Patent Application
20240368659
The present invention relates to genetically modified host cells having increased production of vitamin B compounds through providing for a reduced and/or eliminated binding of cAMP to cAMP receptor protein (CRP); to mutants of native genes and encoded polypeptides providing for a reduced binding of cAMP to CRP; to genetic constructs for expression of such mutants; to cultures of the genetically modified host cells and its use to produce vitamin B compounds; to fermentation liquids comprising vitamin B compounds resulting from such production; to compositions comprising the fermentation liquid; to dietary or pharmaceutical preparations made from such compositions and to the uses of such compositions and preparations.
Vitamin B compounds, such as biotin (B7) are in nature produced by some microbes and plants. The biosynthesis pathways for vitamin B compounds in natural organisms are generally well described in the art. For example, in nature, biotin is synthesized by a linear pathway involving the fatty acid biosynthetic pathway (see
Further, thiamine (B1) is in nature produced by some microbes and plants. The biosynthesis pathways for thiamine in natural organisms are generally well described in the art. For example, in nature, thiamine is synthesized by the pathway shown in
The use of microorganism-based cell factories is a potential route for the biosynthetic production of B vitamins (Acevedo-Rocha, et al. 2019). The advantages of a recombinant microorganisms such as E. coli as a cell factory for production of bio-products are widely recognized due to the fact that: (i) it has unparalleled fast growth kinetics; with a doubling time of about 20 minutes when cultivated in glucose-salts media and under optimal environmental conditions, (ii) it easily achieves a high cell density; where the theoretical density limit of an E. coli liquid culture is estimated to be about 200 g dry cell weight/L or roughly 1×10{circumflex over ( )}13 viable bacteria/mL. Additionally, there are many molecular tools and protocols at hand for genetic modification of E. coli; as well as it being an organism that is amenable to the expression of heterologous proteins; both of which may be essential for obtaining high-level production of desired bio-products.
Cellular production capacity of vitamin B compounds is influenced by many factors and is to a large extent only sparsely described in the art. WO2019/012058 and WO2020/148351 discloses that mutating transcription factor IscR to favor the apoprotein improves conversion of for example desthiobiotin to biotin by activating the SUF and ISC operons producing iron-sulfur cluster (FeS) clusters needed by biotin synthase (BioB) and/or favor the apoprotein improves conversion of for example 5-aminoimidazole ribotide (AIR) to thiamine by activating the SUF and ISC operons producing iron-sulfur cluster (FeS) clusters needed by phosphomethylpyrimidine synthase (ThiC).
Further Ifuku et al. (1995) discloses that BioB over-expression can induce cell death in E. coli strains, possibly due to depletion of FeS clusters from the cell and/or the generation of Reactive Oxygen Species (ROS).
Another factor affecting production of vitamin B compounds in cells is the metabolic state of the cell. There are several cellular regulator proteins that have large effects on the metabolic state of the cell. One such protein is the transcription factor cAMP receptor protein (CRP), also known as catabolite gene activator protein (CAP) which regulates expression of hundreds of genes in E. coli when bound to small molecule adenosine 3′,5′-cyclic monophosphate or cyclic AMP (cAMP, CAS Number 60-92-4). cAMP is synthesized by adenylate cyclase from adenosine triphosphate (ATP).
