillus niger. In a yet further embodiment, the ATP-citrate lyase is selected from Mus musculus NP_001186225.1, Mus musculus NP_598798.1, Aspergillus niger XP_001394055.1, and Aspergillus niger XP_001394057.1, or homolog thereof.
In some embodiments, the one or more modifications associated with comprising reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, the one or more modifications in the PPP comprises one or more of: downregulation of hexose kinase activity; upregulation of one or more oxidative PPP enzyme activity; downregulation of fructose-6-phosphate kinase activity; and/or expression of one or more oxidative PPP enzyme variant. In further embodiments, the upregulation of one or more oxidative PPP enzyme activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a glucose-6-phosphate dehydrogenase (ZWF1), a 6-phosphogluconolactonase (SOL3), or a 6-phosphogluconate dehydrogenase (GND1). In yet a further embodiment, the downregulation of hexose kinase activity and/or fructose-6-phosphate kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme and/or fructose-6-phosphate kinase enzyme. In some embodiments, the one or more oxidative PPP enzyme variant comprises one or more endogenous and/or exogenous nucleic acid molecule encoding an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) and/or an NAD-dependent 6-phosphogluconate dehydrogenase (GND1). In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) from Leuconostoc. In further embodiments, the NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) is selected from Leuconostoc AAA25265.1 and Leuconostoc P11411, or homolog thereof. In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to an NAD-dependent 6-phosphogluconate dehydrogenase (GND1) from Bradyrhizobium or Methylobacillus. In further embodiments, the NAD-dependent 6-phosphogluconate dehydrogenase (GND1) is selected from Bradyrhizobium WP_012029377.1, Bradyrhizobium A4YZZ8, Methylobacillus AAF34407.1, and Methylobacillus Q9L9P8, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises downregulation of mannitol synthesis pathway in the recombinant microorganism. In certain embodiments, downregulation of mannitol synthesis pathway comprises deletion, disruption, and/or mutation of one or more gene encoding an NADPH-dependent mannitol dehydrogenase and/or an aldo-keto reductase. In further embodiments, the one or more gene encoding an NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homolog thereof. In further embodiments, the one or more gene encoding an aldo-keto reductase is selected from YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0C09119g, YALI0D04092g, YALI0B15268g, YALI0C00319g, and YALI0A19910g, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises decoupling and increasing glucose uptake in the recombinant microorganism. In certain embodiments, decoupling and increasing glucose uptake comprises: upregulation of hexose transporter activity; and/or downregulation of hexose kinase activity. In further embodiments, the upregulation of one or more hexose transporter activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter operably linked to one or more heterologous promoters. In some embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encodes for a protein that has at least 70% sequence identity, at least 80% sequence identity, or at least 90% sequence identity to a hexose transporter from Yarrowia lipolytica. In certain embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter is selected from YALI0A14212g, YALI0D01111g, YALI0D00363g, YALI0C16522g, and YALI0F25553g, or homolog thereof. In some embodiments, the downregulation of hexose kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme.
In some embodiments, the one or more modifications associated with reducing equivalent availability, one or more metabolic intermediates availability, or increased product purity comprises downregulation or inhibition of acetyl-CoA carboxylase (ACC) activity in the recombinant microorganism. In certain embodiments, the downregulation or inhibition of ACC activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more ACC enzyme.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more fatty acid co-substrate is a saturated fatty acid. In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more simple carbon co-substrate is selected from glucose, fructose, and glycerol.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the improved production of one or more lipid comprises improved production of one or more mono- or poly-unsaturated C6-C24 fatty acid, fatty alcohol, aldehyde, or acetate. In certain embodiments, the one or more mono- or poly-unsaturated C6-C24 fatty acid, fatty alcohol, aldehyde, or acetate is an insect pheromone. In further embodiments, the insect pheromone is selected from the group consisting of (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadeca-9,1-dienal, (E,E)-8,10-dodecadien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (9Z,12E)-tetradecadienyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, and (Z)-13-octadecenal.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the recombinant microorganism is a eukaryotic microorganism. In certain embodiments, the eukaryotic microorganism is a yeast. In further embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma. In yet a further embodiment, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In certain embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Candida viswanathii, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
In another aspect, the present application provides methods of producing one or more lipid using a recombinant microorganism as described herein. In one embodiment, the method includes cultivating the recombinant microorganism in a culture medium containing a feedstock providing one or more simple carbon and one or more fatty acid co-substrates until the one or more lipid is produced.
In another aspect, the present application provides methods of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, comprising introducing into a microorganism one or more modifications associated with: tricarboxylic acid cycle; lipid synthesis; reducing equivalent availability; one or more metabolic intermediates availability; and/or increased product purity, wherein the introducing one or more modifications yields a recombinant microorganism having improved production of biomass or improved production of one or more lipid compared to a microorganism not comprising the same modifications.
In yet another aspect, the present application provides compositions comprising one or more of the recombinant microorganisms described herein. In certain embodiments, the composition may further comprise one or more lipid produced by the recombinant microorganism. In some embodiments, the composition may further comprise one or more mono- or poly-unsaturated C6-C24 fatty acid, fatty alcohol, aldehyde, or acetate produced by the recombinant microorganism. In some embodiments, the composition may further comprise one or more insect pheromone produced by the recombinant microorganism.
Illustrative embodiments of the disclosure are illustrated in the drawings, in which:
The following definitions and abbreviations are to be used for the interpretation of the disclosure.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pheromone” includes a plurality of such pheromones and reference to “the microorganism” includes reference to one or more microorganisms, and so forth.
As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having, “contains,” “containing,” or any other variation thereof, are intended to cover a non-exclusive inclusion. A composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive “or” and not to an exclusive “or.”
The terms “about” and “around,” as used herein to modify a numerical value, indicate a close range surrounding that explicit value. If “X” were the value, “about X” or “around X” would indicate a value from 0.9X to 1.1X, or, in some embodiments, a value from 0.95X to 1.05X. Any reference to “about X” or “around X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, “about X” and “around X” are intended to teach and provide written description support for a claim limitation of, e.g., “0.98X.”
As used herein, the terms “microbial,” “microbial organism,” and “microorganism” include any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. Therefore, the term is intended to encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea, and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. Also included are cell cultures of any species that can be cultured for the production of a chemical.
As described herein, in some embodiments, the recombinant microorganisms are prokaryotic microorganism. In some embodiments, the prokaryotic microorganisms are bacteria. “Bacteria”, or “eubacteria”, refers to a domain of prokaryotic organisms. Bacteria include at least eleven distinct groups as follows: (1) Gram-positive (gram+) bacteria, of which there are two major subdivisions: (1) high G+C group (Actinomycetes, Mycobacteria, Micrococcus, others) (2) low G+C group (Bacillus, Clostridia, Lactobacillus, Staphylococci, Streptococci, Mycoplasmas); (2) Proteobacteria, e.g., Purple photosynthetic+non-photosynthetic Gram-negative bacteria (includes most “common” Gram-negative bacteria); (3) Cyanobacteria, e.g., oxygenic phototrophs; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroides, Flavobacteria; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophs); (10) Radioresistant micrococci and relatives; (11) Thermotoga and Thermosipho thermophiles.
“Gram-negative bacteria” include cocci, nonenteric rods, and enteric rods. The genera of Gram-negative bacteria include, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.
“Gram positive bacteria” include cocci, nonsporulating rods, and sporulating rods. The genera of gram positive bacteria include, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.
The term “recombinant microorganism” and “recombinant host cell” are used interchangeably herein and refer to microorganisms that have been genetically modified to express or to overexpress endogenous enzymes, to express heterologous enzymes, such as those included in a vector, in an integration construct, or which have an alteration in expression of an endogenous gene. By “alteration” it is meant that the expression of the gene, or level of a RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or activity of one or more polypeptides or polypeptide subunits is up regulated or down regulated, such that expression, level, or activity is greater than or less than that observed in the absence of the alteration. For example, the term “alter” can mean “inhibit,” but the use of the word “alter” is not limited to this definition. It is understood that the terms “recombinant microorganism” and “recombinant host cell” refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein results from transcription and translation of the open reading frame sequence. The level of expression of a desired product in a host cell may be determined on the basis of either the amount of corresponding mRNA that is present in the cell, or the amount of the desired product encoded by the selected sequence. For example, mRNA transcribed from a selected sequence can be quantitated by qRT-PCR or by Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989)). Protein encoded by a selected sequence can be quantitated by various methods, e.g., by ELISA, by assaying for the biological activity of the protein, or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay, using antibodies that recognize and bind the protein. See Sambrook et al., 1989, supra.
The term “polynucleotide” is used herein interchangeably with the term “nucleic acid” and refers to an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof, including but not limited to single stranded or double stranded, sense or antisense deoxyribonucleic acid (DNA) of any length and, where appropriate, single stranded or double stranded, sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or a pyrimidine base and to a phosphate group, and that are the basic structural units of nucleic acids. The term “nucleoside” refers to a compound (as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers, respectively, to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length, DNA, RNA, analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called nucleotidic oligomer or oligonucleotide.
It is understood that the polynucleotides described herein include “genes” and that the nucleic acid molecules described herein include “vectors” or “plasmids.” Accordingly, the term “gene”, also called a “structural gene” refers to a polynucleotide that codes for a particular sequence of amino acids, which comprise all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. The transcribed region of the gene may include untranslated regions, including introns, 5′-untranslated region (UTR), and 3′-UTR, as well as the coding sequence.
The term “enzyme” as used herein refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, which usually includes enzymes totally or partially composed of a polypeptide or polypeptides, but can include enzymes composed of a different molecule including polynucleotides.
As used herein, the term “non-naturally occurring,” when used in reference to a microorganism organism or enzyme activity of the disclosure, is intended to mean that the microorganism organism or enzyme has at least one genetic alteration not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding metabolic polypeptides, other nucleic acid additions, nucleic acid deletions and/or other functional disruption of the microorganism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof, for heterologous, homologous, or both heterologous and homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Exemplary non-naturally occurring microorganism or enzyme activity includes the hydroxylation activity described above.
The term “exogenous” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are not normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
On the other hand, the term “endogenous” or “native” as used herein with reference to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., refers to molecules that are normally or naturally found in and/or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.
The term “heterologous” as used herein in the context of a modified host cell refers to various molecules, e.g., polynucleotides, polypeptides, enzymes, etc., wherein at least one of the following is true: (a) the molecule(s) is/are foreign (“exogenous”) to (i.e., not naturally found in) the host cell; (b) the molecule(s) is/are naturally found in (e.g., is “endogenous to”) a given host microorganism or host cell but is either produced in an unnatural location or in an unnatural amount in the cell; and/or (c) the molecule(s) differ(s) in nucleotide or amino acid sequence from the endogenous nucleotide or amino acid sequence(s).
The term “homolog,” as used herein with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural, or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Homologs most often have functional, structural, or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homologs can be confirmed using functional assays and/or by genomic mapping of the genes.
A protein has “homology” or is “homologous” to a second protein if the amino acid sequence encoded by a gene has a similar amino acid sequence to that of the second gene. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. Thus, the term “homologous proteins” is intended to mean that the two proteins have similar amino acid sequences. In certain instances, the homology between two proteins is indicative of its shared ancestry, related by evolution.
The term “fatty acid” as used herein refers to a compound of structure R—COOH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon and the carboxyl group is at position 1. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon and the carboxyl group is at position 1.
The term “fatty alcohol” as used herein refers to an aliphatic alcohol having the formula R—OH, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon.
The term “fatty acyl-CoA” refers to a compound having the structure R—(CO)—S—R1, wherein R1 is Coenzyme A, and the term “fatty acyl-ACP” refers to a compound having the structure R—(CO)—S—R1, wherein R1 is an acyl carrier protein ACP.
The term “one or more modifications associated with . . . ” refer to one or more modifications in, for example: a biochemical pathway; biological process; growth; cell division; tricarboxylic acid cycle; lipid synthesis; the availability of cofactors, reducing equivalents or metabolic intermediates; and/or product purity in a microorganism or microbial host. The one or more modifications can include, but is not limited to: expression or overexpression of genes or gene variants; deletion, disruption or downregulation of genes; increasing or decreasing activities of gene products; altered localization of a protein within the microorganism, for example, from mitochondria to cytosol or vice versa; alleviation of enzyme feedback inhibition; uptake or secretion of compounds, nutrients, or molecules.
The term “Z11-16 acid selectivity” refers to the degree to which the recombinant microorganism produces Z11-16 acid from palmitate substrate over other lipid variants. Microorganisms that produce higher relative levels of Z11-16 acid over other C6-C24 carbon chain lipids are considered to have higher Z11-16 acid selectivity. Z11-16 acid selectivity can be calculated as done in Example 2, by the formula=[Z11-16Acid]/([Z9-16Acid]+[Z11-16Acid]+[18Acid]+[Z9-18Acid]+[Z11-18Acid]+[Z13-18Acid]+[Z9Z12-18Acid].
The term reduced or eliminated activity refers to enzymes which are either expressed at lower rates than their wild type counterparts, or have been somehow modified to exhibit less enzymatic activity when compared to similarly expressed wild type versions of the enzyme. The reduction in the activity of proteins can be achieved by various methods known to the person skilled in the art such as, for example: (i) through the inhibition or reduction in the expression of the gene coding for the target enzyme; (ii) by partial or complete deletion of the genes coding for the target enzyme, (iii) by expression of non-functional genes that compete against the functional native target enzyme; and/or by inhibition or reduction in the activity of the expressed genes. The inhibition or reduction of the expression of a gene coding for a protein can, for example, be accomplished by inhibition or reduction of the transcription of the coding gene or the translation of the mRNA formed. The deletion of the coding genes can be performed, for example, by a removal of the genes by means of deletion cassettes. The expression of a dysfunctional or activity-reduced gene product can be accomplished, for example, by insertion, substitution or point mutation in the gene coding for the protein. In some embodiments, the deletion of a coding gene is a reduction in activity.
The term “control microorganism” refers to a microorganism that is substantially identical to the referenced recombinant microorganism except for the referenced genetic alteration. References to improvements of the recombinant microorganisms, such as improved lipid production, improved Z11-16 acid selectivity, or improved biomass, should be understood as improvements over a control microorganism lacking the referenced genetic change.
As used herein, the term “bypass pathway culture” refers to a dual substrate culture comprising growth media with a fatty acid precursor substrate and a simple carbon co-substrate. Bypass pathway cultures are designed to bypass de novo lipid synthesis by providing a fatty acid precursor that a microorganism can convert to the final desired desaturated lipid. The simple carbon co-substrate provides the energy input required to maintain the culture, allowing the fatty acid precursor to be used in the final steps of the desired lipid biosynthesis.
Introduction
The present disclosure addresses the need for novel technologies for improved production of biomass or one or more lipid from multiple substrates. The present disclosure solves the problem of carbon utilization to unwanted metabolites in an engineered microbial system. The present disclosure solves the problem of insufficient reducing equivalent pool for high lipid production. Specifically, the present inventors have addressed these problems with the development of recombinant microorganisms having one or more modifications associated with: tricarboxylic acid cycle; lipid synthesis; reducing equivalent availability; one or more metabolic intermediates availability; and/or increased product purity.
In one embodiment, the one or more lipid can be unsaturated C6-C24 fatty acids, alcohols, aldehydes, and acetates including final products or fatty acid precursors of insect pheromones, fragrances, flavors, and polymer intermediates produced from one or more fatty acid and one or more simple carbon co-substrates. Thus, aspects of the disclosure are based on the inventors' discovery that recombinant microorganisms can be engineered to improve production of valuable products such as lipids from one or more fatty acid and one or more simple carbon co-substrates while maintaining or increasing biomass of the recombinant microorganism.
Derivatives of microbial lipids can be harnessed as precursors of fuels, and as chemicals used in detergent formulation, fragrances, and insect control agents. By applying metabolic engineering strategies to increase lipid content in some microbes, several microbial oleochemicals have been produced at commercial scale. Lipid biosynthetic pathways from simple carbon sources such as glucose, fructose, and glycerol (de novo pathway) generate a mixture of fatty acids with different chain lengths and degrees of unsaturation. For certain commercial applications, however, increasing biosynthetic selectivity towards one or a group of some fatty acid species is desirable.
De novo lipid biosynthetic pathways rely on several key enzymes. Fatty acid synthase is a cytosolic enzyme ensemble which catalyzes the polymerization of acyl-CoA with malonyl-CoA. At a certain length, elongation of fatty acyl-ACP polymers is terminated by transacylase activity to produce fatty acyl-CoAs, the precursors of lipids used in diverse functions (e.g. membrane building blocks such as phospholipid, and sphingolipid, or energy storage in the form of mono-, di-, triglycerides, and sterol esters). Prior to conversion into membrane lipid bilayers and storage lipids, fatty acyl-CoAs are processed in the endoplasmic reticulum to undergo desaturations or further elongation. De novo pathways yield a broad range of fatty acid moieties with differing chain lengths and unsaturation. Engineering microbial de novo pathways for the purpose of enriching certain lipid species is challenging, and may compromise host viability because of the interconnectivity of fatty acid enzyme complexes (especially in eukaryotic cells such as yeasts) and the importance of certain fatty acid species on cellular function. De novo lipid pathways are also NADPH intensive. Biosynthetic pathways, such as an insect fatty alcohol pathway to generate pheromone precursors, require NADPH and NADH. Therefore, an improvement in reducing equivalent pool is needed to achieve high level lipid production.
To bypass the limitations in de novo pathway engineering, a strategy was developed to utilize inexpensive plant derived saturated fatty acids in addition to sugar as bioconversion co-substrates to enrich the synthesis of select unsaturated lipid species (bypass pathway). In the presence of multiple substrates, however, metabolic regulations prevent the full utilization of both de novo and bypass pathways for the formation of lipids and biomass. Under a nitrogen-starved and glucose-rich environment, low levels of intracellular AMP reduce the activity of isocitrate dehydrogenase (IDH), a key allosteric enzyme in the TCA cycle in yeast mitochondria. The reduction of IDH activity slows the TCA cycle used for synthesis of biomass and reducing equivalents, and accumulates citrate (the equilibrium form of isocitrate). Build-up of isocitrate in mitochondria creates citrate overflow to the cytosolic compartment. This cytosolic citrate is cleaved into oxaloacetate and acetyl-CoA, the committed precursor of fatty acid synthesis, eliciting lipogenesis and reducing growth. High citrate accumulation has been observed in oleagenic yeast cultivation under nitrogen-limited conditions. Therefore, it is desirable to engineer an oleochemical production host to repurpose citrate for improvement in biomass generation. This can be achieved by an extended activation of the TCA cycle during lipogenesis, while increasing the malonyl-CoA pool, the lipid precursor. Additionally, the number of malonyl-CoA generated for every molecule of glucose consumed can be reduced by the existence of the mannitol synthesis pathway. The NADPH required for 18-carbon triacylglyceride synthesis is balanced if 0.35 moles of mannitol is generated for every mole of glucose consumed resulting in 0.92 moles of malonyl-CoA, 0.04 moles glycerol, and 1.65 moles of NADPH. Alternative sources of NADPH may reduce the required flux through the mannitol pathway and balance NADPH generation. Therefore, in some embodiments, the deletion of the mannitol synthesis pathway can be used to enhance generation of reducing equivalents and improve glucose yields to support production of fatty acid derived products.
Another aspect of the invention relates to increasing the availability of reducing equivalents in the cytosolic compartment. Many heterologous proteins are expressed in the cytosol, or with an active site that is exposed to the cytosol. Of interest is a system which expresses insect desaturases and alcohol forming reductases to generate insect pheromone fatty alcohol precursors. These desaturases and reductases are bound to the endoplasmic reticulum membrane with active sites facing the cytosol. They require cytosolic NADH and NADPH as cofactors to transform a fatty acid precursor into an unsaturated fatty acid, and subsequently into a fatty alcohol. Glycolysis and the pentose phosphate pathway can provide the necessary reducing equivalents. The majority of NADPH required for de novo or bypass fatty acid synthesis in Y. lipolytica comes from the oxidative branch of the pentose phosphate pathway. Under growth conditions which elicit fatty acid synthesis and lipid storage, the majority of glucose flux can be funneled through the pentose phosphate pathway in order to supply the NADPH required for fatty acid synthesis. Depending on various factors such as feeding strategy and cultivation conditions, however, the pool of reducing equivalents in the cytosol may not be sufficient to support heterologous high fatty alcohol production. Therefore, it is desirable to engineer an oleochemical production host with high cytosolic pool of reducing equivalents to allow high level synthesis of lipid products.
Overall, the disclosure describes methods to engineer oleagenic microbes to maximize the conversion efficiency of carbons derived from simple carbons in conjunction with fatty acids for improved microbial production of chemicals such as lipids and fatty acid derivatives by re-directing biosynthetic pathways for reducing overflow metabolites, rebalancing and increasing reducing equivalent, and increasing lipid precursor metabolite.
In one aspect, methods for increasing biomass and precursors of lipid synthesis and microorganisms capable of improved production of biomass and one or more lipid are disclosed.