Deutscher et al. (2006) discloses that when glucose supply is depleted, while other carbon sources are available, the phosphotransferase system (PTS) proteins become phosphorylated, resulting in activation of adenylate cyclase (CyaA) by enzyme IIA-Glc (CRR) and thus cAMP synthesis, ultimately leading to an active CRP-cAMP complex. When glucose is in excess PTS remains unphosphorylated ultimately leading to low cAMP and low levels of CRP-cAMP complex. Shimada et al. (2011) discloses that CRP-cAMP activates (among others) genes involved in the utilization of secondary carbon sources, as well as the transcription of PTS/glucose import genes. Shimada et al. (2011) further discloses that an estimated ˜400-500 operons are under direct control of the CRP-cAMP complex including the following processes: a) carbon metabolism (glycolysis and gluconeogenesis); b) aerobic respiration (TCA cycle); c) nitrogen metabolism; d) regulatory roles (control of many transcription factors); and e) stress response. Barth et al (2009) discloses that a cellular decrease of cAMP derepresses rpoS, a gene that encodes the alternative sigma factor σS, which activates the general stress response in E. coli. Basak & Jiang (2012) discloses that specific mutations to the active site of CRP enables E. coli to tolerate ROS due to expression of stress response genes. Perrenoud & Sauer (2005) showed that deletion of CyaA influences biomass yields, growth rates, glucose consumption rate, acetate production, phosphoenolpyruvate levels and serine levels, while no influence on TCA cycle flux were observed.
Further background art includes US2006/121558 which discloses that mutation of CyaA impacts on cellular production of carotenoids in E. coli; WO2011102305 which discloses that mutation of CyaA impacts on cellular production of amino acids in Enterobacteriaceae; US20140096439 which discloses that mutation of CyaA impacts on cellular production of butanol in yeast; AU2012202630 which discloses that mutation of CyaA impacts on cellular production of isoprenoids in E. coli; WO2013003432 discloses that mutation of CyaA impacts on cellular production of succinate in E. coli; U.S. Pat. No. 9,932,598 which discloses that mutation of CyaA impacts on cellular production of carbohydrates in E. coli; EP3050970 which discloses that mutation of CyaA impacts on cellular production of 1,4-butanediol in E. coli and other organisms; US20180100169 which discloses that mutation of CyaA impacts on cellular production of 2,4-dihydroxybutyrate in E. coli. WO2021122687 which states that increased cyclic AMP levels and mutations causing this increase the production of fine chemicals, especially human milk oligosaccharides, in host cells.
Against this background art, the inventors of the present invention have now found that host cells producing vitamin B compounds genetically modified to reduce binding of cAMP to CRP unexpectedly have increased production of vitamin B compounds. The present inventors have also identified several ways to reduce the binding of cAMP to CRP (see
Specifically, for vitamin B compounds, herein illustrated for example by biotin (B7) or thiamine (B1), it has been found that mutations that eliminate or decrease the amount of CRP-cyclic AMP complex increase the in vivo activity of overexpressed pathway enzymes, such as biotin synthase (BioB) or phosphomethylpyrimidine synthase (ThiC), and thereby increase production of vitamin B compounds such as biotin from glucose in microorganism-based cell factories. Several genes are directly related to regulating or catalyzing the formation of the CRP-cyclic AMP complex and therefore there are several distinct genes that can be modified to arrive at the same phenotype of an improved vitamin B compound production strain. The regulatory effect of eliminating or decreasing the amount of CRP-cyclic AMP complex in the cell is very complex and is known to increase the yield of cell biomass produced from glucose; however, the improvement in vitamin B compound production in genetically modified cell factories has surprisingly been found to exceed the increase in biomass, i.e. production of vitamin B compound, such as biotin or thiamine, per gram of biomass is improved on top of the increase in biomass. While the exact mechanism for improvement of pathway enzymes for vitamin B compounds (e.g., BioB or ThiC) is unknown, it is believed that the change in metabolism brought about by the lack of cyclic AMP complex is beneficial for the formation of functional FeS loaded enzymes, the bottleneck for many enzymatic steps, such as BioB, ThiC, NadA, IlvD and CobG producing biotin, thiamine, quinolate, pantothenate and cobalamin respectively.
CRP is natively activated as a transcriptional regulator by binding to its allosteric activator cyclic AMP (cAMP), which is produced by the enzyme adenylate cyclase (encoded by CyaA) (
Accordingly, in a first aspect the invention provides a genetically modified host cell having increased production of one or more vitamin B compound, wherein the host cell is genetically modified by
In a further aspect the invention provides for mutated polypeptides which are at least 90% identical to
In a still further aspect, the invention provides a polynucleotide construct comprising a mutated polynucleotide sequence of the invention encoding a cAMP receptor protein (CRP), a carbohydrate repression resistance protein (CRR) or a adenylate cyclase protein (CyaA) operably linked to one or more control sequences.
In a still further aspect, the invention provides a cell culture, comprising the genetically modified host cell of the invention and a growth medium.
In a still further aspect, the invention provides a genetically modified host cell comprising the nucleic acid construct.
In a still further aspect, the invention provides a cell culture, comprising the genetically modified host cell of the invention and a growth medium.
In a still further aspect, the invention provides a method for producing a vitamin B compound comprising:
In a still further aspect, the invention provides a fermentation composition comprising the cell culture of the invention and the vitamin B compound comprised therein.
In a still further aspect, the invention provides a composition comprising the fermentation composition of the invention and one or more carriers, agents, adjuvants, additives and/or excipients.
In a final aspect, the invention provides a kit of parts comprising:
The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out. The figures and drawing include:
All publications, patents, and patent applications referred to herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.
Provided for herein are genetically modified host cells having increased production of one or more vitamin B compound, achieved by mutating one or more native genes for reducing formation of a CRP-cAMP complex in the host cell and/or introducing one or more genetic alterations increasing the degradation of cAMP and/or binding of cAMP by a polypeptide which is not CRP in the host cell; whereby the production of the vitamin B compound in the genetically modified host cell is increased compared to a parent host cell. For example, it was unexpectedly found that mutating CyaA and/or CRP and/or CRR in such a way as to delete, disrupt and/or attenuate the protein, e.g., produced a higher BioB or ThiC activity. It is not yet fully known if the cause of this effect is higher BioB or ThiC protein levels or higher BioB or ThiC activity per protein or a combination of both. Additionally, it was observed that deleting, disrupting and/or attenuating mutations in strains also provides for a higher production of the biotin precursor DTB or of thiamines including both thiamine and the precursor thiamine monophosphate (TMP), with the former being the most abundant (>98%) than the latter due to a very efficient phosphatase expression.
Any EC numbers used herein refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including 30 supplements 1-5 published in Eur. J. Bio-chem. 1994, 223, 1-5; Eur. J. Biochem. 1995, 232, 1-6; Eur. J. Biochem. 1996, 237, 1-5; Eur. J. Biochem. 1997, 250, 1-6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. http://enzyme.expasy.org/. The term “PEP” as used herein refers to phosphoenol pyruvate.
The term “CRP” as used herein refers to the cAMP receptor protein (CRP), a transcription factor which binds to cAMP and regulates transcription of a multitude of genes in many cells, such as araB, nadC, aceE, lacZ and many more.
The term “CyaA” as used herein refers to an adenylate cyclase protein (CyaA) converting ATP into cAMP.
The term “CRR” as used herein refers to an Enzyme IIAGIc protein (also referred to as treD; gsr; iex; tgs; EIIAGIc; Enzyme IIIGIc; EIIIGIc; IIIGIc; Enzyme IIAGIc, CRR and Glucose-specific phosphotransferase enzyme IIA component), a phosphotransferase protein.
The term “CpdA” as used herein refers to a 3′,5′-cyclic adenosine monophosphate phosphodiesterase protein (also referred to as cyclic AMP phosphodiesterase, cAMP phosphodiesterase or icc) which hydrolyzes cAMP.
The term “CadD” as used herein refers to a 3′,5′-cyclic adenosine monophosphate deaminase protein (also referred to as ADD, cyclic adenylate deaminase, cyclic AMP deaminase or cAMP deaminase) which deaminates cAMP.
The term “FeS-cluster” as used herein refers to [2Fe-2S] or [4Fe-4S] clusters of the formulas:
The term “FabG” as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] reductase (EC: 1.1.1.100) converting a (3R)-hydroxyacyl-[ACP] into 3-oxoacyl-[ACP].
The term “FabZ” as used herein refers to a 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (EC: 4.2.1.59) converting (3R)-hydroxyacyl-[ACP] into (2E)-enoyl-[ACP].