In some embodiments, microorganisms are modified to alleviate inhibition of isocitrate dehydrogenase (IDH) in the TCA cycle. Specifically, in some embodiments, the locus which corresponds to IDH is replaced with a sequence of an AMP-insensitive IDH variant. Examples of AMP-insensitive IDH variants can be sourced from multiple organisms (Table 1). AMP-insensitive IDH variant can also be engineered in Y. lipolytica IDH1 by mutations of I279A and I280A (Table 1). An AMP-insensitive IDH variant can also be introduced in addition to the native IDH variant to achieve a similar phenotype.
Escherichia
coli
Mycobacterium
smegmatis
Acidithiobacillus
thiooxidans
Yarrowia
lipolytica
In some embodiments, microorganisms are modified to increase mitochondrial pyruvate pool. Pyruvate is a precursor of multiple enzymes involved in the TCA cycle used for biomass and reducing equivalent generation. To increase pyruvate pool in mitochondria, pyruvate flux from cytosol is enhanced by overexpressing select pyruvate transporter proteins. In Saccharomyces cerevisiae, this is achieved via deletion or truncation of mpc2 gene locus, and overexpression of mpc1, and mpc3 loci. In Y. lipolytica, overexpression of the endogenous mpc genes, co-expression or replacement with heterologous mpc genes can achieve a similar phenotype. Heterologous mpc genes can be sourced from multiple organisms (Table 2).
Saccharomyces cerevisiae
Hanseniaspora osmophila
Talaromyces marneffei PM1
In some embodiments, microorganisms are modified to alleviate inhibition of lipid synthesis by removing acetyl-CoA carboxylase (ACC) regulation. Inclusion of a fatty acid as a co-substrate in addition to simple carbon sources (glucose, fructose, etc.) may inhibit de novo lipid biosynthesis due to excess fatty acyl-CoA. A key enzyme in de novo lipid pathway is ACC, an allosterically regulated enzyme to convert acetyl-CoA into malonyl-CoA (lipid precursor). To relieve ACC inhibition, the native gene locus in Y. lipolytica which encodes for ACC is replaced by or co-expressed with a non-native gene fragment that encodes for a feedback-insensitive ACC variant. Heterologous feedback-insensitive ACC genes can be sourced from multiple organisms (Table 3).
Mus musculus
Mus musculus
Rattus norvegicus
Rattus norvegicus
Homo sapiens
Homo sapiens
In another aspect, methods for increasing and balancing reducing equivalents and microorganisms capable of increasing and balancing reducing equivalents are disclosed.
In some embodiments, microorganisms are engineered to assimilate cytosolic citrate or isocitrate into alpha-ketoglutarate, and generation of reducing equivalents. In certain embodiments, an NADP/NAD-dependent IDH is functionally expressed in the cytosol alone or together with an aconitase. To functionally express aconitase and NADP/NAD-linked IDH, a variety of yeast promoter sequences can be used. To redirect expression into the cytosolic compartment, the mitochondrial-targeting peptide of respective enzymes are removed. In the case of NAD/NADP-specific IDHs, cytosolic bacterial proteins can be utilized (Table 1).
In some embodiments, the citrate/isocitrate pool in the cytosol is increased by overexpression of citrate transporter protein in a microorganism to increase and/or rebalance reducing equivalent. In certain embodiments, one or more copies of citrate transporter genes are introduced into a microorganism. Citrate transporter sequences include, but are not limited to, Yarrowia lipolytica YALI0F26323p, Saccharomyces cerevisiae AAC48984.1, Rattus norvegicus AAA18899.1, Caenorhabditis elegans P34519.1, and Caliqus clemensi ACO14982.1.
In some embodiments, microorganisms are engineered to express a decarboxylating malic enzyme to generate reducing equivalent in the cytosol from citrate. An ATP-dependent citrate lyase cleaves cytosolic citrate into acetyl-CoA and oxaloacetate. Subsequently, an NADPH-dependent malate dehydrogenase converts oxaloacetate into malate, a metabolite which is transported back into the mitochondria to enter the TCA cycle. A heterologous decarboxylating malic enzyme is expressed to convert malate into pyruvate and CO2 while generating a reducing equivalent either in the form of NADH or NADPH. To redirect expression into the cytosolic compartment, the mitochondrial-targeting peptide of the malate dehydrogenase is removed. Examples of gene sequences which encode decarboxylating malic enzymes include Arabidopsis thaliana Q9SIU0 (SEQ ID NO: 34), Amaranthus hypochondriacus P37224 (SEQ ID NO: 35), Rhizobium meliloti 030807 (SEQ ID NO: 36), Solanum tuberosum P37221 (SEQ ID NO: 37), Homo sapiens Q16798 (SEQ ID NO: 38), and Escherichia coli P26616 (SEQ ID NO: 29).
In some embodiments, the oxaloacetate pool in the cytosol is increased by upregulation of ATP-citrate lyase to increase and/or rebalance reducing equivalent. Further improvement of reducing equivalent by expressing a malic enzyme is gained by improving oxaloacetate (malic enzyme precursor) via upregulation of ATP-citrate lyase. In some embodiments, upregulation of ATP-citrate lyase in a microorganism comprises replacing the native promoter sequence of the endogenous ATP-citrate lyase with a strong promoter sequence such as one derived from a transaldolase gene. In other embodiments, the activity of ATP-citrate lyase is increased by introducing additional sequences which encode the enzyme. Native or heterologous ATP-citrate lyase sequences such as Mus musculus NP_001186225.1, Mus musculus NP_598798.1, Aspergillus niger XP_001394055.1, and Aspergillus niger XP_001394057.1 can be used for this purpose.
In some embodiments, flux through the pentose phosphate pathway (PPP) is increased upon entering lipogenesis and lipid storage phase after biomass synthesis to increase and/or rebalance reducing equivalent. Several strategies are utilized to funnel carbon into PPP. In some embodiments, hexose kinase activity is downregulated to limit flux of glucose to glucose-6-phosphate. In other embodiments, independent from or in concert with hexose kinase downregulation, the activity of glucose-6-phosphate dehydrogenase (ZWF1), 6-phosphogluconolactonase (SOL3), and 6-phosphogluconate dehydrogenase (GND1) is increased to draw down the pool of glucose-6-phosphate and pull additional fructose-6-phosphate to enter the oxidative pentose phosphate pathway. In certain embodiments, fructose-6-phosphate kinase is downregulated to reduce flux through upper glycolysis. In another aspect, native glucose-6-phosphate dehydrogenase (ZWF1) or 6-phosphogluconate dehydrogenase (GND1) is replaced with recombinant or engineered variants which use NAD+ in place of NADP+ producing NADH instead of NADPH to match cofactor requirements of recombinant pathways. Examples of suitable NAD-dependent variants of GND include Bradyrhizobium WP_012029377.1, Bradyrhizobium A4YZZ8, Methylobacillus AAF34407.1, and Methylobacillus Q9L9P8. Examples of suitable NAD-dependent ZWF include Leuconostoc AAA25265.1 and Leuconostoc P11411.
In some embodiments, deletion or downregulation of mannitol synthesis pathway can increase and/or rebalance reducing equivalent. In certain embodiments, downregulation of mannitol synthesis pathway comprises deletion, disruption, and/or mutation of one or more gene encoding an NADPH-dependent mannitol dehydrogenase and/or an aldo-keto reductase. In some embodiments, the NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g. In other embodiments, the aldo-keto reductase is selected from YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0C09119g, YALI0D04092g, YALI0B15268g, YALI0C00319g, and YALI0A19910g.
In some embodiments, reducing equivalent can be increased and/or rebalanced by decoupling and increasing glucose uptake via amplifying the activity of both high and low affinity hexose transporters. Yeasts possess several variants of hexose transporters that are distinguished based on their specificity and mode of activation. It is desirable to optimize biomass and lipid synthesis regardless of glucose concentration and media composition throughout the fermentation period. It was shown that under distinct fermentation conditions such as nitrogen limitation, glucose uptake rates drop significantly. Thus, glucose depletion might diminish the pool of reducing equivalents necessary for lipid synthesis, desaturation of fatty acids and reduction of fatty acids to fatty alcohols. In some embodiments, hexokinase deletion or downregulation increases both high and low affinity hexose transporters in yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica. The deletion of a hexokinase variant leads to an increase in pyruvate, a precursor of the TCA cycle used for biomass and reducing equivalent generation. An alternative strategy involves the expression of specific glucose transporters using heterologous promoters. The use of heterologous promoters achieves a decoupling of hexose transporter expression from cellular regulation. In some embodiments, a microorganism can be modified to overexpress Yarrowia lipolytica hexose transporter genes selected from YALI0A14212g, YALI0D01111g, YALI0D00363g, YALI0C16522g and YALI0F25553g.
In another aspect, methods of increasing product purity, reducing equivalents, and pathway intermediates and microorganisms capable of increasing product purity, reducing equivalents, and pathway intermediates are disclosed.
In some embodiments, downregulation or inhibition of ACC activity during conversion phase to reduce flux to 18 carbon fatty acids increases product purity, available NADPH, and acetyl-CoA units as potential building blocks for fatty esters or for NADH generation via respiration.
In some embodiments, the recombinant microorganisms of the disclosure can be used to synthesize mono- or poly-unsaturated C6-C24 fatty acids. In other embodiments, the recombinant microorganisms of the disclosure can be used to synthesize mono- or poly-unsaturated C6-C24 fatty alcohols. Mono- or poly-unsaturated C6-C24 fatty alcohols can be further converted into the corresponding aldehydes or acetates. Thus, various embodiments of the present disclosure can be used to synthesize a variety of insect pheromones selected from fatty alcohols, aldehydes, and acetates. Additionally, embodiments described herein can also be used for the synthesis of fragrances, flavors, and polymer intermediates.
Pheromones
As described above, embodiments of the disclosure provide for the production of one or more insect pheromones using a recombinant microorganism of the disclosure. A pheromone is a volatile chemical compound that is secreted by a particular insect for the function of chemical communication within the species. That is, a pheromone is secreted or excreted chemical factor that triggers a social response in members of the same species. There are, inter alia, alarm pheromones, food trail pheromones, sex pheromones, aggregation pheromones, epideictic pheromones, releaser pheromones, primer pheromones, and territorial pheromones, that affect behavior or physiology.
Non-limiting examples of insect pheromones which can be produced using the recombinant microorganisms and methods disclosed herein include linear alcohols, aldehydes, and acetates listed in Table 4.
In some aspects, one or more pheromones that can be produced using a recombinant microorganism of the disclosure include at least one pheromone listed in Table 5 to modulate the behavior of an insect listed in Table 5. In other aspects, non-limiting examples of insect pheromones which can be produced using the recombinant microorganisms and methods disclosed herein include alcohols, aldehydes, and acetates listed in Table 5. However, the microorganisms described herein are not limited to the production of C6-C20 pheromones listed in Table 4 and Table 5. Rather, the disclosed microorganisms can also be utilized in the synthesis of various C6-C24 mono- or poly-unsaturated fatty acids, alcohols, aldehydes, and acetates, including fragrances, flavors, and polymer intermediates.
Agrotis segetum sex pheromone component
Anarsia lineatella sex pheromone component
Anarsia lineatella sex pheromone component
Pseudoplusia includens sex pheromone Agrotis segetum sex pheromone component
Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component
Grapholitha molesta, Ecdytolopha aurantiana sex pheromone component
Grapholitha molesta sex pheromone
component
Eupoecilia ambiguella sex pheromone
Cydia pomonella
Lobesia botrana
Pandemis pyrusana, Naranga aenescens, Agrotis segetum sex pheromone component
Pandemis pyrusana, Choristoneura roseceana sex pheromone component
Choristoneura roseceana, Crocidolomia pavonana sex pheromone component
Diatraea considerata sex pheromone component
Helicoverpa zea, Helicoverpa armigera, Heliothis virescens sex pheromone component
Naranga aenescens sex pheromone component
Platyptila carduidactyla, Heliothis virescens sex pheromone Helicoverpa zea, Helicoverpa armigera, Plutella xylostella, Diatraea considerate, Diatraea grandiosella, Diatraea saccharalis, Acrolepiopsis assectella sex pheromone component
Discestra trifolii sex pheromone Heliothis virescens, Plutella xylostella, Acrolepiopsis assectella, Crocidolomia pavonana, Naranga aenescens sex pheromone component
Amyelosis transitella
Amyelosis transitella
Amyelosis transitella
Diatraea considerata, Diatraea grandiosella sex pheromone component
Amyelosis transitella
Most pheromones comprise a hydrocarbon skeleton with the terminal hydrogen substituted by a functional group (Ryan MF (2002). Insect Chemoreception. Fundamental and Applied. Kluwer Academic Publishers). Table 6 shows some common functional groups, along with their formulas, prefixes and suffixes. The presence of one or more double bonds, generated by the loss of hydrogens from adjacent carbons, determines the degree of unsaturation of the molecule and alters the designation of a hydrocarbon from -ane (no multiple bonds) to -ene. The presence of two and three double bonds is indicated by ending the name with -diene and -triene, respectively. The position of each double bond is represented by a numeral corresponding to that of the carbon from which it begins, with each carbon numbered from that attached to the functional group. The carbon to which the functional group is attached is designated -1-. Pheromones may have, but are not limited to, hydrocarbon chain lengths numbering 10 (deca-), 12 (dodeca-), 14 (tetradeca-), 16 (hexadeca-), or 18 (octadeca-) carbons long. The presence of a double bond has another effect. It precludes rotation of the molecule by fixing it in one of two possible configurations, each representing geometric isomers that are different molecules. These are designated either E (from the German word Entgegen, opposite) or Z (Zusammen, together), when the carbon chains are connected on the opposite (trans) or same (cis) side, respectively, of the double bond.
Pheromones described herein can be referred to using IUPAC nomenclature or various abbreviations or variations known to one skilled in the art. For example, (11Z)-hexadecen-1-al, can also be written as Z-11-hexadecen-1-al, Z-11-hexadecenal, or Z-x-y:Ald, wherein x represents the position of the double bond and y represents the number of carbons in the hydrocarbon skeleton. Abbreviations used herein and known to those skilled in the art to identify functional groups on the hydrocarbon skeleton include “Ald,” indicating an aldehyde, “OH,” indicating an alcohol, and “Ac,” indicating an acetyl. Also, the number of carbons in the chain can be indicated using numerals rather than using the written name. Thus, as used herein, an unsaturated carbon chain comprised of sixteen carbons can be written as hexadecene or 16.
Similar abbreviation and derivations are used herein to describe pheromone precursors. For example, the fatty acyl-CoA precursors of (11Z)-hexadecen-1-al can be identified as (11Z)-hexadecenyl-CoA or Z-11-16:Acyl-CoA.
The present disclosure relates to recombinant microorganisms expressing one or more enzyme or transporter that contributes to improved production of biomass or one or more lipid. The present disclosure also relates to recombinant microorganisms comprising downregulation of one or more enzyme in one or more pathways that contributes to improved production of biomass or one or more lipid.
Isocitrate Dehydrogenase
In some embodiments, the present disclosure teaches microorganisms comprising an AMP-deficient isocitrate dehydrogenase (IDH). IDH catalyzes the oxidation of isocitrate to oxalosuccinate in the TCA pathway. In many microorganisms, including Y. lipolytica, IDH is an allosteric enzyme, sensitive to AMP levels. Under non-optimal growTH conditions, such as nitrogen deprivation, AMP levels are reduced by AMP deaminase (AMD1), thus reducing IDH activity. IDH enzymes produce NADPH/NADH, which are important for native fatty acid synthesis and for methyl palmitate desaturation. Thus in some embodiments, the recombinant microorganisms of the present disclosure exhibit improved lipid production from fatty acid precursors, in part, because they comprise a nucleic acid encoding for an AMP-deficient IDH enzyme.
The present disclosure describes enzymes that catalyze the oxidative decarboxylation of isocitrate, producing alpha-ketoglutarate (α-ketoglutarate) and CO2. This is a two-step process, which involves oxidation of isocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followed by the decarboxylation of the carboxyl group beta to the ketone, forming alpha-ketoglutarate.
Isocitrate dehydrogenase can catalyze the following reactions:
Isocitrate+NAD+↔2-oxoglutarate+CO2+NADH+H+
Isocitrate+NADP+↔2-oxoglutarate+CO2+NADPH+H+
Isocitrate+NADP+Mg2+(metal ion)‰alpha-ketoglutarate+NADPH+H++CO2
The isocitrate dehydrogenase (IDH) enzyme structure in Escherichia coli was the first structure to be elucidated and understood. Most isocitrate dehydrogenases are dimers, to be specific, homodimers (two identical monomer subunits forming one dimeric unit).
Isocitrate dehydrogenase is the first bacterial enzyme shown to be regulated by phosphorylation/dephosphorylation. The modulation of this key enzyme activity enables E. coli to make rapid shifts between TCA and glyoxalate bypass pathways. Fluxes and intercellular concentrations for this junction have been determined. The state of phosphorylation of isocitrate dehydrogenase determines its activity.
There are marked differences in the properties of enzymes from different sources. The E. coli enzyme is not an allosteric protein as isocitrate dehydrogenases from other sources are, and it is cold sensitive. IcdA is observed to have several distinct isoforms. Phosphorylation of the enzyme on a serine residue by isocitrate dehydrogenase kinase/phosphatase inactivates it, and dephosphorylation by the phosphatase reactivates it. Phosphorylation affects the binding of NADP. The enzyme shows allosteric inhibition by phosphoenolpyruvate.
In some embodiments, the one or more modifications associated with tricarboxylic acid cycle comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an AMP-insensitive isocitrate dehydrogenase (IDH) variant in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the at least one nucleic acid molecule is from Yarrowia lipolytica and comprises isoleucine to alanine substitutions at amino acid positions 279 and 280 of XP_503571.2. In some embodiments, the one or more modifications associated with tricarboxylic acid cycle results in extended activation of the tricarboxylic acid cycle.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an NADP/NAD-dependent isocitrate dehydrogenase (IDH) in the cytosol of the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the IDH is selected from Escherichia coli Idh (WP_000444484.1), Mycobacterium smegmatis Icd2 (WP_011727802.1), Acidithiobacillus thiooxidans Idh (PDB: 2D4V_A), and Yarrowia lipolytica Idh1 (XP_503571.2), or homolog thereof.
Pyruvate Transporter
Pyruvate is the end-product of glycolysis, a major substrate for oxidative metabolism, and a branching point for glucose, lactate, fatty acid and amino acid synthesis. The mitochondrial enzymes that metabolize pyruvate are physically separated from cytosolic pyruvate pools and rely on a membrane transport system to shuttle pyruvate across the impermeable inner mitochondrial membrane (IMM). Two proteins, mitochondrial pyruvate carriers MPC1 and MPC2, form a hetero-oligomeric complex in the IMM to facilitate pyruvate transport.
In some embodiments, the one or more modifications associated with comprising tricarboxylic acid cycle or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a pyruvate transporter in the recombinant microorganism. In other embodiments, the one or more metabolic intermediates availability comprises mitochondrial pyruvate availability. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to pyruvate transporter from Saccharomyces cerevisiae, Hanseniaspora osmophila, Yarrowia lipolytica, or Talaromyces marneffei PM1. In further embodiments, the pyruvate transporter is selected from Saccharomyces cerevisiae mpc1, Saccharomyces cerevisiae mpc3 (NP_011759.1), Hanseniaspora osmophila mpc3 (OEJ86292.1), Yarrowia lipolytica mpc, and Talaromyces marneffei PM1 mpc3 (KFX48982.1), or homolog thereof. In yet a further embodiment, the recombinant microorganism is Saccharomyces cerevisiae comprising a deletion, disruption, or loss of function mutation in a gene encoding an mpc2 pyruvate transporter. In some embodiments, the recombinant microorganism is Yarrowia lipolytica.
Aconitase
Aconitase (aconitate hydratase) is an enzyme that catalyzes the stereo-specific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process.
Aconitase has two slightly different structures, depending on whether it is activated or inactivated. In the inactive form, its structure is divided into four domains. Counting from the N-terminus, only the first three of these domains are involved in close interactions with the [3Fe-4S] cluster, but the active site consists of residues from all four domains, including the larger C-terminal domain. The Fe—S cluster and a SO42- anion also reside in the active site. When the enzyme is activated, it gains an additional iron atom, creating a [4Fe-4S] cluster. However, the structure of the rest of the enzyme is nearly unchanged.
In contrast with the majority of iron-sulfur proteins that function as electron carriers, the iron-sulfur cluster of aconitase reacts directly with an enzyme substrate. Aconitase has an active [Fe4S4]2+ cluster, which may convert to an inactive [Fe3S4]+ form. Three cysteine (Cys) residues have been shown to be ligands of the [Fe4S4] center. In the active state, the labile iron ion of the [Fe4S4] cluster is not coordinated by Cys but by water molecules.