The term “Fabl” as used herein refers to an enoyl-[acyl-carrier-protein] reductase (EC: 1.3.1.9) converting 2,3-saturated acyl-[ACP] into (2E)-enoyl-[ACP].
The term “FabB” as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] synthase (EC: 2.3.1.41) converting fatty acyl-[ACP] and malonyl-[ACP] into 3-oxoacyl-[ACP] and holo-[ACP].
The term “FabF” as used herein refers to a 3-oxoacyl-[acyl-carrier-protein] synthase (EC: 2.3.1.179) converting (11Z)-hexadecenoyl-[ACP] and malonyl-[ACP] into 3-oxo-(13Z)-octadecenoyl-[ACP] and holo-[ACP].
The term “BioC” as used herein refers to a malonyl-acyl carrier protein methyltransferase (EC2.1.1.197) converting Malonyl-acyl carrier protein into malonyl-acyl carrier protein methyl ester.
The term “BioF” as used herein refers to an 8-amino-7-oxononanoate synthase (EC2.3.1.47) converting a pimeloyl-acyl carrier protein into KAPA.
The term “BioA” as used herein refers to a Adenosylmethionine-8-amino-7-oxononanoate transaminase (EC2.6.1.62) converting KAPA into DABA using SAM as amino donor.
The term “Biok” as used herein refers to an adenosylmethionine-8-amino-7-oxononanoate transaminase converting KAPA into DAPA using lysine as amino donor.
The term “BioD” as used herein refers to a desthiobiotin synthase (EC6.3.3.3) converting DABA into DTB.
The term “Biol” as used herein refers to a biotin biosynthesis cytochrome P450, (pimeloyl-[acp] synthase (EC1.14.14.46) converting long-chain acyl-[acyl-carrier] protein into pimeloyl-[acp].
The term “BioW” as used herein refers a 6-carboxyhexanoate-CoA ligase (EC6.2.1.14) converting pimelate into pimeloyl-CoA.
The term “KAPA” as used herein refers to 7-keto-8-aminopelargonic acid.
The term “DAPA” as used herein refers to 7,8-Diaminopelargonic Acid.
The term “DTB” as used herein refers to desthiobiotin.
The term “SAM” as used herein refers to S-adenosyl-L-methionine.
The term “SAH” as used herein refers to S-Adenosyl-L-Homocysteine
The term “CoA” as used herein refers to coenzyme A
The term “ACP” as used herein refers to Acyl Carrier Protein
The term “AMTOB” as used herein refers to S-adenosyl-2-oxo-4-thiomethylbutyrate.
The term “5′DOA” as used herein refers to 5′-deoxyadenosine.
The term “TMP-phosphatase” as used herein refers to a thiamine monophosphate phosphatase dephosphorylating thiamine monophosphate to thiamine. It has been shown that for example the bifunctional TH2 protein from Arabidopsis thaliana has this activity (see also WO2017103221).
The term “ThiK” as used herein refers to a thiamine kinase that catalyzes the phosphorylation of thiamine to thiamine-monophosphate (TMP).
The term “ThiL” as used herein refers to a thiamine-monophosphate kinase. It catalyzes the ATP-dependent phosphorylation of thiamine-monophosphate (TMP) to form thiamine-pyrophosphate (TPP), the active form of vitamin B1. It cannot use thiamine as substrate. Is highly specific for ATP as phosphate donor.
The term “ThiM” as used herein refers to a Hydroxyethylthiazole kinase that catalyzes the phosphorylation of the hydroxyl group of 4-methyl-5-beta-hydroxyethylthiazole.
The term “ThiD” as used herein refers to a Hydroxymethylpyrimidine or phosphomethylpyrimidine kinase that catalyzes the phosphorylation of hydroxymethylpyrimidine phosphate (HMP-P) to HMP-PP, and of HMP to HMP-P. ThiD shows no activity with pyridoxal, pyridoxamine or pyridoxine.
The term “ThiC” as used herein refers to a Phosphomethylpyrimidine synthase protein that catalyzes the synthesis of the hydroxymethylpyrimidine phosphate (HMP-P) moiety of thiamine from aminoimidazole ribotide (AIR) in a radical S-adenosyl-L-methionine (SAM)-dependent reaction.
The term “ThiE” as used herein refers to a thiamine-phosphate synthase protein that condenses 4-methyl-5-(beta-hydroxyethyl)-thiazole monophosphate (THZ-P) and 2-methyl-4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) to form thiamine monophosphate (TMP).
The term “ThiF” as used herein refers to a sulfur carrier protein ThiS adenylyltransferase. ThiF catalyzes the adenylation of the carboxy terminus of ThiS and the subsequent displacement of AMP catalyzed by Thil-persulfide to give a ThiS-Thil acyl disulfide ThiS.
The term “ThiS” as used herein refers to thiamine diphosphate biosynthesis gene that is involved in the pathway thiamine diphosphate biosynthesis, which is part of Cofactor biosynthesis.
The term “ThiG” as used herein refers to a Thiazole synthase protein that catalyzes the rearrangement of 1-deoxy-D-xylulose 5-phosphate (DXP) to produce the thiazole phosphate moiety of thiamine. Sulfur is provided by the thiocarboxylate moiety of the carrier protein ThiS.
The term “ThiH” as used herein refers to a 2-iminoacetate synthase protein that catalyzes the radical SAM-mediated cleavage of tyrosine to 2-iminoacetate and 4-cresol.
The term “Thil” as used herein refers to a tRNA sulfurtransferase protein that catalyzes the ATP-dependent transfer of a sulfur to tRNA to produce 4-thiouridine in position 8 of tRNAs, which functions as a near-UV photosensor. Also catalyzes the transfer of sulfur to the sulfur carrier protein ThiS, forming ThiS-thiocarboxylate. This is a step in the synthesis of thiazole, in the thiamine biosynthesis pathway. The sulfur is donated as persulfide by IscS.
The term “Dxs protein” as used herein refers to a 1-deoxy-D-xylulose-5-phosphate synthase that catalyzes the acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield 1-deoxy-D-xylulose-5-phosphate (DXP).
The term “ThiO” as used herein refers to a glycine oxidase that catalyzes the FAD-dependent oxidative deamination of various amines and D-amino acids to yield the corresponding alpha-keto acids, ammonia/amine, and hydrogen peroxide. It is essential for thiamine biosynthesis in organisms that generally lack ThiH since the oxidation of glycine catalyzed by ThiO also generates the glycine imine intermediate (dehydroglycine) required for the biosynthesis of the thiazole ring of thiamine pyrophosphate.
The term “IscR” as used herein refers to HTH-type transcriptional regulator IscR that regulates the transcription of several operons and genes involved in the biogenesis of Fe—S clusters and Fe—S-containing proteins. Transcriptional repressor of the iscRSUA operon, which is involved in the assembly of Fe—S clusters into Fe—S proteins.
The term “host cell” refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
The term “polynucleotide construct” refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.
The term “operably linked” refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.
The terms “nucleotide sequence” and “polynucleotide” are used herein interchangeably.
The term “coding sequence” refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
The term “control sequence” as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene or organism) or heterologous or foreign (i.e., from a different or organism) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.
The term “expression vector” refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.
The term “expression” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
The terms “heterologous” or “recombinant” or “genetically modified” and their grammatical equivalents as used herein interchangeably refers to entities “derived from a different species or cell”. For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e., the gene is from a different species or cell type than the host cell. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes.
The term “metabolic pathway” as used herein is intended to mean two or more enzymes acting sequentially in a live cell to convert chemical substrate(s) into chemical product(s). Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s).
An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds such as proteins for example enzymes (co-enzymes). A cytochrome P450 reductase (CPR) that reduces cofactors such as NADPH in certain cytochrome P450 enzymes is an example of an enzymatic co-factor. The term “operative biosynthetic metabolic pathway” refers to a metabolic pathway that occurs in a live recombinant host, as described herein.