The iron-responsive element-binding protein (IRE-BP) and 3-isopropylmalate dehydratase (α-isopropylmalate isomerase), an enzyme catalyzing the second step in the biosynthesis of leucine, are known aconitase homologues. Iron regulatory elements (IREs) constitute a family of 28-nucleotide, non-coding, stem-loop structures that regulate iron storage, heme synthesis and iron uptake. They also participate in ribosome binding and control the mRNA turnover (degradation). The specific regulator protein, the IRE-BP, binds to IREs in both 5′ and 3′ regions, but only to RNA in the apo form, without the Fe—S cluster. Expression of IRE-BP in cultured cells has revealed that the protein functions either as an active aconitase, when cells are iron-replete, or as an active RNA-binding protein, when cells are iron-depleted. Mutant IRE-BPs, in which any or all of the three Cys residues involved in Fe—S formation are replaced by serine, have no aconitase activity, but retain RNA-binding properties.
Aconitase is inhibited by fluoroacetate, therefore fluoroacetate is poisonous. Fluoroacetate, in the citric acid cycle, can innocently enter as fluorocitrate. However, aconitase cannot bind this substrate and thus the citric acid cycle is halted. The iron sulfur cluster is highly sensitive to oxidation by superoxide.
In some embodiments, the one or more modifications associated with reducing equivalent availability further comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an aconitase in the cytosol of the recombinant microorganism. In certain embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding the aconitase lack a sequence encoding a mitochondrial-targeting peptide.
Citrate Transporter
Citrate is a key intermediate in both catabolism and anabolism and thus occupies a prominent position in eukaryotic energy metabolism. When the cell has excess energy, citrate is transported out of the mitochondrial matrix across the inner membrane via the mitochondrial citrate transport protein (CTP). Citrate can then passively diffuse through an anion selective channel across the outer mitochondrial membrane into the cytoplasm. Once in the cytoplasm, citrate is broken down to acetyl CoA and oxaloacetate, the former providing the immediate carbon source to fuel fatty acid, triacylglycerol, and cholesterol biosyntheses.
The mitochondrial CTP catalyzes an obligatory exchange of tricarboxylates (i.e., citrate, isocitrate) either for each other or for the dicarboxylate malate or for phosphoenolpyruvate. In higher eukaryotes the transporter catalyzes citrate/malate exchange with citrate moving outwardly across the inner membrane. In yeast, the CTP is thought to catalyze a citrate/isocitrate exchange. In both cases, the CTP catalyzes a facilitated-diffusion with dianions being the transported species. The CTP is a member of the mitochondrial transporter family. Most members of this family display several common characteristics including: a size of approximately 300 amino acids and a basic isoelectric point, the presence of 3 homologous sequence domains, and a signature sequence motif of Px(D,E)x(V,I,A,M)(K,R)x(R,K,Q,A)(L,M,F,I) which repeats two-three times.
In some embodiments, the one or more modifications associated with reducing equivalent availability or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding a citrate transporter in the recombinant microorganism. In certain embodiments, the one or more metabolic intermediates availability comprises cytosolic citrate/isocitrate. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a citrate transporter from Yarrowia lipolytica, Saccharomyces cerevisiae, Rattus norvegicus, Caenorhabditis elegans, or Caliqus clemensi. In yet further embodiments, the citrate transporter is selected from Yarrowia lipolytica YALI0F26323p, Saccharomyces cerevisiae AAC48984.1, Rattus norvegicus AAA18899.1, Caenorhabditis elegans P34519.1, and Caliqus clemensi ACO14982.1, or homolog thereof.
Decarboxylating Malic Enzyme
In some embodiments, the present disclosure teaches recombinant microorganisms comprising a nucleic acid encoding for a heterologous malic enzyme. In some embodiments, the recombinant microorganisms of the present disclosure exhibit improved production of lipids from fatty acid substrates. In some embodiments, the recombinant microorganisms of the present disclosure exhibit improved Z11-16 acid selectivity.
Microorganisms of the present disclosure produce citrate as co-product under nitrogen-limited conditions. This is because during nitrogen starvation many organisms, including Y. lipolytica down-regulate respiration to divert carbon/energy storage via lipid synthesis. Citrate is first exported from mitochondria into the cytosol and subsequently from the cell. Exported citrate can be re-assimilated, especially when alternative carbon sources are scarce. Alternatively, the combination of the enzymes ATP citrate lyase (ACL), malate dehydrogenase (e.g., MDH2) and cytosolic malic enzyme can turn cytosolic citrate into pyruvate, to feed back into the TCA cycle to feed further lipid synthesis (
The inventors further hypothesized that expression of a heterologous NADP+ dependent cytosolic malic enzyme may increase fatty acid production if the primary rate limitation is cofactor supply. Surprisingly, the inventors also discovered that expression of a heterologous malic enzyme improved the Z11-16 acid selectivity of the recombinant microorganisms.
Malate dehydrogenase (decarboxylating) or NAD-malic enzyme (NAD-ME) is an enzyme that catalyzes the chemical reaction:
(S)-malate+NAD+⇄pyruvate+CO2+NADH
Thus, the two substrates of this enzyme are (S)-malate and NAD+, whereas its three products are pyruvate, CO2, and NADH. Malate is oxidized to pyruvate and CO2, and NAD+ is reduced to NADH.
This enzyme belongs to the family of oxidoreductases, to be specific, those acting on the CH—OH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is (S)-malate:NAD+ oxidoreductase (decarboxylating). This enzyme participates in pyruvate metabolism and carbon fixation. NAD-malic enzyme is one of three decarboxylation enzymes used in the inorganic carbon concentrating mechanisms of C4 and CAM plants. The others are NADP-malic enzyme and PEP carboxykinase.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one exogenous nucleic acid molecule encoding a decarboxylating malic enzyme in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a decarboxylating malic enzyme from Arabidopsis thaliana, Amaranthus hypochondriacus, Rhizobium meliloti, Solanum tuberosum, Homo sapiens, or Escherichia coli. In further embodiments, the decarboxylating malic enzyme is selected from Arabidopsis thaliana Q9SIU0, Amaranthus hypochondriacus P37224, Rhizobium meliloti 030807, Solanum tuberosum P37221, Homo sapiens Q16798, and Escherichia coli P26616, or homolog thereof. In yet a further embodiment, the decarboxylating malic enzyme lacks a sequence encoding a mitochondrial-targeting peptide.
ATP-Citrate Lyase
ATP citrate lyase is an enzyme that represents an important step in fatty acid biosynthesis. ATP citrate lyase is important in that, by converting citrate to acetyl CoA, it links the metabolism of carbohydrates, which yields citrate as an intermediate, and the production of fatty acids, which requires acetyl CoA. ATP-citrate lyase is responsible for catalyzing the conversion of citrate and CoA into acetyl-CoA and oxaloacetate, along with the hydrolysis of ATP.
ATP citrate lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. The enzyme is a tetramer of apparently identical subunits. The product, acetyl-CoA, serves several important biosynthetic pathways, including lipogenesis and cholesterogenesis. It is activated by insulin.
In some embodiments, the one or more modifications associated with one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an ATP-citrate lyase in the recombinant microorganism. In certain embodiments, the one or more metabolic intermediates availability comprises cytosolic oxaloacetate availability. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ATP-citrate lyase from Saccharomyces cerevisiae, Yarrowia lipolytica, Mus musculus, and Aspergillus niger. In a yet further embodiment, the ATP-citrate lyase is selected from Mus musculus NP_001186225.1, Mus musculus NP_598798.1, Aspergillus niger XP_001394055.1, and Aspergillus niger XP_001394057.1, or homolog thereof.
Hexose Transporter
The hexose transporters belong to a transporter superfamily termed the major facilitator superfamily. The members of this superfamily include a variety of sugar transporters and transporters of other carbon compounds in eukaryotes as well as prokaryotes. The yeast hexose transporters form a subfamily. Twenty hexose transporter proteins are found in S. cerevisiae.
Glucose-dependent modulation of the affinity of glucose transport in wild-type cells can depend upon a number of factors, including the regulation of expression of various sets of Hxtp proteins with significantly different affinities to the sugar, the removal and inactivation of transporter proteins under certain conditions, the modulation of the affinity of specific transporters and, possibly, interactions between the different transporter proteins.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises decoupling and increasing glucose uptake in the recombinant microorganism. In certain embodiments, decoupling and increasing glucose uptake comprises: upregulation of hexose transporter activity. In further embodiments, the upregulation of one or more hexose transporter activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter operably linked to one or more heterologous promoters. In some embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a hexose transporter from Yarrowia lipolytica. In certain embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter is selected from YALI0A14212g, YALI0D01111g, YALI0D00363g, YALI0C16522g, and YALI0F25553g, or homolog thereof.
Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase 1 catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first committed and rate-limiting step in de novo fatty acid biosynthesis. Malonyl-CoA is used as a building block to extend the chain length of fatty acids by fatty acid synthase.
Two isoforms exist in mammals, acetyl-CoA carboxylase 1 and acetyl-CoA carboxylase 2. Both are dimeric, multifunctional enzymes composed of four distinct domains; a biotin carboxylase (BC) domain, a biotin carboxyl carrier protein (BCCP) domain, the ACC central region, and a carboxyltransferase (CT) domain.
The function of ACC is to regulate the metabolism of fatty acids. When the enzyme is active, the product, malonyl-CoA, is produced which is a building block for new fatty acids and can inhibit the transfer of the fatty acyl group from acyl CoA to carnitine with carnitine acyltransferase, which inhibits the beta-oxidation of fatty acids in the mitochondria.
The regulation of mammalian ACC is complex, in order to control two distinct pools of malonyl CoA that direct either the inhibition of beta oxidation or the activation of lipid biosynthesis.
Mammalian ACC1 and ACC2 are regulated transcriptionally by multiple promoters which mediate ACC abundance in response to the cells nutritional status. Activation of gene expression through different promoters results in alternative splicing. The sensitivity to nutritional status results from the control of these promoters by transcription factors such as SREBP1c, controlled by insulin at the transcriptional level, and ChREBP, which increases in expression with high carbohydrates diets.
Through a feedforward loop, citrate allosterically activates ACC. Citrate may increase ACC polymerization to increase enzymatic activity. Other allosteric activators include glutamate and other dicarboxylic acids. Long and short chain fatty acyl CoAs are negative feedback inhibitors of ACC.
Phosphorylation can result when the hormones glucagon or epinephrine bind to cell surface receptors, but the main cause of phosphorylation is due to a rise in AMP levels when the energy status of the cell is low, leading to the activation of the AMP-activated protein kinase (AMPK). AMPK is the main kinase regulator of ACC, able to phosphorylate a number of serine residues on both isoforms of ACC. On ACC1, AMPK phosphorylates Ser79, Ser1200, and Ser1215. Protein kinase A also has the ability to phosphorylate ACC, with a much greater ability to phosphorylate ACC2 than ACC1. Researchers hypothesize there are other ACC kinases important to its regulation as there are many other possible phosphorylation sites on ACC.
When insulin binds to its receptors on the cellular membrane, it activates a phosphatase enzyme called protein phosphatase 2A (PP2A) to dephosphorylate the enzyme, thereby removing the inhibitory effect. Furthermore, insulin induces a phosphodiesterase that lowers the level of cAMP in the cell, thus inhibiting PKA, and also inhibits AMPK directly.
In some embodiments, the one or more modifications associated with lipid synthesis comprises alleviation of acetyl-CoA carboxylase (ACC) inhibition. In certain embodiments, alleviation of ACC inhibition comprises the replacement of the endogenous ACC, or overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a feedback-insensitive ACC variant in the recombinant microorganism. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ACC from Mus musculus, Rattus norvegicus, or Homo sapiens.
In some embodiments, the one or more modifications associated with reducing equivalent availability, one or more metabolic intermediates availability, or increased product purity comprises downregulation or inhibition of acetyl-CoA carboxylase (ACC) activity in the recombinant microorganism. In certain embodiments, the downregulation or inhibition of ACC activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more ACC enzyme.
Hexose Kinase
A hexose kinase is an enzyme that phosphorylates hexoses (six-carbon sugars), forming hexose phosphate. In most organisms, glucose is the most important substrate of hexose kinases, and glucose-6-phosphate is the most important product. Hexose kinase also possesses the ability of transferring an inorganic phosphate group from ATP to a substrate.
They are categorized as actin fold proteins, sharing a common ATP binding site core that is surrounded by more variable sequences which determine substrate affinities and other properties.
Most bacterial hexose kinases are approximately 50 kD in size. Multicellular organisms such as plants and animals often have more than one hexose kinase isoform. Most are about 100 kD in size and consist of two halves (N and C terminal), which share much sequence homology.
By catalyzing the phosphorylation of glucose to yield glucose 6-phosphate, hexose kinases maintain the downhill concentration gradient that favors the facilitated transport of glucose into cells. This reaction also initiates all physiologically relevant pathways of glucose utilization, including glycolysis and the pentose phosphate pathway. The addition of a charged phosphate group at the 6-position of hexoses also ensures ‘trapping’ of glucose and 2-deoxyhexose glucose analogs (e.g. 2-deoxyglucose, and 2-fluoro-2-deoxyglucose) within cells, as charged hexose phosphates cannot easily cross the cell membrane.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, one or more modifications in the PPP comprises downregulation of hexose kinase activity. In yet a further embodiment, the downregulation of hexose kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises decoupling and increasing glucose uptake in the recombinant microorganism. In certain embodiments, decoupling and increasing glucose uptake comprises downregulation of hexose kinase activity. In some embodiments, the downregulation of hexose kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme.
Fructose-6-Phosphate Kinase
Fructose-6-phosphate kinase (phosphofructokinase) is a kinase enzyme that phosphorylates fructose 6-phosphate in glycolysis.
The enzyme-catalyzed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. Fructose-6-phosphate kinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, a key regulatory step in the glycolytic pathway. It is allosterically inhibited by ATP and allosterically activated by AMP, thus indicating the cell's energetic needs when it undergoes the glycolytic pathway. PFK exists as a homotetramer in bacteria and mammals (where each monomer possesses 2 similar domains) and as an octamer in yeast (where there are 4 alpha-(PFK1) and 4 beta-chains (PFK2), the latter, like the mammalian monomers, possessing 2 similar domains). This protein may use the morpheein model of allosteric regulation.
PFK is about 300 amino acids in length, and structural studies of the bacterial enzyme have shown it comprises two similar (alpha/beta) lobes: one involved in ATP binding and the other housing both the substrate-binding site and the allosteric site.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, the one or more modifications in the PPP comprises downregulation of fructose-6-phosphate kinase activity. In yet a further embodiment, the downregulation of fructose-6-phosphate kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more fructose-6-phosphate kinase enzyme.
NADPH-Dependent Mannitol Dehydrogenase
Mannitol 2-dehydrogenase (NADP+) is an enzyme that catalyzes the chemical reaction:
D-mannitol+NADP+⇄D-fructose+NADPH+H+
Thus, the two substrates of this enzyme are D-mannitol and NADP+, whereas its 3 products are D-fructose, NADPH, and H+.
This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH—OH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is D-mannitol:NADP+2-oxidoreductase. This enzyme is also called mannitol 2-dehydrogenase (NADP+). This enzyme participates in fructose and mannose metabolism.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises downregulation of mannitol synthesis pathway in the recombinant microorganism. In certain embodiments, downregulation of mannitol synthesis pathway comprises deletion, disruption, and/or mutation of one or more gene encoding an NADPH-dependent mannitol dehydrogenase. In further embodiments, the one or more gene encoding an NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homolog thereof
Aldo-Keto Reductase
Aldo-keto reductases (AKR) comprise a superfamily of structurally-similar proteins that catalyze the NADPH-dependent conversion of various carbonyl compounds into their corresponding alcohol products.
The mannitol synthesis pathway comprises three unique enzymatic steps: (a) isomerization of fructose-6-phosphate to mannose-6-phosphate by mannose-6-phosphate isomerase; (b) reduction of mannose-6-phosphate to mannitol-1-phosphate by mannose-6-phosphate reductase; and (c) dephosphorylation of mannitol-1-phosphate to mannitol by mannitol-1-phosphate phosphatase. Radiotracer studies and kinetic analyses suggest that mannose-6-phosphate reductase, an aldo-keto reductase, plays a regulatory role in this pathway. In Yarrowia, a fructose-6-phosphate phosphatase produces fructose which is reduced by mannitol dehydrogenase into mannitol, and NADP.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises downregulation of mannitol synthesis pathway in the recombinant microorganism. In certain embodiments, downregulation of mannitol synthesis pathway comprises deletion, disruption, and/or mutation of one or more gene encoding an aldo-keto reductase. In further embodiments, the one or more gene encoding an NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homolog thereof. In further embodiments, the one or more gene encoding an aldo-keto reductase is selected from YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0009119g, YALI0D04092g, YALI0B15268g, YALI0000319g, and YALI0A19910g, or homolog thereof.
Pentose Phosphate Pathway
The pentose phosphate pathway (PPP) is a central and widely conserved metabolic pathway of carbohydrate metabolism located in the cytoplasm in eukaryotic cells. This pathway serves two major functions: production of precursors for biosynthesis of macromolecules and production of reducing equivalents in the form of NADPH. Accordingly, these two roles are reflected in the two major phases of the PPP: in the “oxidative phase,” glucose 6-phosphate (G6P) is converted into ribulose 5-phosphate (RuSP) through the sequential action of: glucose-6-phosphate dehydrogenase (ZWF1) which converts G6P to 6-phospho D-glucono-1,5-lactone with generation of NADPH; 6-phosphogluconolactonase (SOL3, SOL4) which converts 6-phospho D-glucono-1,5-lactone to D-gluconate 6-phosphate; 6-phosphogluconate dehydrogenase (GND1, GND2) which converts D-gluconate 6-phosphate to RuSP with generation of NADPH. The “non-oxidative phase” carries out the isomerization of RuSP to ribose 5-phosphate (R5P), the epimerization of RuSP to xylulose 5-phosphate (X5P) and, through the actions of transketolase (TKL1, TKL2) and transaldolase (TAL1, NQM1), a series of carbon skeleton transfers that can interconvert pentose phosphate into fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (GAP)—both glycolytic intermediates—and erythrose 4-phosphate (E4P). The net effect of the non-oxidative phase is to produce an equilibrium between the pentoses needed for biosynthesis of macromolecules and the hexoses needed for energy management, allowing the two pools of sugars easily to interconvert.
The oxidative branch is considered to be largely irreversible under normal cellular conditions, whilst the non-oxidative branch is reversible. The PPP is not a simple linear pathway since several carbon atoms are recycled back into glycolysis. Furthermore, the enzyme transketolase catalyses two different reactions in the pathway, resulting in the substrates of these reactions being competitive inhibitors of one another. The PPP has three main products: reduced equivalents in the form of NADPH, produced in the oxidative phase, needed in biosynthetic pathways and for maintenance of the oxidative level of cells; RSP, for the biosynthesis of all nucleic acids; and E4P, for biosynthesis of the three aromatic amino acids. Different physiological states require operation of this biochemical network in different modes: in actively growing cells, such as during culture growth in reactors, the pathway must produce a sufficient amount of all three products, since all are required in the construction of new cells. Under stress conditions growth slows and the only product in considerable demand is NADPH.
The inventors hypothesized that the recombinant microorganism's lipid production could further be increased by overexpressing the genes of the upper (oxidative) pentose phosphate pathway (ZWF1, SOL3, and GND1), to increase NADPH supply for fatty acid biosynthesis. Surprisingly, the inventors discovered that upregulation of ZWF1, SOL3, and/or GND1 resulted in improved Z11-16 selectivity in recombinant microorganisms.
Enzymes to Express to Increase Levels of One or More Coenzymes
Nicotinamide adenine dinucleotide (NAD, including NAD+ and NADH) and nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: NAD mediates energy metabolism and mitochondrial functions; NADPH is a key component in cellular antioxidation systems; NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Additionally, the in situ regeneration of reduced nicotinamide cofactors (NAD(P)H) is necessary for practical synthesis of many important chemicals in recombinant microorganisms.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, the one or more modifications in the PPP comprises upregulation of one or more oxidative PPP enzyme activity. In certain embodiments, the one or more modifications in the PPP comprises expression of one or more oxidative PPP enzyme variant. In further embodiments, the upregulation of one or more oxidative PPP enzyme activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a glucose-6-phosphate dehydrogenase (ZWF1), a 6-phosphogluconolactonase (SOL3), or a 6-phosphogluconate dehydrogenase (GND1).
In some embodiments, the one or more oxidative PPP enzyme variant comprises one or more endogenous and/or exogenous nucleic acid molecule encoding an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) and/or an NAD-dependent 6-phosphogluconate dehydrogenase (GND1). In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) from Leuconostoc. In further embodiments, the NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) is selected from Leuconostoc AAA25265.1 and Leuconostoc P11411, or homolog thereof. In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent 6-phosphogluconate dehydrogenase (GND1) from Bradyrhizobium or Methylobacillus. In further embodiments, the NAD-dependent 6-phosphogluconate dehydrogenase (GND1) is selected from Bradyrhizobium WP_012029377.1, Bradyrhizobium A4YZZ8, Methylobacillus AAF34407.1, and Methylobacillus Q9L9P8, or homolog thereof.