The term “in vivo”, as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.
The term “in vitro”, as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like.
The term “substrate” or “precursor”, as used herein refers to any compound that can be converted into a different compound. For example, desthiobiotin can be a substrate for biotin synthase and can be converted into biotin. For clarity, substrates and/or precursors include both compounds generated in situ by an enzymatic reaction in a cell or exogenously provided compounds, such as exogenously provided organic molecules which the host cell can metabolize into a desired compound.
Term “endogenous” or “native” as used herein refers to a gene or a polypeptide in a host cell which originates from the same host cell.
The term “deletion” as used herein refers to manipulation of a gene so that it is no longer expressed in a host cell.
The term “disruption” as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it is no longer expressed in a host cell.
The term “attenuation” as used herein refers to manipulation of a gene or any of the machinery participating in the expression the gene, so that it the expression of the gene is reduced as compared to expression without the manipulation.
The terms “substantially” or “approximately” or “about”, as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.
The term “and/or” as used herein is intended to represent an inclusive “or”. The wording X and/or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.
The term “isolated” as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the invention for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the invention may be isolated into a pure or substantially pure form. In this context, a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1%, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment, the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100% pure by weight.
The term “% identity” is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences. “% identity” as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
The term “% identity” as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
The protein sequences of the present invention can further be used as a “query sequence” to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:
Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.
The term “mature polypeptide” or “mature enzyme” as used herein refers to a polypeptide in its final active form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
The term “cDNA” refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
The terms “comprise” and “include” as used throughout the specification and the accompanying items as well as variations such as “comprises”, “comprising”, “includes” and “including” are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
The articles “a” and “an” are used herein refers to one or to more than one (i.e., to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element.
Terms like “preferably”, “commonly”, “particularly”, and “typically” are not utilized herein to limit the scope of the itemed invention or to imply that certain features are critical, essential, or even important to the structure or function of the itemed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
The term “cell culture” as used herein refers to a culture medium comprising a plurality of host cells of the invention. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., one or more of (i) trace metals; (ii) vitamins; (iii) salts (such as salts of phosphate, magnesium, potassium, zinc, iron); (iv) nitrogen sources (such as YNB, ammonium sulfate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and/or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and/or acetate); (vi) nucleobases; (vii) aminoglycosides; and/or (viii) antibiotics (such as G418 and hygromycin B).
The term “radical SAM” as used herein refers to a superfamily of enzymes that use a [4Fe-4S]+ cluster to reductively cleave S-adenosyl-L-methionine (SAM) to generate a radical, usually a 5′-deoxyadenosyl radical, as a critical intermediate. The vast majority of known radical SAM enzymes have a cysteine-rich motif that matches or resembles CxxxCxxC.
The genetical modification of the host cells provided for in the first aspect having increased production of vitamin B compounds, include in some embodiments one or more mutations in native polynucleotide constructs encoding one or more proteins selected from protein cAMP receptor protein (CRP), carbohydrate repression resistance protein (CRR) and adenylate cyclase protein (CyaA).
The increase in the genetically modified host cells capacity to produce vitamin B compounds can in some embodiments be at least 50%, such as at least 100%, such as least 150%, such as at least 200%.
Mutations in the native polynucleotide constructs preferably include deletions, disruption, and/or an attenuation of the gene and particularly the deletions, disruptions and/or attenuations comprise a full or partial deletion of the gene, a translational knockout through introduction of a stop codon or a frameshift mutation. In one embodiment, the mutation is a deletion through complete removal of the gene or a translational knockout by introducing one or more stop codons or frameshift mutations preventing expression of an active peptide. In another embodiment the deletion, disruption and/or attenuation may be a point mutation in the polynucleotide constructs made in a promoter for the protein encoding gene, in the RBS region and/or in protein encoding sequence. In one particular embodiment, such point mutation is made in the sequence encoding the active site of the CyaA enzyme and reduces the activity of CyaA while in another embodiment such point mutation is made in the sequence encoding the cAMP binding moieties of CRP to reduce the affinity of CRP for cAMP. In another embodiment the phosphorylation site of CRR is mutated, preventing the formation of phosphorylated EIIAGIc, and thereby activation of CyaA.