Dehydrogenase
In some embodiments, the recombinant microorganisms of the present disclosure are engineered to reduce or eliminate expression of one or more endogenous fatty alcohol dehydrogenases (i.e. FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7). In some embodiments, the recombinant microorganisms of the present disclosure are engineered comprise deletions of one or more endogenous fatty alcohol dehydrogenases (i.e. FADH, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7). For example, in some embodiments, the recombinant microorganism is a Y. lipolytica strain, wherein the recombinant microorganism comprises reduced or eliminated activities of relevant (fatty) alcohol dehydrogenases selected from FADH: YALI0F09603g, ADH1: YALI0D25630g, ADH2: YALI0E17787g, ADH3: YALI0A16379g, ADH4: YALI0E15818g, ADH5: YALI0D02167g, ADH6: YALI0A15147g and/or ADH7: YALI0E07766g.
The present disclosure describes enzymes that catalyze the conversion of a fatty aldehyde to a fatty alcohol in various organisms. In some embodiments, an alcohol dehydrogenase (ADH, Table 6.1) is used to catalyze the conversion of a fatty aldehyde to a fatty alcohol. A number of ADHs identified from alkanotrophic organisms, Pseudomonas fluorescens NRRL B-1244 (Hou et al. 1983), Pseudomonas butanovora ATCC 43655 (Vangnai and Arp 2001), and Acinetobacter sp. strain M-1 (Tani et al. 2000), have shown to be active on short to medium-chain alkyl alcohols (C2 to C14). Additionally, commercially available ADHs from Sigma, Horse liver ADH and Baker's yeast ADH have detectable activity for substrates with length Cm and greater. The reported activities for the longer fatty alcohols may be impacted by the difficulties in solubilizing the substrates. For the yeast ADH from Sigma, little to no activity is observed for C12 to C14 aldehydes by (Tani et al. 2000), however, activity for C12 and C16 hydroxy-ω-fatty acids has been observed (Lu et al. 2010). Recently, two ADHs were characterized from Geobacillus thermodenitrificans NG80-2, an organism that degrades C15 to C36 alkanes using the LadA hydroxylase. Activity was detected from methanol to 1-triacontanol (C30) for both ADHs, with 1-octanol being the preferred substrate for ADH2 and ethanol for ADH1 (Liu et al. 2009).
The use of ADHs in whole-cell bioconversions has been mostly focused on the production of chiral alcohols from ketones (Ernst et al. 2005) (Schroer et al. 2007). Using the ADH from Lactobacillus brevis and coupled cofactor regeneration with isopropanol, Schroer et al. reported the production of 797 g of (R)-methyl-3 hydroxybutanoate from methyl acetoacetate, with a space time yield of 29 g/L/h (Schroer et al. 2007). Examples of aliphatic alcohol oxidation in whole-cell transformations have been reported with commercially obtained S. cerevisiae for the conversion of hexanol to hexanal (Presecki et al. 2012) and 2-heptanol to 2-heptanone (Cappaert and Larroche 2004).
Bactrocera oleae (Olive fruit fly) (Dacus oleae)
Cupriavidus necator (Alcaligenes eutrophus)
Drosophila adiastola (Fruit fly) (Idiomyia
adiastola)
Drosophila affinidisjuncta (Fruit fly) (Idiomyia
affini disjuncta)
Drosophila ambigua (Fruit fly)
Drosophila borealis (Fruit fly)
Drosophila differens (Fruit fly)
Drosophila equinoxialis (Fruit fly)
Drosophila flavomontana (Fruit fly)
Drosophila guanche (Fruit fly)
Drosophila hawaiiensis (Fruit fly)
Drosophila heteroneura (Fruit fly)
Drosophila immigrans (Fruit fly)
Drosophila insularis (Fruit fly)
Drosophila lebanonensis (Fruit fly)
Drosophila mauritiana (Fruit fly)
Drosophila madeirensis (Fruit fly)
Drosophila mimica (Fruit fly) (Idiomyia mimica)
Drosophila nigra (Fruit fly) (Idiomyia nigra)
Drosophila orena (Fruit fly)
Drosophila pseudoobscura bogotana (Fruit fly)
Drosophila picticomis (Fruit fly) (Idiomyia
picticomis)
Drosophila planitibia (Fruit fly)
Drosophila paulistorum (Fruit fly)
Drosophila silvestris (Fruit fly)
Drosophila subobscura (Fruit fly)
Drosophila teissieri (Fruit fly)
Drosophila tsacasi (Fruit fly)
Fragaria ananassa (Strawberry)
Malus domestica (Apple) (Pyrus malus)
Scaptomyza albovittata (Fruit fly)
Scaptomyza crassifemur (Fruit fly) (Drosophila
crassifemur)
Sulfolobus sp. (strain RC3)
Zaprionus tuberculatus (Vinegar fly)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Drosophila mayaguana (Fruit fly)
Drosophila melanogaster (Fruit fly)
Drosophila pseudoobscura pseudoobscura (Fruit
Drosophila simulans (Fruit fly)
Drosophila yakuba (Fruit fly)
Drosophila ananassae (Fruit fly)
Drosophila erecta (Fruit fly)
Drosophila grimshawi (Fruit fly) (Idiomyia
grimshawi)
Drosophila willistoni (Fruit fly)
Drosophila persimilis (Fruit fly)
Drosophila sechellia (Fruit fly)
Cupriavidus necator (strain ATCC 17699 / H16 /
Mycobacterium tuberculosis (strain CDC 1551 /
Oshkosh)
Staphylococcus aureus (strain MW2)
Mycobacterium tuberculosis (strain ATCC 25618 /
Staphylococcus aureus (strain N315)
Staphylococcus aureus (strain bovine RF122 /
Sulfolobus acidocaldarius (strain ATCC 33909 /
Staphylococcus aureus (strain COL)
Staphylococcus aureus (strain NCTC 8325)
Staphylococcus aureus (strain MRSA252)
Staphylococcus aureus (strain M55A476)
Staphylococcus aureus (strain USA300)
Staphylococcus aureus (strain Mu50 / ATCC
Staphylococcus epidermidis (strain ATCC 12228)
Staphylococcus epidermidis (strain ATCC 35984 /
Sulfolobus solfataricus (strain ATCC 35092 / DSM
Sulfolobus tokodaii (strain DSM 16993 / JCM
Anas platyrhynchos (Domestic duck) (Anas
boschas)
Apteryx australis (Brown kiwi)
Ceratitis capitata (Mediterranean fruit fly)
Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
Gallus gallus (Chicken)
Columba livia (Domestic pigeon)
Coturnix coturnix japonica (Japanese quail)
Drosophila hydei (Fruit fly)
Drosophila montana (Fruit fly)
Drosophila mettleri (Fruit fly)
Drosophila mulleri (Fruit fly)
Drosophila navojoa (Fruit fly)
Geomys attwateri (Attwater's pocket gopher)
Geomys bursarius (Plains pocket gopher)
Geomys knoxjonesi (Knox Jones's pocket gopher)
Hordeum vulgare (Barley)
Kluyveromyces marxianus (Yeast) (Candida kefyr)
Zea mays (Maize)
Mesocricetus auratus (Golden hamster)
Pennisetum americanum (Pearl millet) (Pennisetum
glaucum)
Petunia hybrida (Petunia)
Oryctolagus cuniculus (Rabbit)
Solanum tuberosum (Potato)
Struthio camelus (Ostrich)
Trifolium repens (Creeping white clover)
Zea luxurians (Guatemalan teosinte) (Euchlaena
luxurians)
Saccharomyces cerevisiae (strain ATCC 204508 /
Arabidopsis thaliana (Mouse-ear cress)
Schizosaccharomyces pombe (strain 972 / ATCC
Drosophila lacicola (Fruit fly)
Mus musculus (Mouse)
Peromyscus maniculatus (North American deer
Rattus norvegicus (Rat)
Drosophila virilis (Fruit fly)
Scheffersomyces stipitis (strain ATCC 58785 /
Aspergillus flavus (strain ATCC 200026 / FGSC
Neurospora crassa (strain ATCC 24698 / 74-
Candida albicans (Yeast)
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585 / CBS
Oryza sativa subsp. indica (Rice)
Pongo abelii (Sumatran orangutan) (Pongo
pygmaeus abelii)
Homo sapiens (Human)
Macaca mulatta (Rhesus macaque)
Pan troglodytes (Chimpanzee)
Papio hamadryas (Hamadryas baboon)
Homo sapiens (Human)
Homo sapiens (Human)
Papio hamadryas (Hamadryas baboon)
Ceratitis capitata (Mediterranean fruit fly)
Ceratitis cosyra (Mango fruit fly) (Trypeta cosyra)
Ceratitis rosa (Natal fruit fly) (Pterandrus rosa)
Drosophila arizonae (Fruit fly)
Drosophila buzzatii (Fruit fly)
Drosophila hydei (Fruit fly)
Drosophila montana (Fruit fly)
Drosophila mulleri (Fruit fly)
Drosophila wheeleri (Fruit fly)
Entamoeba histolytica
Hordeum vulgare (Barley)
Kluyveromyces marxianus (Yeast) (Candida kefyr)
Zea mays (Maize)
Oryza sativa subsp. indica (Rice)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum tuberosum (Potato)
Scheffersomyces stipitis (strain ATCC 58785 /
Arabidopsis thaliana (Mouse-ear cress)
Saccharomyces cerevisiae (strain ATCC 204508 /
Candida albicans (strain SC5314 / ATCC MYA-
Oryza sativa subsp. japonica (Rice)
Drosophila mojavensis (Fruit fly)
Kluyveromyces lactis (strain ATCC 8585 / CBS
Oryctolagus cuniculus (Rabbit)
Oryctolagus cuniculus (Rabbit)
Hordeum vulgare (Barley)
Solanum tuberosum (Potato)
Kluyveromyces lactis (strain ATCC 8585 / CBS
Saccharomyces cerevisiae (strain ATCC 204508 /
Homo sapiens (Human)
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Struthio camelus (Ostrich)
Kluyveromyces lactis (strain ATCC 8585 / CBS
Schizosaccharomyces pombe (strain 972 / ATCC
Saccharomyces cerevisiae (strain YJM789)
Saccharomyces cerevisiae (strain ATCC 204508 /
Saccharomyces pastorianus (Lager yeast)
eubayanus)
Bos taurus (Bovine)
Equus caballus (Horse)
Mus musculus (Mouse)
Rattus norvegicus (Rat)
Oryctolagus cuniculus (Rabbit)
Homo sapiens (Human)
Dictyostelium discoideum (Slime mold)
Saccharomyces cerevisiae (strain ATCC 204508 /
Homo sapiens (Human)
Peromyscus maniculatus (North American deer
Pongo abelii (Sumatran orangutan) (Pongo
pygmaeus abelii)
Rattus norvegicus (Rat)
Homo sapiens (Human)
Rattus norvegicus (Rat)
Mus musculus (Mouse)
Mycobacterium tuberculosis (strain CDC 1551 /
Rhizobium meliloti (strain 1021) (Ensifer meliloti)
Mycobacterium tuberculosis (strain ATCC 25618 /
Zymomonas mobilis subsp. mobilis (strain ATCC
Mycobacterium bovis (strain ATCC BAA-935 /
Mycobacterium tuberculosis (strain CDC 1551 /
Oshkosh)
Mycobacterium tuberculosis (strain ATCC 25618 /
Zymomonas mobilis subsp. mobilis (strain ATCC
Zymomonas mobilis subsp. mobilis (strain ATCC
Mycobacterium tuberculosis (strain CDC 1551 /
Mycobacterium tuberculosis (strain ATCC 25618 /
Clostridium acetobutylicum (strain ATCC 824 /
Escherichia coli (strain K12)
Escherichia coli O157:H7
Rhodobacter sphaeroides (strain ATCC 17023 /
Oryza sativa subsp. indica (Rice)
Escherichia coli (strain K12)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Emericella nidulans (strain FGSC A4 / ATCC
Emericella nidulans (strain FGSC A4 / ATCC
Emericella nidulans (strain FGSC A4 / ATCC
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Zea mays (Maize)
Drosophila melanogaster (Fruit fly)
Bacillus subtilis (strain 168)
Caenorhabditis elegans
Oryza
sativa subsp. japonica (Rice)
Mycobacterium tuberculosis (strain ATCC 25618 /
Caenorhabditis elegans
Caenorhabditis elegans
Pseudomonas sp.
Escherichia coli (strain K12)
Moraxella sp. (strain TAE123)
Alligator mississippiensis (American alligator)
Catharanthus roseus (Madagascar periwinkle)
Gadus morhua subsp. callarias (Baltic cod)
Naja naja (Indian cobra)
Pisum sativum (Garden pea)
Pelophylax perezi (Perez's frog) (Rana perezi)
Saara hardwickii (Indian spiny-tailed lizard)
Saara hardwickii (Indian spiny-tailed lizard)
Equus caballus (Horse)
Equus caballus (Horse)
Geobacillus stearothermophilus (Bacillus
stearothermophilus)
Gadus morhua (Atlantic cod)
Gadus morhua (Atlantic cod)
Myxine glutinosa (Atlantic hagfish)
Octopus vulgaris (Common octopus)
Pisum sativum (Garden pea)
Saara hardwickii (Indian spiny-tailed lizard)
Scyliorhinus canicula (Small-spotted catshark)
Sparus aurata (Gilthead sea bream)
Acyl-CoA Oxidases
In some embodiments, the present disclosure teaches recombinant microorganisms that are engineered to reduce or eliminate the expression or activity of one or more of the endogenous acyl-CoA oxidases (i.e. POX1, POX2, POX3, POX4, POX5, and POX6). In some embodiments, recombinant microorganisms of the present disclosure are engineered to comprise deletions of endogenous acyl-CoA oxidases (i.e. POX1, POX2, POX3, POX4, POX5, and POX6). For example, in some embodiments, the recombinant microorganism is a Y. lipolytica strain exhibiting a reduction in the activities of acyl-CoA oxidases (namely POX1: YALI0E32835g, POX2: YALI0F10857g, POX3: YALI0D24750g, POX4: YALI0E27654g, POX5: YALI0C23859g, POX6: YALI0E06567g).
Alcohol Oxidase
In some embodiments, the present disclosure teaches recombinant microorganisms that are engineered to reduce or eliminate the expression or activity of endogenous fatty alcohol oxidase (FAO1). In some embodiments, recombinant microorganisms of the present disclosure are engineered to comprise deletions of of endogenous fatty alcohol oxidase (FAO1). For example, in some embodiments, the recombinant microorganism is Y. lipolytica, and the microorganism comprises reduced activity of relevant (fatty) alcohol oxidases, namely FAO1: YALI0B14014g).
In some embodiments, an alcohol oxidase (AOX) is used to catalyze the conversion of a fatty alcohol to a fatty aldehyde. Alcohol oxidases catalyze the conversion of alcohols into corresponding aldehydes (or ketones) with electron transfer via the use of molecular oxygen to form hydrogen peroxide as a by-product. AOX enzymes utilize flavin adenine dinucleotide (FAD) as an essential cofactor and regenerate with the help of oxygen in the reaction medium. Catalase enzymes may be coupled with the AOX to avoid accumulation of the hydrogen peroxide via catalytic conversion into water and oxygen.
Based on the substrate specificities, AOXs may be categorized into four groups: (a) short chain alcohol oxidase, (b) long chain alcohol oxidase, (c) aromatic alcohol oxidase, and (d) secondary alcohol oxidase (Goswami et al. 2013). Depending on the chain length of the desired substrate, some members of these four groups are better suited than others as candidates for evaluation.
Short chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.13, Table 6.2) catalyze the oxidation of lower chain length alcohol substrates in the range of C1-C8 carbons (van der Klei et al. 1991) (Ozimek et al. 2005). Aliphatic alcohol oxidases from methylotrophic yeasts such as Candida boidinii and Komagataella pastoris (formerly Pichia pastoris) catalyze the oxidation of primary alkanols to the corresponding aldehydes with a preference for unbranched short-chain aliphatic alcohols. The most broad substrate specificity is found for alcohol oxidase from the Pichia pastoris including propargyl alcohol, 2-chloroethanol, 2-cyanoethanol (Dienys et al. 2003). The major challenge encountered in alcohol oxidation is the high reactivity of the aldehyde product. Utilization of a two liquid phase system (water/solvent) can provide in-situ removal of the aldehyde product from the reaction phase before it is further converted to the acid. For example, hexanal production from hexanol using Pichia pastoris alcohol oxidase coupled with bovine liver catalase was achieved in a bi-phasic system by taking advantage of the presence of a stable alcohol oxidase in aqueous phase (Karra-Chaabouni et al. 2003). For example, alcohol oxidase from Pichia pastoris was able to oxidize aliphatic alcohols of C6 to C11 when used biphasic organic reaction system (Murray and Duff 1990). Methods for using alcohol oxidases in a biphasic system according to (Karra-Chaabouni et al. 2003) and (Murray and Duff 1990) are incorporated by reference in their entirety.
Long chain alcohol oxidases (including but not limited to those currently classified as EC 1.1.3.20; Table 6.3) include fatty alcohol oxidases, long chain fatty acid oxidases, and long chain fatty alcohol oxidases that oxidize alcohol substrates with carbon chain length of greater than six (Goswami et al. 2013). Banthorpe et al. reported a long chain alcohol oxidase purified from the leaves of Tanacetum vulgare that was able to oxidize saturated and unsaturated long chain alcohol substrates including hex-trans-2-en-1-ol and octan-1-ol (Banthorpe 1976) (Cardemil 1978). Other plant species, including Simmondsia chinensis (Moreau, R. A., Huang 1979), Arabidopsis thaliana (Cheng et al. 2004), and Lotus japonicas (Zhao et al. 2008) have also been reported as sources of long chain alcohol oxidases. Fatty alcohol oxidases are mostly reported from yeast species (Hommel and Ratledge 1990) (Vanhanen et al. 2000) (Hommel et al. 1994) (Kemp et al. 1990) and these enzymes play an important role in long chain fatty acid metabolism (Cheng et al. 2005). Fatty alcohol oxidases from yeast species that degrade and grow on long chain alkanes and fatty acid catalyze the oxidation of fatty alcohols. Fatty alcohol oxidase from Candida tropicalis has been isolated as microsomal cell fractions and characterized for a range of substrates (Eirich et al. 2004) (Kemp et al. 1988) (Kemp et al. 1991) (Mauersberger et al. 1992). Significant activity is observed for primary alcohols of length C8 to C16 with reported KM in the 10-50 μM range (Eirich et al. 2004). Alcohol oxidases described may be used for the conversion of medium chain aliphatic alcohols to aldehydes as described, for example, for whole-cells Candida boidinii (Gabelman and Luzio 1997), and Pichia pastoris (Duff and Murray 1988) (Murray and Duff 1990). Long chain alcohol oxidases from filamentous fungi were produced during growth on hydrocarbon substrates (Kumar and Goswami 2006) (Savitha and Ratledge 1991). The long chain fatty alcohol oxidase (LjFAO1) from Lotus japonicas has been heterologously expressed in E. coli and exhibited broad substrate specificity for alcohol oxidation including 1-dodecanol and 1-hexadecanol (Zhao et al. 2008).