The CRP, native or mutated, is preferably at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CRP comprised in SEQ ID NO: 39 or 97. The CRR, native or mutated is preferably at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CRR comprised in SEQ ID NO: 41 or 99. The CyaA, native or mutated is preferably at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CyaA comprised in SEQ ID NO: 43 or 101.
In some embodiments the gene encoding the CRP, native or mutated, is preferably least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 40 or 98 or genomic DNA thereof encoding the CRP comprised in SEQ ID NO: 39 or 97. The CRR, native or mutated, is preferably least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 42 or 100 or genomic DNA thereof encoding the CRR comprised in SEQ ID NO: 41 or 99. The CyaA, native or mutated, is preferably least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 44 or 102 or genomic DNA thereof encoding the CyaA comprised in SEQ ID NO: 43 or 101.
In some embodiments the encoded mutant CRP comprises a mutation in one or more positions corresponding to T12, D138, T146, F69, R82, V139, G57, K58, E59, M60, 161, L62, S63, G72, E73, L74, R83, S84, T128, S129, A136, F137, Q171, E172, 1173, G174, Q175, 1176, V177, G178, C179, S180, R181, E182, T183, V184, G185, R186 of SEQ ID NO: 39 or 97.
In some embodiments the encoded mutant CRR comprises a mutation in one or more positions corresponding to H76, H91 of SEQ ID NO: 41 or 99;
In some embodiments the encoded mutant CyaA comprise a mutation in the position corresponding to G60, K59, L63, T65, R188, G195, K196, R192, S103, S113, D114, D116, W118, E185, T189, K260, K264, K332, W200, D300 of SEQ ID NO: 43 or 101.
In the alternative the one or more genetic alterations increasing the non-CRP polypeptide binding of cAMP and/or degradation of cAMP in the host cell, may comprise:
In particular the one or more non-CRP cAMP binding proteins and/or cAMP degrading enzymes of a) can be selected among heterologous cAMP phosphodiesterase (CpdA) and cAMP deaminase (CadD). Where the host cell is E. coli or many other host cells, the CpdA and/or CadD must be introduced heterologously to the host cell using methods known in the art. In some embodiments wherein the over-expression of b) can comprise a cis-modification in the genome or a trans-modification in a plasmid, while in other embodiments the mutation of c) is a point mutation in a promoter for the protein encoding sequence, in the RBS region and/or in protein encoding sequence. Such point mutations can be made in the sequence encoding the active site of the for example CpdA and or CadD increasing the activity of CpdA and/or CadD. The CpdA may be at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CpdA comprised in SEQ ID NO: 45 or 103; while the CadD may be at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CadD comprised in SEQ ID NO: 47 or 105. More particularly the gene encoding the CpdA may be least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 46 or 104 or genomic DNA thereof encoding the CpdA comprised in SEQ ID NO: 45 or 105; while the gene encoding the CadD may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 48 or 106 or genomic DNA thereof encoding the CadD comprised in SEQ ID NO: 47 or 105.
The host cell of the invention further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the vitamin B compound, and in particular such pathway elements comprise one or more one or more FeS cluster dependent enzymes, in particular radical SAM enzymes.