Komagataella pastoris (strain ATCC 76273 / CBS 7435 /
Komagataella pastoris (strain GS115 / ATCC 20864)
Komagataella pastoris (strain ATCC 76273 / CBS 7435 /
Komagataella pastoris (strain GS115 / ATCC 20864)
Candida boidinii (Yeast)
Pichia angusta (Yeast) (Hansenula polymorpha)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Thanatephorus cucumeris (strain AG1-IB / isolate 7/3/14)
Ogataea henricii
Candida methanosorbosa
Candida methanolovescens
Candida succiphila
Aspergillus niger (strain CBS 513.88 / FGSC A1513)
Aspergillus niger (strain CBS 513.88 / FGSC A1513)
Moniliophthora pemiciosa (Witches'-broom disease fungus)
Candida cariosilignicola
Candida pignaliae
Candida pignaliae
Candida sonorensis
Candida sonorensis
Pichia naganishii
Ogataea minuta
Ogataea philodendra
Ogataea wickerhamii
Kuraishia capsulate
Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 /
Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 /
Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 /
Talaromyces stipitatus (strain ATCC 10500 / CBS 375.48 /
Ogataea glucozyma
Ogataea parapolymorpha (strain DL-1 / ATCC 26012 /
Gloeophyllum trabeum (Brown rot fungus)
Pichia angusta (Yeast) (Hansenula polymorpha)
Pichia trehalophila
Pichia angusta (Yeast) (Hansenula polymorpha)
Pichia angusta (Yeast) (Hansenula polymorpha)
Ixodes scapularis (Black-legged tick) (Deer tick)
Lotus japonicus (Lotus comiculatus var.
japonicus)
Arabidopsis thaliana (Mouse-ear cress)
Lotus japonicus (Lotus comiculatus var.
japonicus)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Arabidopsis thaliana (Mouse-ear cress)
Microbotryum violaceum (strain p1A1 Lamole)
Ajellomyces dermatitidis ATCC 26199
Gibberella zeae (strain PH-1 / ATCC MYA-4620 /
fungus) (Fusarium graminearum)
Pichia sorbitophila (strain ATCC MYA-4447 /
Emericella nidulans (strain FGSC A4 / ATCC
Pyrenophora tritici-repentis (strain Pt-1C-BFP)
repentis)
Paracoccidioides lutzii (strain ATCC MYA-826 /
Candida parapsilosis (strain CDC 317 / ATCC
Pseudozyma brasiliensis (strain GHG001) (Yeast)
Candida parapsilosis (strain CDC 317 / ATCC
Sclerotinia borealis F-4157
Sordaria macrospora (strain ATCC MYA-333 /
Sordaria macrospora (strain ATCC MYA-333 /
Meyerozyma guilliermondii (strain ATCC 6260 /
Trichophyton rubrum CBS 202.88
Arthrobotrys oligospora (strain ATCC 24927 /
fungus) (Didymozoophaga oligospora)
Scheffersomyces stipitis (strain ATCC 58785 /
Scheffersomyces stipitis (strain ATCC 58785 /
Aspergillus oryzae (strain 3.042) (Yellow koji
Fusarium oxysporum (strain Fo5176) (Fusarium
vascular wilt)
Rhizopus delemar (strain RA 99-880 / ATCC
Rhizopus
delemar (strain RA 99-880 / ATCC
Fusarium oxysporum (strain Fo5176) (Fusarium
vascular wilt)
Penicillium roqueforti
Aspergillus clavatus (strain ATCC 1007 / CBS
Arthroderma otae (strain ATCC MYA-4605 /
Trichophyton tonsurans (strain CBS 112818)
Colletotrichum higginsianum (strain IMI 349063)
Ajellomyces capsulatus (strain H143) (Darling's
disease
fungus) (Histoplasma capsulatum)
Trichophyton rubrum (strain ATCC MYA-4607 /
Cochliobolus heterostrophus (strain C5 / ATCC
fungus) (Bipolaris maydis)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Candida orthopsilosis (strain 90-125) (Yeast)
Pseudozyma aphidis DSM 70725
Coccidioides posadasii (strain C735) (Valley
fever
fungus)
Magnaporthe oryzae (strain P131) (Rice blast
fungus) (Pyricularia oryzae)
Neurospora tetrasperma (strain FGSC 2508 /
Hypocrea virens (strain Gv29-8 / FGSC 10586)
Hypocrea virens (strain Gv29-8 / FGSC 10586)
Aspergillus niger (strain CBS 513.88 / FGSC
Verticillium dahliae (strain VdLs.17 / ATCC
Ustilago maydis (strain 521 / FGSC 9021) (Corn
smut fungus)
Fusarium oxysporum
f. sp. lycopersici MN25
Fusarium oxysporum
f. sp. lycopersici MN25
Candida tropicalis (Yeast)
Magnaporthe oryzae (strain 70-15 / ATCC MYA-
Candida tropicalis (Yeast)
Candida tropicalis (Yeast)
Phaeosphaeria nodorum (strain SN15 / ATCC
fungus) (Septoria nodorum)
Candida tropicalis (Yeast)
Pestalotiopsis fici W106-1
Magnaporthe oryzae (strain Y34) (Rice blast
fungus) (Pyricularia oryzae)
Pseudogymnoascus destructans (strain ATCC
syndrome fungus) (Geomyces destructans)
Pseudogymnoascus destructans (strain ATCC
syndrome fungus) (Geomyces destructans)
Mycosphaerella fijiensis (strain CIRAD86)
Bipolaris oryzae ATCC 44560
Fusarium oxysporum
f. sp. melonis 26406
Fusarium oxysporum
f. sp. melonis 26406
Cyphellophora europaea CBS 101466
Aspergillus kawachii (strain NBRC 4308) (White
Aspergillus terreus (strain NIH 2624 / FGSC
Coccidioides immitis (strain RS) (Valley fever
fungus)
Ajellomyces dermatitidis (strain ER-3 / ATCC
Fusarium oxysporum
f. sp. cubense (strain race 1)
Rhodotorula glutinis (strain ATCC 204091 / IIP
Aspergillus niger (strain ATCC 1015 / CBS
Candida cloacae
Candida cloacae
Fusarium oxysporum
f. sp. cubense (strain race 1)
Candida albicans (strain 5C5314 / ATCC MYA-
Candida albicans (strain 5C5314 / ATCC MYA-
Chaetomium thermophilum (strain DSM 1495 /
Mucor circinelloides
f. circinelloides (strain
circinelloides)
Mucor circinelloides
f. circinelloides (strain
circinelloides)
Mucor circinelloides
f. circinelloides (strain
circinelloides)
Botryotinia fuckeliana (strain BcDW1) (Noble rot
fungus) (Botrytis cinerea)
Podospora anserina (strain S / ATCC MYA-4624 /
Neosartorya fumigata (strain ATCC MYA-4609 /
Fusarium oxysporum
f. sp. vasinfectum 25433
Fusarium oxysporum
f. sp. vasinfectum 25433
Trichophyton interdigitale H6
Beauveria bassiana (strain ARSEF 2860) (White
muscardine disease fungus) (Tritirachium
shiotae)
Fusarium oxysporum f. sp. radicis-lycopersici
Fusarium oxysporum f. sp. radicis-lycopersici
Neurospora tetrasperma (strain FGSC 2509 /
Pseudozyma hubeiensis (strain SY62) (Yeast)
Lodderomyces elongisporus (strain ATCC 11503 /
Malassezia globosa (strain ATCC MYA-4612 /
Byssochlamys spectabilis (strain No. 5 / NBRC
Ajellomyces capsulatus (strain H88) (Darling's
disease fungus) (Histoplasma capsulatum)
Trichosporon asahii var. asahii (strain ATCC
Penicillium oxalicum (strain 114-2 / CGMCC
Fusarium oxysporum f. sp. conglutinans race 2
Fusarium oxysporum
f. sp. conglutinans race 2
Fusarium oxysporum f. sp. raphani 54005
Fusarium oxysporum f. sp. raphani 54005
Metarhizium acridum (strain CQMa 102)
Arthroderma benhamiae (strain ATCC MYA-
mentagrophytes)
Fusarium oxysporum f. sp. cubense tropical race
Fusarium oxysporum f. sp. cubense tropical race
Cochliobolus heterostrophus (strain C4 / ATCC
Trichosporon asahii var. asahii (strain CBS 8904)
Mycosphaerella graminicola (strain CBS 115943 /
tritici)
Botryotinia fuckeliana (strain T4) (Noble rot
Metarhizium anisopliae (strain ARSEF 23 /
Cladophialophora carrionii CBS 160.54
Coccidioides posadasii (strain RMSCC 757 /
Rhodosporidium toruloides (strain NP11) (Yeast)
Puccinia graminis f. sp. tritici (strain CRL 75-36-
Trichophyton rubrum CBS 288.86
Colletotrichum fioriniae PJ7
Trichophyton rubrum CBS 289.86
Cladophialophora yegresii CBS 114405
Colletotrichum orbiculare (strain 104-T / ATCC
Drechslerella stenobrocha 248
Neosartorya fumigata (strain CEA10 / CBS
Thielavia terrestris (strain ATCC 38088 / NRRL
Gibberella fujikuroi (strain CBS 195.34 / IMI
disease fungus) (Fusarium fujikuroi)
Gibberella fujikuroi (strain CBS 195.34 / IMI
disease fungus) (Fusarium fujikuroi)
Aspergillus flavus (strain ATCC 200026 / FGSC
Togninia minima (strain UCR-PA7) (Esca disease
fungus) (Phaeoacremonium aleophilum)
Ajellomyces dermatitidis (strain ATCC 18188 /
Macrophomina phaseolina (strain M56)
Neurospora crassa (strain ATCC 24698 / 74-
Neosartorya fischeri (strain ATCC 1020 / DSM
fischerianus)
Fusarium pseudograminearum (strain C53096)
Spathaspora passalidarum (strain NRRL
Spathaspora passalidarum (strain NRRL
Trichophyton verrucosum (strain HKI 0517)
Arthroderma gypseum (strain ATCC MYA-4604 /
Hypocrea jecorina (strain QM6a) (Trichoderma
reesei)
Trichophyton rubrum MR1448
Aspergillus ruber CBS 135680
Glarea lozoyensis (strain ATCC 20868 /
Setosphaeria turcica (strain 28A) (Northern leaf
blight fungus) (Exserohilum turcicum)
Paracoccidioides brasiliensis (strain Pb18)
Fusarium oxysporum Fo47
Fusarium oxysporum Fo47
Trichophyton rubrum MR1459
Penicillium marneffei (strain ATCC 18224 / CBS
Sphaerulina musiva (strain SO2202) (Poplar stem
canker fungus) (Septoria musiva)
Gibberella moniliformis (strain M3125 / FGSC
verticillioides)
Gibberella moniliformis (strain M3125 / FGSC
verticillioides)
Pseudozyma antarctica (strain T-34) (Yeast)
Paracoccidioides brasiliensis (strain Pb03)
Rhizophagus irregularis (strain DAOM 181602 /
mycorrhizal fungus) (Glomus intraradices)
Penicillium chrysogenum (strain ATCC 28089 /
notatum)
Baudoinia compniacensis (strain UAMH 10762)
Hypocrea atroviridis (strain ATCC 20476 / IMI
Colletotrichum gloeosporioides (strain Cg-14)
Cordyceps militaris (strain CM01) (Caterpillar
fungus)
Pyronema omphalodes (strain CBS 100304)
Colletotrichum graminicola (strain M1.001 / M2 /
Glarea lozoyensis (strain ATCC 74030 /
Fusarium oxysporum f. sp. cubense (strain race 4)
Fusarium oxysporum f. sp. cubense (strain race 4)
Cochliobolus sativus (strain ND90Pr / ATCC
fungus) (Bipolaris sorokiniana)
Mixia osmundae (strain CBS 9802 / IAM 14324 /
Mycosphaerella pini (strain NZE10 / CBS
Grosmarmia clavigera (strain kw1407 / UAMH
clavigera)
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Fusarium oxysporum FOSC 3-a
Nectria haematococca (strain 77-13-4 / ATCC
solani
subsp. pisi)
Nectria haematococca (strain 77-13-4 / ATCC
solani
subsp. pisi)
Tuber melanosporum (strain Me128) (Perigord
black truffle)
Ajellomyces dermatitidis (strain SLH14081)
Chaetomium globosum (strain ATCC 6205 / CBS
Candida tenuis (strain ATCC 10573 / BCRC
Trichophyton rubrum CBS 100081
Pyrenophora teres f. teres (strain 0-1) (Barley net
blotch fungus) (Drechslera teres f. teres)
Colletotrichum gloeosporioides (strain Nara gc5)
Gibberella zeae (Wheat head blight fungus)
Trichophyton soudanense CBS 452.61
Sclerotinia sclerotiorum (strain ATCC 18683 /
sclerotiorum)
Fusarium oxysporum f. sp. pisi HDV247
Fusarium oxysporum f. sp. pisi HDV247
Ustilago hordei (strain Uh4875-4) (Barley
covered smut fungus)
Sporisorium reilianum (strain SRZ2) (Maize head
smut fungus)
Bipolaris zeicola 26-R-13
Melampsora larici-populina (strain 98AG31 /
Fusarium oxysporum f. sp. lycopersici (strain
Fusarium oxysporum f. sp. lycopersici (strain
Bipolaris victoriae FI3
Debaryomyces hansenii (strain ATCC 36239 /
Clavispora lusitaniae (strain ATCC 42720)
Candida albicans (strain WO-1) (Yeast)
Trichophyton rubrum MR850
Candida dubliniensis (strain CD36 / ATCC
Starmerella bombicola
Thielavia heterothallica (strain ATCC 42464 /
thermophila)
Claviceps purpurea (strain 20.1) (Ergot fungus)
Aspergillus oryzae (strain ATCC 42149 / RIB 40)
Dictyostelium discoideum (Slime mold)
Triticum urartu (Red wild einkorn) (Crithodium
urartu)
Solanum tuberosum (Potato)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Zea mays (Maize)
Citrus clementina
Citrus clementina
Citrus clementina
Citrus clementina
Morus notabilis
Morus notabilis
Medicago truncatula (Barrel medic) (Medicago
tribuloides)
Arabidopsis thaliana (Mouse-ear cress)
Medicago truncatula (Barrel medic) (Medicago
tribuloides)
Simmondsia chinensis (Jojoba) (Buxus chinensis)
Prunus persica (Peach) (Amygdalus persica)
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Aphanomyces astaci
Phaeodactylum tricornutum (strain CCAP
Hordeum vulgare var. distichum (Two-rowed
barley)
Hordeum vulgare var. distichum (Two-rowed
barley)
Hordeum vulgare var. distichum (Two-rowed
barley)
Hordeum vulgare var. distichum (Two-rowed
barley)
Hordeum vulgare var. distichum (Two-rowed
barley)
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis)
Ricinus communis (Castor bean)
Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis)
Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis)
Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis)
Brassica rapa subsp. pekinensis (Chinese
cabbage) (Brassica pekinensis)
Ricinus communis (Castor bean)
Zea mays (Maize)
Oryza glaberrima (African rice)
Zea mays (Maize)
Zea mays (Maize)
Aegilops tauschii (Tausch's goatgrass) (Aegilops
squarrosa)
Solanum habrochaites (Wild tomato)
Physcomitrella patens subsp. patens (Moss)
Physcomitrella patens subsp. patens (Moss)
Physcomitrella patens subsp. patens (Moss)
Solanum pennellii (Tomato) (Lycopersicon
pennellii)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Vitis vinifera (Grape)
Capsella rubella
Capsella rubella
Capsella rubella
Capsella rubella
Capsella rubella
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Eutrema salsugineum (Saltwater cress)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Selaginella moellendorffii (Spikemoss)
Sorghum bicolor (Sorghum) (Sorghum vulgare)
Sorghum bicolor (Sorghum) (Sorghum vulgare)
Sorghum bicolor (Sorghum) (Sorghum vulgare)
Sorghum bicolor (Sorghum) (Sorghum vulgare)
Sorghum bicolor (Sorghum) (Sorghum vulgare)
Solanum pimpinellifolium (Currant tomato)
Phaseolus vulgaris (Kidney bean) (French bean)
Phaseolus vulgaris (Kidney bean) (French bean)
Phaseolus vulgaris (Kidney bean) (French bean)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Solanum tuberosum (Potato)
Glycine max (Soybean) (Glycine hispida)
Glycine max (Soybean) (Glycine hispida)
Populus trichocarpa (Western balsam poplar)
Picea sitchensis (Sitka spruce) (Pinus sitchensis)
Populus trichocarpa (Western balsam poplar)
Populus trichocarpa (Western balsam poplar)
Glycine max (Soybean) (Glycine hispida)
Glycine max (Soybean) (Glycine hispida)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Setaria italica (Foxtail millet) (Panicum italicum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Solanum lycopersicum (Tomato) (Lycopersicon
esculentum)
Setaria italica (Foxtail millet) (Panicum italicum)
Setaria italica (Foxtail millet) (Panicum italicum)
Mimulus guttatus (Spotted monkey flower)
Mimulus guttatus (Spotted monkey flower)
Mimulus guttatus (Spotted monkey flower)
Mimulus guttatus (Spotted monkey flower)
Mimulus guttatus (Spotted monkey flower)
Musa acuminata subsp. malaccensis (Wild
Musa acuminata subsp. malaccensis (Wild
Musa acuminata subsp. malaccensis (Wild
Saprolegnia diclina V520
Brachypodium distachyon (Purple false brome)
Brachypodium distachyon (Purple false brome)
Brachypodium distachyon (Purple false brome)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. indica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Oryza sativa subsp. japonica (Rice)
Arabidopsis lyrata subsp. lyrata (Lyre-leaved
Arabidopsis lyrata subsp. lyrata (Lyre-leaved
Arabidopsis lyrata subsp. lyrata (Lyre-leaved
Arabidopsis lyrata subsp. lyrata (Lyre-leaved
Desaturase
The present disclosure describes enzymes that desaturate fatty acyl substrates to corresponding unsaturated fatty acyl substrates.
In some embodiments, a desaturase is used to catalyze the conversion of a fatty acyl-CoA or acyl-ACP to a corresponding unsaturated fatty acyl-CoA or acyl-ACP. A desaturase is an enzyme that catalyzes the formation of a carbon-carbon double bond in a saturated fatty acid or fatty acid derivative, e.g., fatty acyl-CoA or fatty acyl-ACP (collectively referred to herein as “fatty acyl”), by removing at least two hydrogen atoms to produce a corresponding unsaturated fatty acid/acyl. Desaturases are classified with respect to the ability of the enzyme to selectively catalyze double bond formation at a subterminal carbon relative to the methyl end of the fatty acid/acyl or a subterminal carbon relative to the carbonyl end of the fatty acid/acyl. Omega (ω) desaturases catalyze the formation of a carbon-carbon double bond at a fixed subterminal carbon relative to the methyl end of a fatty acid/acyl. For example, an ω3 desaturase catalyzes the formation of a double bond between the third and fourth carbon relative the methyl end of a fatty acid/acyl. Delta (Δ) desaturases catalyze the formation of a carbon-carbon double bond at a specific position relative to the carboxyl group of a fatty acid or the carbonyl group of a fatty acyl CoA. For example, a Δ9 desaturase catalyzes the formation of a double bond between the C9 and C10 carbons with respect to the carboxyl end of the fatty acid or the carbonyl group of a fatty acyl CoA.
As used herein, a desaturase can be described with reference to the location in which the desaturase catalyzes the formation of a double bond and the resultant geometric configuration (i.e., E/Z) of the unsaturated hydrocarbon. Accordingly, as used herein, a Z9 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C9 and C10 carbons with respect to the carbonyl end of a fatty acid/acyl, thereby orienting two hydrocarbons on opposing sides of the carbon-carbon double bonds in the cis or Z configuration. Similarly, as used herein, a Z11 desaturase refers to a Δ desaturase that catalyzes the formation of a double bond between the C11 and C12 carbons with respect to the carbonyl end of a fatty acid/acyl.
Desaturases have a conserved structural motif. This sequence motif of transmembrane desaturases is characterized by [HX3-4HX7-41(3 non-His)HX2-3(1 nonHis)HHX61-189(40 non-His)HX2-3(1 non-His)HH]. The sequence motif of soluble desaturases is characterized by two occurrences of [D/EEXXH].
In some embodiments, the desaturase is a fatty acyl-CoA desaturase that catalyzes the formation of a double bond in a fatty acyl-CoA. In some such embodiments, the fatty acyl-CoA desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal CoA on the unsaturated fatty acyl-CoA. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-CoA.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-CoA has a cis or trans (i.e., Z or E) geometric configuration.
In some embodiments, the desaturase is a fatty acyl-ACP desaturase that catalyzes the formation of a double bond in a fatty acyl-ACP. In some embodiments, the fatty acyl-ACP desaturase described herein is capable of utilizing a fatty acyl-CoA as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. Thus, the desaturase used in the recombinant microorganism can be selected based on the chain length of the substrate.
In some embodiments, the fatty acyl-ACP desaturase described herein is capable of catalyzing the formation of a double bond at a desired carbon relative to the terminal carbonyl on the unsaturated fatty acyl-ACP. Thus, in some embodiments, a desaturase can be selected for use in the recombinant microorganism which catalyzes double bond insertion at the 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 position with respect to the carbonyl group on a fatty acyl-ACP.
In some embodiments, the fatty acyl desaturase described herein is capable of catalyzing the formation of a double bond in a saturated fatty acyl-CoA such that the resultant unsaturated fatty acyl-ACP has a cis or trans (i.e., Z or E) geometric configuration.
In one embodiment, the fatty acyl desaturase is a Z11 desaturase. In some embodiments, a nucleic acid sequence encoding a Z11 desaturase from organisms of the species Agrotis segetum, Amyelois transitella, Argyrotaenia velutiana, Choristoneura rosaceana, Lampronia capitella, Trichoplusia ni, Helicoverpa zea, or Thalassiosira pseudonana is codon optimized. In some embodiments, the Z11 desaturase comprises a sequence set forth in SEQ ID NO: 32 or 33 from Helicoverpa zea.
Fatty Acyl Reductase
The present disclosure describes enzymes that reduce fatty acyl substrates to corresponding fatty alcohols or aldehydes.
In some embodiments, a fatty alcohol forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-CoA to a corresponding fatty alcohol. In some embodiments, a fatty aldehyde forming fatty acyl-reductase is used to catalyze the conversion of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl reductase is an enzyme that catalyzes the reduction of a fatty acyl-CoA to a corresponding fatty alcohol or the reduction of a fatty acyl-ACP to a corresponding fatty aldehyde. A fatty acyl-CoA and fatty acyl-ACP has a structure of R—(CO)—S—R1, wherein R is a C6 to C24 saturated, unsaturated, linear, branched or cyclic hydrocarbon, and R1 represents CoA or ACP. In a particular embodiment, R is a C6 to C24 saturated or unsaturated linear hydrocarbon. “CoA” is a non-protein acyl carrier group involved in the synthesis and oxidation of fatty acids. “ACP” is an acyl carrier protein, i.e., a polypeptide or protein subunit, of fatty acid synthase used in the synthesis of fatty acids.
Thus, in some embodiments, the disclosure provides for a fatty alcohol forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-CoA to the corresponding fatty alcohol. For example, R—(CO)—S-CoA is converted to R—CH2OH and CoA-SH when two molecules of NAD(P)H are oxidized to NAD(P)+. Accordingly, in some such embodiments, a recombinant microorganism described herein can include a heterologous fatty alcohol forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-CoA to the corresponding fatty alcohol. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty alcohol forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-CoA into the corresponding mono- or poly-unsaturated C6-C24 fatty alcohol.
In other embodiments, the disclosure provides for a fatty aldehyde forming fatty acyl-reductase which catalyzes the reduction of a fatty acyl-ACP to the corresponding fatty aldehyde. For example, R—(CO)—S-ACP is converted to R—(CO)—H and ACP-SH when one molecule of NAD(P)H is oxidized to NAD(P)+. In some such embodiments, a recombinant microorganism described herein can include a heterologous fatty aldehyde forming fatty acyl-reductase, which catalyzes the reduction a fatty acyl-ACP to the corresponding fatty aldehyde. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule encoding a fatty aldehyde forming fatty-acyl reductase which catalyzes the conversion of a mono- or poly-unsaturated C6-C24 fatty acyl-ACP into the corresponding mono- or poly-unsaturated C6-C24 fatty aldehyde.
In some embodiments, a nucleic acid sequence encoding a fatty-acyl reductase from organisms of the species Agrotis segetum, Spodoptera littoralis, or Helicoverpa amigera is codon optimized.
Acyl-ACP Synthetase
The present disclosure describes enzymes that ligate a fatty acid to the corresponding fatty acyl-ACP.
In some embodiments, an acyl-ACP synthetase is used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. An acyl-ACP synthetase is an enzyme capable of ligating a fatty acid to ACP to produce a fatty acid acyl-ACP. In some embodiments, an acyl-ACP synthetase can be used to catalyze the conversion of a fatty acid to a corresponding fatty acyl-ACP. In some embodiments, the acyl-ACP synthetase is a synthetase capable of utilizing a fatty acid as a substrate that has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In one such embodiment, a recombinant microorganism described herein can include a heterologous acyl-ACP synthetase, which catalyzes the conversion of a fatty acid to a corresponding fatty acyl-ACP. In an exemplary embodiment, a recombinant microorganism disclosed herein includes at least one exogenous nucleic acid molecule which encodes an acyl-ACP synthetase that catalyzes the conversion of a saturated C6-C24 fatty acid to a corresponding saturated C6-C24 fatty acyl-ACP.
Improved Production of Biomass and One or More Lipid Using a Recombinant Microorganism
As discussed above, in a first aspect, the present disclosure relates to a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, wherein the recombinant microorganism comprises one or more modifications associated with: tricarboxylic acid cycle; lipid synthesis; reducing equivalent availability; one or more metabolic intermediates availability; and/or increased product purity, wherein the recombinant microorganism has improved production of biomass or improved production of one or more lipid compared to a microorganism without the same modifications.
In some embodiments, the one or more modifications associated with tricarboxylic acid cycle comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an AMP-insensitive isocitrate dehydrogenase (IDH) variant in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to AMP-insensitive IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the at least one nucleic acid molecule is from Yarrowia lipolytica and comprises isoleucine to alanine substitutions at amino acid positions 279 and 280 of XP_503571.2. In some embodiments, the one or more modifications associated with tricarboxylic acid cycle results in extended activation of the tricarboxylic acid cycle.
In some embodiments, the one or more modifications associated with tricarboxylic acid cycle or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a pyruvate transporter in the recombinant microorganism. In other embodiments, the one or more metabolic intermediates availability comprises mitochondrial pyruvate availability. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to pyruvate transporter from Saccharomyces cerevisiae, Hanseniaspora osmophila, Yarrowia lipolytica, or Talaromyces marneffei PM1. In further embodiments, the pyruvate transporter is selected from Saccharomyces cerevisiae mpc1, Saccharomyces cerevisiae mpc3 (NP_011759.1), Hanseniaspora osmophila mpc3 (OEJ86292.1), Yarrowia lipolytica mpc, and Talaromyces marneffei PM1 mpc3 (KFX48982.1), or homolog thereof. In yet a further embodiment, the recombinant microorganism is Saccharomyces cerevisiae comprising a deletion, disruption, or loss of function mutation in a gene encoding an mpc2 pyruvate transporter. In some embodiments, the recombinant microorganism is Yarrowia lipolytica.
In some embodiments, the one or more modifications associated with lipid synthesis comprises alleviation of acetyl-CoA carboxylase (ACC) inhibition. In certain embodiments, alleviation of ACC inhibition comprises the replacement of the endogenous ACC, or overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding a feedback-insensitive ACC variant in the recombinant microorganism. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ACC from Mus musculus, Rattus norvegicus, or Homo sapiens.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an NADP/NAD-dependent isocitrate dehydrogenase (IDH) in the cytosol of the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the IDH is selected from Escherichia coli Idh (WP_000444484.1), Mycobacterium smegmatis Icd2 (WP_011727802.1), Acidithiobacillus thiooxidans Idh (PDB: 2D4V_A), and Yarrowia lipolytica Idh1 (XP_503571.2), or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability further comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an aconitase in the cytosol of the recombinant microorganism. In certain embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding the IDH and the at least one endogenous and/or exogenous nucleic acid molecule encoding the aconitase lack a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the one or more modifications associated with reducing equivalent availability or one or more metabolic intermediates availability comprises the overexpression of at least one endogenous or exogenous nucleic acid encoding a citrate transporter in the recombinant microorganism. In certain embodiments, the one or more intermediate comprises cytosolic citrate/isocitrate. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a citrate transporter from Yarrowia lipolytica, Saccharomyces cerevisiae, Rattus norvegicus, Caenorhabditis elegans, or Caliqus clemensi. In yet further embodiments, the citrate transporter is selected from Yarrowia lipolytica YALI0F26323p, Saccharomyces cerevisiae AAC48984.1, Rattus norvegicus AAA18899.1, Caenorhabditis elegans P34519.1, and Caliqus clemensi ACO14982.1, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises the overexpression of at least one exogenous nucleic acid molecule encoding a decarboxylating malic enzyme in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a decarboxylating malic enzyme from Arabidopsis thaliana, Amaranthus hypochondriacus, Rhizobium meliloti, Solanum tuberosum, Homo sapiens, or Escherichia coli. In further embodiments, the decarboxylating malic enzyme is selected from Arabidopsis thaliana Q9SIU0 (SEQ ID NO: 34), Amaranthus hypochondriacus P37224 (SEQ ID NO: 35), Rhizobium meliloti 030807 (SEQ ID NO: 36), Solanum tuberosum P37221 (SEQ ID NO: 37), Homo sapiens Q16798 (SEQ ID NO: 38), and Escherichia coli P26616 (SEQ ID NO: 29), or homolog thereof. In yet a further embodiment, the decarboxylating malic enzyme lacks a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the one or more modifications associated with one or more metabolic intermediates availability comprises the overexpression of at least one endogenous and/or exogenous nucleic acid encoding an ATP-citrate lyase in the recombinant microorganism. In certain embodiments, the one or more intermediates availability comprises cytosolic oxaloacetate availability. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ATP-citrate lyase from Saccharomyces cerevisiae, Yarrowia lipolytica, Mus musculus, and Aspergillus niger. In a yet further embodiment, the ATP-citrate lyase is selected from Mus musculus NP_001186225.1, Mus musculus NP_598798.1, Aspergillus niger XP_001394055.1, and Aspergillus niger XP_001394057.1, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, the one or more modifications in the PPP comprises one or more of: downregulation of hexose kinase activity; upregulation of one or more oxidative PPP enzyme activity; downregulation of fructose-6-phosphate kinase activity; and/or expression of one or more oxidative PPP enzyme variant. In further embodiments, the upregulation of one or more oxidative PPP enzyme activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a glucose-6-phosphate dehydrogenase (ZWF1), a 6-phosphogluconolactonase (SOL3), or a 6-phosphogluconate dehydrogenase (GND1). In yet a further embodiment, the downregulation of hexose kinase activity and/or fructose-6-phosphate kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme and/or fructose-6-phosphate kinase enzyme. In some embodiments, the one or more oxidative PPP enzyme variant comprises one or more endogenous and/or exogenous nucleic acid molecule encoding an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) and/or an NAD-dependent 6-phosphogluconate dehydrogenase (GND1). In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) from Leuconostoc. In further embodiments, the NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) is selected from Leuconostoc AAA25265.1 and Leuconostoc P11411, or homolog thereof. In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent 6-phosphogluconate dehydrogenase (GND1) from Bradyrhizobium or Methylobacillus. In further embodiments, the NAD-dependent 6-phosphogluconate dehydrogenase (GND1) is selected from Bradyrhizobium WP_012029377.1, Bradyrhizobium A4YZZ8, Methylobacillus AAF34407.1, and Methylobacillus Q9L9P8, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises downregulation of mannitol synthesis pathway in the recombinant microorganism. In certain embodiments, downregulation of mannitol synthesis pathway comprises deletion, disruption, and/or mutation of one or more gene encoding an NADPH-dependent mannitol dehydrogenase and/or an aldo-keto reductase. In further embodiments, the one or more gene encoding an NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homolog thereof. In further embodiments, the one or more gene encoding an aldo-keto reductase is selected from YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0C09119g, YALI0D04092g, YALI0B15268g, YALI0C00319g, and YALI0A19910g, or homolog thereof.
In some embodiments, the one or more modifications associated with reducing equivalent availability comprises decoupling and increasing glucose uptake in the recombinant microorganism. In certain embodiments, decoupling and increasing glucose uptake comprises: upregulation of hexose transporter activity; and/or downregulation of hexose kinase activity. In further embodiments, the upregulation of one or more hexose transporter activity comprises the overexpression of one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter operably linked to one or more heterologous promoters. In some embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a hexose transporter from Yarrowia lipolytica. In certain embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter is selected from YALI0A14212g, YALI0D01111g, YALI0D00363g, YALI0C16522g, and YALI0F25553g, or homolog thereof. In some embodiments, the downregulation of hexose kinase activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme.
In some embodiments, the one or more modifications associated with reducing equivalent availability, one or more metabolic intermediates availability, or increased product purity comprises downregulation or inhibition of acetyl-CoA carboxylase (ACC) activity in the recombinant microorganism. In certain embodiments, the downregulation or inhibition of ACC activity comprises deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more ACC enzyme.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more fatty acid co-substrate is a saturated fatty acid. In some embodiments of a recombinant microorganism having improved production of biomass or improved production one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more simple carbon co-substrate is selected from glucose, fructose, and glycerol.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the improved production of one or more lipid comprises improved production of one or more mono- or poly-unsaturated C6-C24 fatty acid, fatty alcohol, aldehyde, or acetate. In certain embodiments, the one or more mono- or poly-unsaturated C6-C24 fatty acid, fatty alcohol, aldehyde, or acetate is an insect pheromone. In further embodiments, the insect pheromone is selected from the group consisting of (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadeca-9,1-dienal, (E,E)-8,10-dodecadien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (9Z,12E)-tetradecadienyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, and (Z)-13-octadecenal.
In some embodiments of a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the recombinant microorganism is a eukaryotic microorganism. In certain embodiments, the eukaryotic microorganism is a yeast. In further embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma. In yet a further embodiment, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In certain embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Candida viswanathii, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T. cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
Recombinant Microorganism
The disclosure provides microorganisms modified to have improved production of biomass or valuable products, such as one or more lipids or metabolic intermediates. In one embodiment, the valuable product is one or more lipids. In some embodiments, the valuable product is fatty acid, fatty alcohol, fatty aldehyde, and/or fatty acetate. In some embodiments, the valuable product is one or more pheromones. In some embodiments, the valuable product is one or more fatty acid precursors of one or more pheromones.
In various embodiments described herein, the recombinant microorganism is a eukaryotic microorganism. In some embodiments, the eukaryotic microorganism is a yeast. In exemplary embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma.
The present inventors have discovered that oleaginous yeast, such as Candida and Yarrowia, have a surprisingly high tolerance to the C6-C24 fatty alcohol substrates and products. Accordingly, in one such exemplary embodiment, the recombinant microorganism of the invention is an oleaginous yeast. In further embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In even further embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Candida viswanathii, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
In some embodiments, the recombinant microorganism is a prokaryotic microorganism. In exemplary embodiments, the prokaryotic microorganism is a member of a genus selected from the group consisting of Escherichia, Clostridium, Zymomonas, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.
In some embodiments, the recombinant microorganism is used to produce a mono- or poly-unsaturated C6-C24 fatty acid, alcohol, aldehyde, or acetate disclosed herein.
Accordingly, in another aspect, the present disclosure provides a method of producing one or more lipid using a recombinant microorganism described herein. In one embodiment, the method comprises cultivating the recombinant microorganism in a culture medium containing a feedstock providing one or more carbon source until the one or more lipid is produced. In some embodiments, the one or more lipid comprises one or more mono- or poly-unsaturated C6-C24 fatty acid, alcohol, aldehyde, or acetate. In further embodiments, the one or more mono- or poly-unsaturated C6-C24 fatty acid, alcohol, aldehyde, or acetate is one or more insect pheromone. In further embodiments, the insect pheromone is selected from the group consisting of (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadeca-9,1-dienal, (E,E)-8,10-dodecadien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (9Z,12E)-tetradecadienyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, and (Z)-13-octadecenal. In a further embodiment, the one or more lipid is recovered. Recovery can be by methods known in the art, such as distillation, membrane-based separation gas stripping, solvent extraction, and expanded bed adsorption.
In some embodiments, the feedstock comprises a carbon source. In various embodiments described herein, the carbon source may be selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide. In a further embodiment, the sugar is selected from the group consisting of glucose, fructose, and sucrose. In some embodiments, the one or more carbon source comprises one or more simple carbon and one or more fatty acid. In certain embodiments, the one or more simple carbon is selected from glucose, fructose and glycerol.
In some embodiments, the recombinant microorganism is a microalgae. Non-limiting examples of microalgae that can be used with the methods and compositions of the disclosure are members of the following divisions: cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow green algae (Xanthophyceae), golden algae (Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoflagellates (Dinophyceae), and “pico-plankton” (Prasinophyceae and Eustigmatophyceae). In some embodiments, the preferred algae for use in connection with the production of Fas are green algae (fresh water), Cyanobacteria, and Diotoms (Marine). In certain embodiments, the microalgae used with the methods of the disclosure are members of one of the following classes: Bacillariophyceae, Eustigmatophyceae, and Chrysophyceae. In certain embodiments, the microalgae used with the methods of the disclosure are members of one of the following genera: Nannochloropsis, Chlorella, Dunaliella, Scenedesmus, Selenastrum, Oscillatoria, Phormidium, Spirulina, Amphora, and Ochromonas.
Non-limiting examples of microalgae species that can be used in connection with the present disclosure include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorelladesiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorellaluteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella noctuma, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorellasaccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorellastigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliellapeircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis off galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrine, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis carter ae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.
Methods of Producing a Recombinant Microorganism Having Improved Production of Biomass and One or More Lipid
As discussed above, in another aspect, the present disclosure relates to a method of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, comprising introducing into a microorganism one or more modifications associated with: tricarboxylic acid cycle; lipid synthesis; reducing equivalent availability; one or more metabolic intermediates availability; and/or increased product purity, wherein the introducing one or more modifications yields a recombinant microorganism having improved production of biomass or improved production of one or more lipid compared to a microorganism not comprising the same modifications.
In some embodiments, the introducing one or more modifications associated with tricarboxylic acid cycle comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid molecule encoding an AMP-insensitive isocitrate dehydrogenase (IDH) variant. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the at least one nucleic acid molecule is from Yarrowia lipolytica and comprises isoleucine to alanine substitutions at amino acid positions 279 and 280 of XP_503571.2. In some embodiments, the one or more modifications associated with tricarboxylic acid cycle results in extended activation of the tricarboxylic acid cycle.
In some embodiments, the introducing one or more modifications associated with tricarboxylic acid cycle or one or more metabolic intermediates availability comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid molecule encoding a pyruvate transporter. In other embodiments, the one or more metabolic intermediates availability comprises mitochondrial pyruvate availability. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to pyruvate transporter from Saccharomyces cerevisiae, Hanseniaspora osmophila, Yarrowia lipolytica, or Talaromyces marneffei PM1. In further embodiments, the pyruvate transporter is selected from Saccharomyces cerevisiae mpc1, Saccharomyces cerevisiae mpc3 (NP_011759.1), Hanseniaspora osmophila mpc3 (OEJ86292.1), Yarrowia lipolytica mpc, and Talaromyces marneffei PM1 mpc3 (KFX48982.1), or homolog thereof. In yet a further embodiment, the recombinant microorganism is Saccharomyces cerevisiae comprising a deletion, disruption, or loss of function mutation in a gene encoding an mpc2 pyruvate transporter. In some embodiments, the recombinant microorganism is Yarrowia lipolytica.
In some embodiments, the introducing one or more modifications associated with lipid synthesis comprises alleviation of acetyl-CoA carboxylase (ACC) inhibition. In certain embodiments, alleviation of ACC inhibition comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid molecule encoding a feedback-insensitive ACC variant in the recombinant microorganism. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ACC from Mus musculus, Rattus norvegicus, or Homo sapiens.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid molecule encoding an NADP/NAD-dependent isocitrate dehydrogenase (IDH) in the cytosol of the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IDH from Escherichia coli, Mycobacterium smegmatis, Acidithiobacillus thiooxidans, or Yarrowia lipolytica. In further embodiments, the IDH is selected from Escherichia coli Idh (WP_000444484.1), Mycobacterium smegmatis Icd2 (WP_011727802.1), Acidithiobacillus thiooxidans Idh (PDB: 2D4V_A), and Yarrowia lipolytica Idh1 (XP_503571.2), or homolog thereof.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability further comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid encoding an aconitase in the cytosol of the recombinant microorganism. In certain embodiments, the at least one endogenous and/or exogenous nucleic acid molecule encoding the IDH and the at least one endogenous and/or exogenous nucleic acid molecule encoding the aconitase lack a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability or one or more metabolic intermediates availability comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid encoding a citrate transporter in the recombinant microorganism. In certain embodiments, the one or more intermediate comprises cytosolic citrate/isocitrate. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a citrate transporter from Yarrowia lipolytica, Saccharomyces cerevisiae, Rattus norvegicus, Caenorhabditis elegans, or Caliqus clemensi. In yet further embodiments, the citrate transporter is selected from Yarrowia lipolytica YALI0F26323p, Saccharomyces cerevisiae AAC48984.1, Rattus norvegicus AAA18899.1, Caenorhabditis elegans P34519.1, and Caliqus clemensi ACO14982.1, or homolog thereof.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability comprises introducing into and/or overexpressing in the recombinant microorganism at least one exogenous nucleic acid molecule encoding a decarboxylating malic enzyme in the recombinant microorganism. In certain embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a decarboxylating malic enzyme from Arabidopsis thaliana, Amaranthus hypochondriacus, Rhizobium meliloti, Solanum tuberosum, Homo sapiens, or Escherichia coli. In further embodiments, the decarboxylating malic enzyme is selected from Arabidopsis thaliana Q9SIU0, Amaranthus hypochondriacus P37224, Rhizobium meliloti 030807, Solanum tuberosum P37221, Homo sapiens Q16798, and Escherichia coli P26616, or homolog thereof. In yet a further embodiment, the decarboxylating malic enzyme lacks a sequence encoding a mitochondrial-targeting peptide.
In some embodiments, the introducing one or more modifications associated with one or more metabolic intermediates availability comprises introducing into and/or overexpressing in the recombinant microorganism at least one endogenous and/or exogenous nucleic acid encoding an ATP-citrate lyase in the recombinant microorganism. In certain embodiments, the one or more metabolic intermediates availability comprises cytosolic oxaloacetate availability. In further embodiments, the at least one nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ATP-citrate lyase from Saccharomyces cerevisiae, Yarrowia lipolytica, Mus musculus, and Aspergillus niger. In a yet further embodiment, the ATP-citrate lyase is selected from Mus musculus NP_001186225.1, Mus musculus NP_598798.1, Aspergillus niger XP_001394055.1, and Aspergillus niger XP_001394057.1, or homolog thereof.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability comprises one or more modifications in the pentose phosphate pathway (PPP) in the recombinant microorganism. In certain embodiments, the one or more modifications in the PPP comprises one or more of: downregulation of hexose kinase activity; upregulation of one or more oxidative PPP enzyme activity; downregulation of fructose-6-phosphate kinase activity; and/or expression of one or more oxidative PPP enzyme variant. In further embodiments, the upregulation of one or more oxidative PPP enzyme activity comprises introducing into and/or overexpressing in the recombinant microorganism one or more endogenous and/or exogenous nucleic acid molecule encoding a glucose-6-phosphate dehydrogenase (ZWF1), a 6-phosphogluconolactonase (SOL3), or a 6-phosphogluconate dehydrogenase (GND1). In yet a further embodiment, the downregulation of hexose kinase activity and/or fructose-6-phosphate kinase activity comprises introducing into the recombinant microorganism a deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme and/or fructose-6-phosphate kinase enzyme. In some embodiments, the one or more oxidative PPP enzyme variant comprises one or more endogenous and/or exogenous nucleic acid molecule encoding an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) and/or an NAD-dependent 6-phosphogluconate dehydrogenase (GND1). In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) from Leuconostoc. In further embodiments, the NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) is selected from Leuconostoc AAA25265.1 and Leuconostoc P11411, or homolog thereof. In certain embodiments, the one or more nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an NAD-dependent 6-phosphogluconate dehydrogenase (GND1) from Bradyrhizobium or Methylobacillus. In further embodiments, the NAD-dependent 6-phosphogluconate dehydrogenase (GND1) is selected from Bradyrhizobium WP_012029377.1, Bradyrhizobium A4YZZ8, Methylobacillus AAF34407.1, and Methylobacillus Q9L9P8, or homolog thereof.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability comprises downregulation of mannitol synthesis pathway in the recombinant microorganism. In certain embodiments, downregulation of mannitol synthesis pathway comprises introducing into the recombinant microorganism a deletion, disruption, and/or mutation of one or more gene encoding an NADPH-dependent mannitol dehydrogenase and/or an aldo-keto reductase. In further embodiments, the one or more gene encoding an NADPH-dependent mannitol dehydrogenase is selected from YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homolog thereof. In further embodiments, the one or more gene encoding an aldo-keto reductase is selected from YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0009119g, YALI0D04092g, YALI0B15268g, YALI0C00319g, and YALI0A19910g, or homolog thereof.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability comprises decoupling and increasing glucose uptake in the recombinant microorganism. In certain embodiments, decoupling and increasing glucose uptake comprises: upregulation of hexose transporter activity; and/or downregulation of hexose kinase activity. In further embodiments, the upregulation of one or more hexose transporter activity comprises introducing into and/or overexpressing in the recombinant microorganism one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter operably linked to one or more heterologous promoters. In some embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encodes for a protein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a hexose transporter from Yarrowia lipolytica. In certain embodiments, the one or more endogenous and/or exogenous nucleic acid molecule encoding a hexose transporter is selected from YALI0A14212g, YALI0D01111g, YALI0D00363g, YALI0C16522g, and YALI0F25553g, or homolog thereof. In some embodiments, the downregulation of hexose kinase activity comprises introducing into the recombinant microorganism a deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more hexose kinase enzyme.
In some embodiments, the introducing one or more modifications associated with reducing equivalent availability, one or more metabolic intermediates availability, and/or increased product purity comprises downregulation or inhibition of acetyl-CoA carboxylase (ACC) activity in the recombinant microorganism. In certain embodiments, the downregulation or inhibition of ACC activity comprises introducing into the recombinant microorganism a deletion, disruption, and/or mutation of one or more endogenous gene encoding one or more ACC enzyme.
In some embodiments of a method of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more fatty acid co-substrate is a saturated fatty acid. In some embodiments of a method of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the one or more simple carbon co-substrate is selected from glucose, fructose, and glycerol.
In some embodiments of a method of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the improved production of one or more lipid comprises improved production of one or more mono- or poly-unsaturated C6-C24 fatty acid, alcohol, aldehyde, or acetate. In certain embodiments, the one or more mono- or poly-unsaturated C6-C24 fatty acid, alcohol, aldehyde, or acetate is an insect pheromone. In further embodiments, the insect pheromone is selected from the group consisting of (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, (Z)-9-tetradecenyl acetate, (Z,Z)-11,13-hexadecadienal, (9Z,11E)-hexadeca-9,1-dienal, (E,E)-8,10-dodecadien-1-ol, (7E,9Z)-dodecadienyl acetate, (Z)-3-nonen-1-ol, (Z)-5-decen-1-ol, (Z)-5-decenyl acetate, (E)-5-decen-1-ol, (E)-5-decenyl acetate, (Z)-7-dodecen-1-ol, (Z)-7-dodecenyl acetate, (E)-8-dodecen-1-ol, (E)-8-dodecenyl acetate, (Z)-8-dodecen-1-ol, (Z)-8-dodecenyl acetate, (Z)-9-dodecen-1-ol, (Z)-9-dodecenyl acetate, (Z)-9-tetradecen-1-ol, (Z)-11-tetraceden-1-ol, (Z)-11-tetracedenyl acetate, (E)-11-tetradecen-1-ol, (E)-11-tetradecenyl acetate, (9Z,12E)-tetradecadienyl acetate, (Z)-7-hexadecen-1-ol, (Z)-7-hexadecenal, (Z)-9-hexadecen-1-ol, (Z)-9-hexadecenal, (Z)-9-hexadecenyl acetate, (Z)-11-hexadecen-1-ol, (Z)-13-octadecen-1-ol, and (Z)-13-octadecenal.
In some embodiments of a method of producing a recombinant microorganism having improved production of biomass or improved production of one or more lipid from one or more fatty acid and one or more simple carbon co-substrates, the recombinant microorganism is a eukaryotic microorganism. In certain embodiments, the eukaryotic microorganism is a yeast. In further embodiments, the yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Saccharomyces, Pichia, Hansenula, Kluyveromyces, Issatchenkia, Zygosaccharomyces, Debaryomyces, Schizosaccharomyces, Pachysolen, Cryptococcus, Trichosporon, Rhodotorula, and Myxozyma. In yet a further embodiment, the yeast is an oleaginous yeast. In some embodiments, the oleaginous yeast is a member of a genus selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. In certain embodiments, the oleaginous yeast is a member of a species selected from Yarrowia lipolytica, Candida tropicalis, Candida viswanathii, Rhodosporidium toruloides, Lipomyces starkey, L. lipoferus, C. revkaufi, C. pulcherrima, C. utilis, Rhodotorula minuta, Trichosporon pullans, T cutaneum, Cryptococcus curvatus, R. glutinis, and R. graminis.
Enzyme Engineering
The enzymes in the recombinant microorganism can be engineered to improve one or more aspects of the substrate to product conversion. Non-limiting examples of enzymes that can be further engineered for use in methods of the disclosure include an isocitrate dehydrogenase, a pyruvate transporter, an acetyl-CoA carboxylase, an aconitase, a citrate transporter, a decarboxylating malic enzyme, an ATP-citrate lyase, a glucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a hexose transporter and combinations thereof. These enzymes can be engineered for improved catalytic activity, improved selectivity, improved stability, improved tolerance to various fermentations conditions (temperature, pH, etc.), altered selectivity of one or more substrate, altered selectivity of one or more co-factor, altered cellular localization, or improved tolerance to various metabolic substrates, products, by-products, intermediates, etc.
In some embodiments, an isocitrate dehydrogenase (IDH) enzyme can be engineered for insensitivity to AMP. Under a nitrogen-starved and glucose-rich environment, low levels of intracellular AMP reduce the activity of isocitrate dehydrogenase (IDH), a key allosteric enzyme in the TCA cycle in yeast mitochondria. The reduction of IDH activity slows the TCA cycle used for synthesis of biomass and reducing equivalents, and accumulates citrate (the equilibrium form of isocitrate). Therefore, in some embodiments, it is desirable to engineer an oleochemical production host to repurpose citrate for improvement in biomass generation. In certain embodiments, the AMP-insensitive IDH is from Yarrowia lipolytica and comprises isoleucine to alanine substitutions at amino acid positions 279 and 280 of XP_503571.2. In further non-limiting examples, an IDH can be engineered to lack a sequence encoding a mitochondrial-targeting peptide. In some embodiments, the IDH lacking a mitochondrial-targeting peptide localizes to the cytosol of a cell. In a further example, an aconitase can be engineered to lack a sequence encoding a mitochondrial-targeting peptide. In some embodiments, the aconitase lacking a mitochondrial-targeting peptide localizes to the cytosol of a cell. In a further example, a malate dehydrogenase can be engineered to lack a sequence encoding a mitochondrial-targeting peptide. In some embodiments, the malate dehydrogenase lacking a mitochondrial-targeting peptide localizes to the cytosol of a cell. In a further example, an acetyl-CoA carboxylase (ACC) enzyme can be engineered to be feedback-insensitive. In some embodiments, the feedback-insensitive ACC leads to increased lipid biosynthesis. In a further example, glucose-6-phosphate dehydrogenase (ZWF1) can be engineered to use NAD+ in place of NADP+ producing NADH instead of NADPH to match cofactor requirements of recombinant biosynthesis pathways. In a further example, 6-phosphogluconate dehydrogenase (GND1) can be engineered to use NAD+ in place of NADP+ producing NADH instead of NADPH to match cofactor requirements of recombinant biosynthesis pathways.
The term “improved catalytic activity” as used herein with respect to a particular enzymatic activity refers to a higher level of enzymatic activity than that measured relative to a comparable non-engineered enzyme, such as a non-engineered isocitrate dehydrogenase, an acetyl-CoA carboxylase, an aconitase, a decarboxylating malic enzyme, an ATP-citrate lyase, a glucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase, or a 6-phosphogluconate dehydrogenase. For example, overexpression of a specific enzyme can lead to an increased level of activity in the cells for that enzyme. Mutations can be introduced into an isocitrate dehydrogenase, an acetyl-CoA carboxylase, an aconitase, a decarboxylating malic enzyme, an ATP-citrate lyase, a glucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase, or a 6-phosphogluconate dehydrogenase enzyme resulting in engineered enzymes with improved catalytic activity. Methods to increase enzymatic activity are known to those skilled in the art. Such techniques can include increasing the expression of the enzyme by increasing plasmid copy number and/or use of a stronger promoter and/or use of activating riboswitches, introduction of mutations to relieve negative regulation of the enzyme, introduction of specific mutations to increase specific activity and/or decrease the KM for the substrate, or by directed evolution. See, e.g., Methods in Molecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press (2003).
Metabolic Engineering—Enzyme Overexpression and Gene Deletion/Downregulation for Increased Pathway Flux
In various embodiments described herein, the exogenous and endogenous enzymes in the recombinant microorganism participating in the pathways described herein may be overexpressed.
The terms “overexpressed” or “overexpression” refers to an elevated level (e.g., aberrant level) of mRNAs encoding for a protein(s), and/or to elevated levels of protein(s) in cells as compared to similar corresponding unmodified cells expressing basal levels of mRNAs or having basal levels of proteins. In particular embodiments, mRNA(s) or protein(s) may be overexpressed by at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more in microorganisms engineered to exhibit increased gene mRNA, protein, and/or activity.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous isocitrate dehydrogenase enzymes to extend activation of the tricarboxylic acid (TCA) cycle and increase flux from citrate, thereby resulting in increased biomass generation. In some embodiments, extended activation of the tricarboxylic acid cycle comprises the overexpression of at least one endogenous and/or exogenous nucleic acid molecule encoding an AMP-insensitive isocitrate dehydrogenase (IDH) variant in the recombinant microorganism. In some embodiments, the overexpression of an NADP/NAD-dependent isocitrate dehydrogenase (IDH) in the cytosol of the recombinant microorganism increases the availability or amount of reducing equivalents.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous pyruvate transporters to enhance pyruvate flux from the cytosol to the mitochondria. In some embodiments, it may be useful to increase the expression of endogenous or exogenous pyruvate transporters to increase the mitochondrial pyruvate pool. In some embodiments, one or more modifications associated with tricarboxylic acid cycle and/or one or more metabolic intermediates availability comprises the overexpression of a pyruvate transporter in the recombinant microorganism. In other embodiments, the one or more metabolic intermediates availability comprises mitochondrial pyruvate availability.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous acetyl-CoA carboxylase enzymes to increase lipid synthesis. In some embodiments, it may be useful to increase the expression of endogenous or exogenous acetyl-CoA carboxylase feedback-insensitive variant enzymes to alleviate inhibition of lipid synthesis.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous aconitase enzymes to increase and/or rebalance reducing equivalents. In further embodiments, the overexpression of an aconitase in the cytosol of the recombinant microorganism increases and/or rebalances reducing equivalents.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous citrate transporters to increase and/or rebalance reducing equivalents. In some embodiments, it may be useful to increase the expression of endogenous or exogenous citrate transporters to increase the amount of one or more metabolic intermediate. In certain embodiments, the one or more intermediate comprises cytosolic citrate/isocitrate.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous decarboxylating malic enzymes to increase and/or rebalance reducing equivalents. In one embodiment, the malic enzyme is NAD+ dependent. In another embodiment, the malic enzyme is NADP+ dependent. In yet a further embodiment, it may be useful to increase the expression of endogenous or exogenous malate dehydrogenases in the cytosol of a recombinant microorganism.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous ATP-citrate lyase to increase the amount of one or more metabolic intermediate. In certain embodiments, the one or more intermediate comprises cytosolic oxaloacetate.
In some embodiments, it may be useful to increase the expression of endogenous or exogenous oxidative pentose phosphate pathway (PPP) enzymes to increase flux through the PPP. In some embodiments, increasing the expression of oxidative PPP enzymes draws down the pool of glucose-6-phosphate and pulls additional fructose-6-phosphate to enter the oxidative PPP. In certain embodiments, increasing the flux through the PPP increases and/or rebalances reducing equivalents. In further embodiments, the upregulation of one or more oxidative PPP enzyme activity comprises the overexpression of a glucose-6-phosphate dehydrogenase (ZWF1), a 6-phosphogluconolactonase (SOL3), or a 6-phosphogluconate dehydrogenase (GND1). In some embodiments, it may be useful to increase the expression of endogenous or exogenous PPP enzyme variants which use NAD+ in place of NADP+ producing NADH instead of NADPH to match cofactor requirements of recombinant pathways. In some embodiments, the one or more oxidative PPP enzyme variant comprises an NAD-dependent glucose-6-phosphate dehydrogenase (ZWF1) and/or an NAD-dependent 6-phosphogluconate dehydrogenase (GND1).
In some embodiments, it may be useful to increase the expression of endogenous or exogenous hexose transporters to increase and/or rebalance reducing equivalents. In certain embodiments, the hexose transporter is high affinity. In some embodiments, the hexose transporter is low affinity.
Improved biomass or lipid production can be accomplished by, for example, overexpression of nucleic acids encoding one or more of the above-described enzymes or transporters. Overexpression of the above-described enzymes or proteins can occur, for example, through increased expression of an endogenous gene or genes, or through the expression, or increased expression, of an exogenous gene or genes. Therefore, naturally occurring organisms can be readily modified to generate non-natural microorganisms having improved biomass and lipid production through overexpression of one or more nucleic acid molecules encoding, for example, an isocitrate dehydrogenase, a pyruvate transporter, an acetyl-CoA carboxylase, an aconitase, a citrate transporter, a decarboxylating malic enzyme, an ATP-citrate lyase, a glucose-6-phosphate dehydrogenase, a 6-phosphogluconolactonase, a 6-phosphogluconate dehydrogenase, a hexose transporter, or combination thereof.
In addition, a non-naturally occurring organism can be generated by mutagenesis of an endogenous gene that results in an increase in activity of an enzyme or transporter as described herein.
Equipped with the present disclosure, the skilled artisan will be able to readily construct the recombinant microorganisms described herein, as the recombinant microorganisms of the disclosure can be constructed using methods well known in the art as exemplified above to exogenously express at least one nucleic acid encoding an enzyme or transporter described herein in sufficient amounts to produce lipid and generate biomass.
Methods for constructing and testing the expression levels of a non-naturally occurring lipid and biomass-producing host can be performed, for example, by recombinant and detection methods well known in the art. Such methods can be found described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubo et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1999).
A variety of mechanisms known in the art can be used to express, or overexpress, exogenous or endogenous genes. For example, an expression vector or vectors can be constructed to harbor one or more enzyme or transporter encoding nucleic acids as exemplified herein operably linked to expression control sequences functional in the host organism. Expression vectors applicable for use in the microbial host organisms of the invention include, for example, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, including vectors and selection sequences or markers operable for stable integration into a host chromosome. Selectable marker genes also can be included that, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more exogenous encoding nucleic acids are to be co-expressed, both nucleic acids can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The transformation of exogenous nucleic acid sequences involved in a metabolic or synthetic pathway can be confirmed using methods well known in the art.
Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell. Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
In various embodiments, an expression control sequence may be operably linked to a polynucleotide sequence. By “operably linked” is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s). Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation. Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes to increase and/or rebalance reducing equivalents. In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes to increase product purity. In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes to modify availability of metabolic intermediates.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of glucose to glucose-6-phosphate. In some such embodiments, the enzymes that catalyze the conversion of glucose to glucose-6-phosphate are hexose kinases. In some embodiments, the deletion, disruption, mutation, and/or reduction in activity of one or more hexose kinase decouples and increases glucose uptake. In some embodiments, the deletion, disruption, mutation, and/or reduction in activity of one or more hexose kinase increases and/or rebalances reducing equivalents.
In other embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate to reduce flux through upper glycolysis. In some such embodiments, the enzymes that catalyze the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate are fructose-6-phosphate kinases. In some embodiments, the deletion, disruption, mutation, and/or reduction in activity of one or more fructose-6-phosphate kinase increases and/or rebalances reducing equivalents.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more enzymes involved in the mannitol synthesis pathway. In some embodiments, disruption of the mannitol synthesis pathway can be used to enhance generation of reducing equivalents and improve glucose yields to support production of fatty acid derived products. In certain embodiments, the one or more enzyme involved in the mannitol synthesis pathway is an NADPH-dependent mannitol dehydrogenase. In certain embodiments, the NADPH-dependent mannitol dehydrogenase is selected from the group consisting of YALI0B16192g, YALI0D18964g, and YALI0E12463g, or homologs thereof. In certain embodiments, the one or more enzyme involved in the mannitol synthesis pathway is an aldo-keto reductase. In certain embodiments, the aldo-keto reductase is selected from the group consisting of YALI0D07634g, YALI0F18590g, YALI0C13508g, YALI0F06974g, YALI0A15906g, YALI0B21780g, YALI0E18348g, YALI0B07117g, YALI0009119g, YALI0D04092g, YALI0B15268g, YALI0000319g, YALI0A19910g, or homologs thereof.
In some embodiments, the recombinant microorganism is manipulated to delete, disrupt, mutate, and/or reduce the activity of one or more endogenous enzymes that catalyzes carboxylation of acetyl-CoA to malonyl-CoA. In one embodiment, the one or more endogenous enzymes comprise one or more acetyl-CoA carboxylase. In some embodiments, the deletion, disruption, mutation, and/or reduction in activity of one or more acetyl-CoA carboxylase increases availability or amount of reducing equivalents, increases amount of one or more metabolic intermediate, and/or increases product purity.
Pheromone Compositions and Uses Thereof
As described above, products made via the methods described herein may be pheromones. Pheromones prepared according to the methods of the invention can be formulated for use as insect control compositions. The pheromone compositions can include a carrier, and/or be contained in a dispenser. The carrier can be, but is not limited to, an inert liquid or solid. In some embodiments, the pheromone composition is combined with an active chemical agent such that a synergistic effect results. In some embodiments, the pheromone composition can include one or more insecticides, one or more solubilizing agents, one or more fillers, one or more solvents, one or more solubilizing agents, one or more binders, one or more surface-active agents, one or more wetting agents, one or more dispersing agents, one or more polymeric surfactants, one or more emulsifying agents, one or more gelling agents, one or more anti-foam agents, and/or one or more preservative. According to another embodiment of the disclosure, the pheromone composition may include one or more insect feeding stimulants. According to another embodiment of the disclosure, the pheromone composition may include one or more insect growth regulators (“IGRs”). According to another embodiment of the disclosure, the attractant-composition may include one or more insect sterilants that sterilize the trapped insects or otherwise block their reproductive capacity, thereby reducing the population in the following generation.
In some embodiments, the pheromone compositions disclosed herein can be formulated as a sprayable composition (i.e., a sprayable pheromone composition). In some embodiments, the pheromone compositions disclosed herein can be formulated as a microencapsulated pheromone, such as disclosed in Ill'lchev, A L et al., J. Econ. Entomol. 2006; 99(6):2048-54; and Stelinki, L L et al., J. Econ. Entomol. 2007; 100(4):1360-9. Pheromone compositions can be formulated so as to provide slow release into the atmosphere, and/or so as to be protected from degradation following release. The pheromone compositions of the disclosure may be used in traps or lures. Pheromone compositions of the present disclosure can be used in conjunction with a dispenser for release of the composition in a particular environment.
Pheromone compositions prepared according to the methods disclosed herein can be used to control or modulate the behavior of insects. Thus, in some embodiments, the pheromones can be used to attract insects away from vulnerable crop areas. Pheromones prepared according to the methods of the disclosure can also be used to disrupt mating. Mating disruption is a pest management technique designed to control insect pests by introducing artificial stimuli (e.g., a pheromone composition as disclosed herein) that confuses the insects and disrupts mating localization and/or courtship, thereby preventing mating and blocking the reproductive cycle. In some embodiments, the pheromone compositions may be used in attract and kill. The attract and kill method utilizes an attractant, such as a sex pheromone, to lure insects of the target species to an insecticidal chemical, surface, device, etc., for mass-killing and ultimate population suppression.
Thus, in some embodiments, the present disclosure teaches edibles produced from the Specialty Cannabis and/or cannabinoid compositions disclosed herein.
This invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.
Background and Rationale
Many microorganisms down-regulate respiration under non-optimal growth conditions to encourage carbon/energy storage via citrate and lipid synthesis. Under nitrogen limited conditions, intracellular AMP levels are depleted by an AMP deaminase (AMD1). Reduced AMP levels in these microorganisms, inactivate the mitochondrial isocitrate dehydrogenase complex (IDH1/2) through allosteric regulation, which results in accumulation of isocitrate, and reduced TCA cycle throughput.
Both native fatty acid synthesis, and the bypass methyl palmitate based synthesis of Z11-16 acid, require reducing equivalents in the form of NADPH and NADH. The inventors hypothesized that increasing the oxidation rate of citrate (through IDH activation) could increase the available reduced cofactor pool leading to increased production of unsaturated fatty acids.
Experimental Design
Two different loci were targeted to test the impact of individual IDHs on glucose consumption, growth, citrate, and fatty acid production. One cassette was designed to target the Y. lipolytica IDH1 locus replacing the native IDH1 variant with recombinant AMP-insensitive IDH enzymes (
A second cassette was designed to add a recombinant copy of IDH while retaining the wild type Y. lipolytica IDH1 copy. This cassette targets the FAO1 locus and uses the constitutive TEF promoter to drive expression (
The 19 constructs described above were transformed into SPV739, a marker rescued descendent of SPV458. SPV458 was created by integrating a single copy of the H. zea Z11 desaturase into the XPR2 locus of SPV300 (H222 Δpox1 Δpox2 Δpox3 Δpox4 Δpox5 Δpox6 Δadh1 Δadh2 Δadh3 Δadh4 Δadh5 Δadh6 Δadh7 Δfao1 Δura3, herein also referred to as “H222 ΔP ΔA ΔF ura3”). H222 corresponds Y. lipolytica H222 (Mauersberger, S., H. J. Wang, et al. (2001), J Bacteriol 183(17): 5102-5109), according to Barth and Gaillardin (Barth G & Gaillardin C (1996) Yarrowia lipolytica. Springer-Verlag, Berlin, Heidelberg, New York).
Y. Lipolytica
Y. lipolytica IDH1
H. sapiens
Y. lipolytica pTEF
E. Coli
Y. lipolytica IDH1
H. sapiens
Y. lipolytica pTEF
H. sapiens
H. sapiens
A. Thiooxidans
Y. lipolytica IDH1
H. sapiens
Y. lipolytica pTEF
H. sapiens
H. sapiens
M. smegmatis
Y. lipolytica IDH1
H. sapiens
Y. lipolytica pTEF
H. sapiens
H. sapiens
R. reniformis
Y. lipolytica IDH1
H. sapiens
Four clonal isolates of each construct were selected for screening in a 24-deepwell plate assay (76 strains total) (See Materials and Methods for full description of protocol). Initial seeds were grown in YPD medium before transfer to a nitrogen limited medium supplemented with methyl palmitate as a saturated fatty acid substrate. To evaluate the impact of each IDH, extracellular glucose and citrate and fatty acid content were quantified (See Materials and Methods for sampling procedures).
Experimental Results
Analysis of Strains with Type 1 Cassettes (Single IDH Copy, Replaced Native IDH1)
Fatty Acid Analysis
Strain IDH002 (Y. lipolytica IDH1 D279A, I280A codon optimized), displayed increased OD-normalized Z11-16Acid titer at both 48 hours (˜40%) and 72 hours (˜20%). In addition, IDH002 produced increased titers of other native Y. lipolytica fatty acids including the major native fatty acid Z9-18Acid. Two other constructs, IDH001 (Y. lipolytica IDH1 D279A, I280A native sequence) and IDH004 (A. thiooxidans IDH) produced increased titers of other native Y. lipolytica fatty acids. As expected, the idh1::GFP knockout strains (IDH006) produced less fatty acids generally and ˜50% less Z11-16Acid.
Glucose Analysis
In agreement with increased TCA cycle flux, IDH001 and IDH002 also demonstrated increased glucose consumption over the 72-hour assay. IDH004 consumed an equivalent quantity of glucose to the SPV458 control. Two constructs which displayed the largest increase in fatty acid production also showed increased glucose consumption at 48 and 72 hours (IDH002, IDH011). The idh1::GFP knockout (IDH006) displayed reduced glucose consumption at both 48 and 72 hours. Initial concentration of 60 g/L glucose.
Citrate Analysis
Citrate titers followed multiple trends depending on the IDH variant. Y. lipolytica IDH1 D279A, I280A constructs (IDH001, IDH002) did not display reduced citrate titer (g/L-OD). The A. thiooxidans IDH construct, IDH004 however, displayed lower normalized citrate titer at 72.
Analysis of Strains with Type 2 Cassettes (Native+Recombinant IDH at FAO1 Locus)
Fatty Acid Analysis
Two FAO1 cassette constructs, IDH011 (A. thiooxidans IDH, mitochondria targeted, codon optimized) and IDH012 (M. smegmatis IDH, cytosol targeted, codon optimized), produced increased Z11-16 acid titer and OD-normalized titer at 48 hours. Increases were 20% for IDH011 and 10% for IDH012. Both these constructs also produced higher titers of native fatty acid species.
Increased native fatty acid titers were also observed for three other FAO1 cassette constructs: native coded E. coli constructs which were targeted to either the cytosol (IDH014) or mitochondria (IDH015) and codon optimized M. smegmatis IDH targeted to the mitochondria (IDH013). Of the two E. coli constructs, IDH014 (cytosol) sustained the increase in fatty acid production at the 72-hour time point while titers were stagnant for IDH015 (mitochondria).
Glucose Analysis
IDH009 (E. coli IDH, mitochondria targeted, codon optimized), IDH010 (A. thiooxidans IDH, cytosol targeted, codon optimized), IDH0011 (A. thiooxidans IDH, mitochondria targeted, codon optimized) and IDH019 (M. smegmatis IDH, mitochondira targeted, native sequence) displayed increased glucose consumption.
Citrate Analysis
As with IDH1 cassettes, citrate titers followed multiple trends depending on the IDH variant. Of the E. coli IDH constructs, only IDH009 produced less citrate than the SPV458. Both A. thiooxidans IDH constructs which consumed additional glucose, IDH010, and IDH011, also produced less citrate. M. smegmatis constructs which either showed increased fatty acid production (IDH012, IDH013) or increased glucose consumption (IDH019) also produced less citrate. Of the M. smegmatis constructs, the most significant reduction in citrate was observed for IDH019.
Summary of the 24 Well Assay
An additional trend emerges when citrate titers are examined as a function of expression cassette design for each of the recombinant IDHs. IDH1 targeted cassettes with the IDH1 promoters produced the highest citrate titers while FAO1 cassettes with the TEF promoter and the Y. lipolytica IDH1 mitochondrial targeting sequence produced the lowest citrate titers. This dependence on cassette design is consistent with high mitochondrial expression of IDH leading to increased TCA cycle flux. Finally, as expected, the IDH006 strains (idh1::GFP) grew slowly and produced the highest normalized citrate titers (g/L-OD). The increase was especially noticeable at 48 hours. This increase in citrate titer confirms the negative control hypothesis that reduced TCA cycle flux in the mitochondria leads to further accumulation of citrate.
Strains expressing codon optimized A. thiooxidans IDH using the TEF promoter and targeted to the mitochondria showed reduced citrate titer (g/L) and specific citrate production (g/L-OD) at 72 hours in the 24-deepwell plate assay. Cell density, as measured by OD600, increased by ˜35% at 72 hours. Fatty acid titers were increased over the SPV458 control as follows:
Strains expressing a copy of the Y. lipolytica IDH1 mutant D279, I280A under the TEF promoter did not display reduced citrate titer or increased cell density in the 24-deepwell plate assay, but did produce increased fatty acid titers as follows:
For each construct 4 clonal isolates were inoculated in 1 ml of YPD seed culture in 24-deepwell plates for 24 hours at 28° C. and 1000 rpm (Infors plate incubator). Seed cultures were pelleted and supernatant removed before resuspension in 2 ml of S2 medium. After cell pellets were resuspended, 24 μl of 37° C. methyl palmitate was added to each well (˜10 g/L final concentration). The plates were then incubated for 48 hours at 28° C. and 1000 rpm (Infors plate incubator) before 250 μl samples were transferred to glass, crimp-top GC vials. Plates were incubated an additional 24 hours and a second set of 250 μl samples were taken at 72 hours. Vials were frozen at −80° C. before further processing. Cell density was measured with a Tecan M200 Pro plate reader at each sampling.
GC Sample Processing-Lyophilized Samples
250 uL of culture were lyophilized in open glass crimp top vials for at least 3 hours. 500 uL of TMSH were added to the vials and sealed with a crimp cap. These vials were arrayed in racks, which were placed in a 28° C. plate shaker for 2 hours at 250 rpm. After mixing these dried cells with the derivatizing agent, the vials were incubated in a heat block for 1 hour at 85° C. to lyse the cell membranes. Finally, the liquid portion of the methylated sample was transferred to a clean GC vial with glass insert to prevent solid debris from clogging the column during GC analysis. Samples were run on GC-FID.
Background and Rationale
Microorganisms of the present disclosure produce citrate as co-product under nitrogen-limited conditions. This is because during nitrogen starvation many organisms, including Y. lipolytica down-regulate respiration to divert carbon/energy storage via lipid synthesis. Citrate is first exported from mitochondria into the cytosol and subsequently from the cell. Exported citrate can be re-assimilated, especially when alternative carbon sources are scarce. Alternatively, the combination of the enzymes ATP citrate lyase (ACL), malate dehydrogenase (e.g., MDH2) and cytosolic malic enzyme can turn cytosolic citrate into pyruvate, to feed back into the TCA cycle to power further lipid synthesis (
The inventors further hypothesized that expression of a heterologous NADP+ dependent cytosolic malic enzyme may increase fatty acid production if the primary rate limitation is cofactor supply.
In many organisms, including Y. lipolytica, the pentose phosphate pathway (PPP) is the primary source of NADPH reducing equivalents for fatty acid synthesis. The inventors hypothesized that the recombinant microorganisms lipid production and selectivity could further be increased by overexpressing the genes of the upper (oxidative) pentose phosphate pathway (ZWF1, SOL3, and GND1), potentially offering another route to increase biomass and/or fatty acid synthesis.
Experimental Design
Nucleic acids encoding for heterologous malic enzymes, and overexpressing endogenous PPP enzymes, ZWF1, GND1, and SOL3, were introduced into a Y. lipolytica microorganism. Three or four clonal isolates of each construct were characterized in a 24-well bioconversion assay, feeding co-substrates glucose/glycerol and methyl palmitate. Fatty acid profiles for all constructs were quantified using GC analysis. Measurements for initial biomass in YPD and final biomass in nitrogen-limited media were taken. Growth of each microorganism was tracked and analyzed for fatty acid profiles in nitrogen-limited media with Solulys95 to confirm that access to methyl palmitate as a co-substrate is necessary to realize improvements in Z11-16Acid selectivity, total fatty acid production, and/or biomass generation. Table 8 provides a summary of the recombinant microorganisms tested. Note that the L. starkeyi construct was excluded from the bioconversion results due to technical issues during the experiment resulting in no growth of the microorganism.
L. starkeyi
R. toruloides
M. musculus
R.
norvegicus
H. sapiens
E. coli
Y. lipolytica
Y. lipolytica
Y. lipolytica
Three or four clonal isolates of each construct were selected for screening in a 24-deepwell plate assay (See Materials and Methods for full description of protocol). Initial seeds were grown in YPD medium before transfer to a nitrogen limited medium supplemented with methyl palmitate as a saturated FAME substrate. To evaluate the impact of malic enzyme and PPP overexpression, fatty acid content and biomass generation were quantified (See Materials and Methods).
Experimental Results
Overexpression of the NADP+ dependent L. starkeyi and M. musculus cytosolic malic enzyme, as well as the PPP gene GND1, led to increased titers of total fatty acid compared to the reference strain SPV458 when feeding glucose/glycerol and methyl palmitate co-substrates after 42 hours of bioconversion (
While three strains produced higher Z11-16Acid titer when fed methyl palmitate, the strains did not exhibit significant improvements in de novo lipid production (
Surprisingly, all tested strains exhibited an improvement in Z11-16Acid selectivity (
We tested the strains in bioprocess media with glucose and excess nitrogen to assess differences in growth and lean biomass accumulation. As seen in
The bioconversion assay did not uncover a significant increase in biomass production for any of our strains either, further corroborating the hypothesis that improved Z11-16Acid production and selectivity is not a related to increased biomass generation, but primarily to increased bioconversion rate and/or efficiency.
Materials & Methods
24-Deepwell Assay
For each construct three or four clonal isolates were inoculated in 1 ml of YPD seed culture in 17 mm glass vials in 24-deepwell plates for 24 hours at 28° C. and 1000 rpm (Infors plate incubator). Seed cultures were pelleted and supernatant removed before resuspension in 1 mL FERMI media. Cultures were incubated at 28° C. and 1000 rpm (Infors plate shaker) for an additional 6 hours in FERMI media. Then, methyl palmitate was added at 20 g/L with a P200 multi-channel. After an additional 42 hours of bioconversion (72 hours total), 250 μl sample was transferred to a crimp-top GC vial for analysis. Vials were frozen at −80° C. before further processing. Cell density was measured with a Tecan M200 Pro plate reader at feeding and at sampling.
Biolector Growth Assay
For each construct three or four clonal isolates were inoculated in 2 ml of YPD seed culture in 24-deepwell plates for 24 hours at 28° C. and 1000 rpm (Infors plate incubator). 7-8 uL of each seed culture were used to inoculate 750 uL of Bioprocess media in each Biolector plate well. Cultures were incubated at 32° C. and 1500 rpm (Biolector) for an additional 72 hours in Bioprocess media, either with excess nitrogen (10 g/L) or limited nitrogen (2 g/L). After 72 hours of growth, 250 μl sample for the nitrogen-limited condition was transferred to a crimp-top GC vial for analysis. Vials were frozen at −80° C. before further processing. Cell density was measured with a Tecan M200 Pro plate reader at feeding and at sampling. Growth curves for the excess nitrogen condition were assessed to compare lean biomass generation for each strain.
GC Sample Work-up
250 uL of culture were lyophilized in open glass crimp top vials for at least 3 hours. 500 uL of methanol was added to the vials followed by addition of 35 ul 10M potassium hydroxide. Vials were then sealed with a crimp cap, arrayed in 54-well racks, mixed for 10 minutes at 2000 RPM using a Mixmate plate shaker, and heated at 60 C for 40 minutes in a convection oven. Vials were cooled to room temperature and decapped. 29 ul 24N sulfuric acid was added to vials. Vials were then sealed with a crimp cap, arrayed in 54-well racks, mixed for 2 minutes at 2000 RPM using a Mixmate plate shaker, and heated at 60 C for 40 minutes in a convection oven. Vials were cooled to room temperature and decapped. 1 ml hexanes was added to vials. Vials were then sealed with a crimp cap, arrayed in 54-well racks, and mixed for 10 minutes at 2000 RPM using a Mixmate plate shaker. Vials then spun down for 5 minutes at 1000 RPM using an Avanti centrifuge. Vials were run on GC-FID.
Background and Rationale
ACC catalyzes the irreversible conversion of acetyl-CoA to malonyl-CoA, one of the key substrates in lipid biosynthesis. To regulate lipid biosynthesis, kinases inhibit ACC via post-translational modifications. Kinases phosphorylate ACC serine residues, which, when phosphorylated, interact with two downstream arginine residues to inhibit the activity. In order to counteract the microorganism's native regulation of lipid biosynthesis, a few engineering approaches can be taken to increase lipid accumulation in the microorganism, aided by the fact that neither of the residues involved in ACC inhibition are also involved in the conversion of acetyl-CoA to malonyl-CoA. The first approach is to overexpress heterologous ACC variants which may not be phosphorylated by host kinases and are thus not inhibited by a phosphorylated serine. To further improve lipid production, the heterologous ACC variants are further engineered by eliminating key serine residues through replacement with alternative amino acids. The serine residue is replaced by either a point mutation or through replacement of a loop region of the amino acid sequence which contains the key serine residue. In another strategy to further improve lipid synthesis, the heterologous ACC is mutated by replacing the arginine residues involved in inhibiting ACC with a residue, like alanine, that does not interact with phosphorylated serine to deactivate the enzyme. In addition to overexpression of heterologous feedback-resistant ACC variants, the host's native ACC can be deleted or down-regulated. The overexpression of modified ACC variants relieved of post-translational phosphorylation is expected to lead to enhanced flux towards malonyl-CoA and enhanced fatty acid production.
Experimental Design
An expression cassette containing the nucleic acid encoding select heterologous acetyl-CoA carboxylase (see sequences below) will be introduced into the chromosome of the Y. lipolytica host that has been previously engineered to produce insect fatty acid pheromone precursors. Expression of the ACC gene variant will be mediated by a strong promoter such as the yeast trancription elongation factor promoter sequence. In the alternative, ACC variants will be expressed by the microorganism's native ACC promoter sequence. Each transformed gene will contain at least one of the three modifications:
Genome integration of the cassette will utilize homologous recombination methods used in Examples 1 and 2. Inclusion of an amino acid biosynthetic or antibiotic resistance gene in the cassette allows selection of colonies harboring the cassette by plating on selective agar media. Successful integration of the cassette is verified via PCR and sequencing. Three or four positive clonal isolates harboring each heterologous ACC construct will be characterized in a 24-well bioconversion assay, feeding co-substrates glucose/glycerol and methyl palmitate. Fatty acid profiles for all constructs will be quantified using GC analysis. Measurements for initial biomass in YPD and final biomass in nitrogen-limited media will be taken. Growth of each microorganism will be tracked and analyzed for fatty acid profiles in nitrogen-limited media with Solulys95 to confirm that access to methyl palmitate as a co-substrate is necessary to realize improvements in Z11-16Acid selectivity, total fatty acid production, and/or biomass generation.
Experimental Results
Expression of the heterologous ACC genes including the mutations described above are expected to increase accumulation of total fatty acids. All samples will be analyzed for the following fatty acids: Z9-16Acid, Z11-16Acid, 18Acid, Z9-18Acid, Z11-18Acid, Z13-18Acid, Z9Z12-18Acid.
Particular subject matter contemplated by the present disclosure is set out in the below numbered embodiments.
1. A recombinant microorganism having improved production of biomass or improved production of one or more lipids from one or more fatty acid and one or more simple carbon co-substrates, wherein the recombinant microorganism comprises one or more modifications in one or more fields comprising:
Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:
1. A recombinant microorganism with improved lipid production from one or more fatty acid, and one or more simple carbon co-substrates, wherein the recombinant microorganism comprises:
The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood there from as modifications will be obvious to those skilled in the art.
While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.
Y. lipolytica IDH1 D279A, I280A amino acid sequence
E. coli IDH amino acid sequence WP_000444484.1
A. thiooxidans IDH amino acid sequence PDB: 2D4V_A
M. smegmatis IDH amino acid sequence WP_011727802.1
L. starkeyi malic enzyme amino acid sequence (mitochondrial, NAD+,
R. toruloides malic enzyme amino acid sequence (mitochondrial, NAD+,
H. sapiens malic enzyme amino acid sequence (mitochondrial, NAD+,
R. norvegicus malic enzyme amino acid sequence (cytoplasm, NADP+,
M. musculus malic enzyme amino acid sequence (cytoplasm, NADP+,
E. coli malic enzyme amino acid sequence (NAD+, unmodified) P26616
Helicoverpa zea Z11 desaturase
Helicoverpa zea Z11 desaturase
osmophila]
Bacillus subtilis IDH S104A mutant, AMP-insensitive
norvegicus OX=10116 GN=Acaca PE=1 SV=1
cerevisiae (strain ATCC 204508 / 5288c) OX=559292 GN=ACC1 PE=1
sapiens OX=9606 GN=ACACA PE=1 SV=2
All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes.
However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
This application is a divisional of U.S. patent application Ser. No. 16/401,690, filed May 2, 2019, which issued on Jan. 11, 2022 as U.S. Pat. No. 11,220,675, and which claims priority to U.S. Provisional Patent Application No. 62/665,809, filed on May 2, 2018, each of which is incorporated by reference herein in its entirety for all purposes.
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| WO-2016207339 | Dec 2016 | WO |
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| Number | Date | Country | |
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| 20220177857 A1 | Jun 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62665809 | May 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16401690 | May 2019 | US |
| Child | 17550212 | US |