In one preferred embodiment the vitamin B compound is biotin and the host cell comprise an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the biotin. In this context the term “pathway elements” include any step or element relevant for the functioning of the pathway, including proteins, polypeptides, peptides, enzymes, co-factors or the like. The pathway elements for producing biotin can include one or more pathway elements are selected from:
In some embodiments of the biotin pathway
The gene encoding the BioC of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 2 or genomic DNA thereof encoding the BioC comprised in SEQ ID NO: 1; the gene encoding the BioH of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 4 or genomic DNA thereof encoding the BioH comprised in SEQ ID NO: 3; the gene encoding the BioF of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 6 or genomic DNA thereof encoding the BioF comprised in SEQ ID NO: 5; the gene encoding the BioA of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 8 or genomic DNA thereof encoding the BioA comprised in SEQ ID NO: 7; the gene encoding the Biok of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 10 or genomic DNA thereof encoding the Biok comprised in SEQ ID NO: 9; the gene encoding the BioD of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 12 or genomic DNA thereof encoding the BioD comprised in SEQ ID NO: 11; the gene encoding the Biol of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 14 or genomic DNA thereof encoding the Biol comprised in SEQ ID NO: 13; the gene encoding the BioW of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 16 or genomic DNA thereof encoding the BioW comprised in SEQ ID NO: 15; the gene encoding the IscR of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 or genomic DNA thereof encoding the IscR comprised in SEQ ID NO: 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 respectively; and the gene encoding the BioB of the biotin pathway may be at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the gene comprised in SEQ ID NO: 38 or genomic DNA thereof encoding the BioB comprised in SEQ ID NO: 37.
In another preferred embodiment the vitamin B compound is thiamine and the host cell further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing the thiamine, and in particular such pathway elements comprise one or more thiamine mono-phosphate phosphatase enzymes. In this context the term “pathway elements” include any element relevant for the functioning of the pathway, including proteins, polypeptides, peptides, enzymes, co-factors or the like. In some embodiments the pathway elements for producing thiamine can include one or more pathway elements are selected from:
In some embodiments of the thiamine pathway
In other embodiments the vitamin B compound is one or more vitamins in the B3 complex and the host cell disclosed herein further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing ome or more B3 vitamins. In this context the term “pathway elements” include any step or element relevant for the functioning of the pathway, including proteins, polypeptides, peptides, enzymes, co-factors or the like. B3 complex includes B3 vitamins nicotinamide riboside (NR), nicotinic acid (NA), nicotinamide (NA), nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD), or the intermediate quinolate.
In some embodiments the pathway elements for producing B3 vitamins can include one or more pathway elements are selected from:
In some embodiments of the B3 pathway
The NadA may be transgene or an up-regulated nadA endogenous gene. The NadE nicotinic acid mononucleotide amidase can synthesize both quinolate and NR (
An increase in the levels of those polypeptides that catalyze steps in the B3 (NR) pathway enhances the synthesis of both intermediates and end products of the NR pathway in the host cell. When the gene(s) encoding quinolate synthase together with one or more additional polypeptides that catalyze additional steps in the quinolate and B3 vitamins pathways are transgenes, they are preferably located in the genome of the genetically modified prokaryotic cell, either integrated into the host cell chromosome or on a self-replicating plasmid. The transgene encoding NadA and one or more of the transgenes (nadB, and nadE) encoding enzymes in the NR pathway enzymes may be present in the genome within one or more operons.
The promoter driving expression of the transgene encoding NadB and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When expression driven by the promoter is constitutive, then a suitable promoter includes apFab family SEQ ID NO: 138 while a suitable inducible promoter includes the lac promoter lac p, which is regulated by repressor lacl SEQ ID NO: 135. Suitable terminators include members of the apFAB terminator family including SEQ ID NO: 139. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
B3 vitamins and quinolate can be produced using genetically modified host cells such as described in example 4 of WO2020148351 further modified as described herein.
In other embodiments the vitamin B compound is vitamin B12 cobalamin, and the host cell disclosed herein further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing vitamin B12. In this context the term “pathway elements” include any step or element relevant for the functioning of the pathway, including proteins, polypeptides, peptides, enzymes, co-factors or the like. Host cells described herein genetically modified to lack or down-regulate CRP-cAMP complex and modified with a transgene or up-regulated native cobG gene and additional transgenes or up-regulated native genes have an enhanced flux through the B12 pathway, and enhanced production of the intermediate hydrogenobyrinic acid (HBA).
Host cells further modified to comprise transgenes encoding polypeptides that catalyze the subsequent steps in the cob synthesis pathway (see
Accordingly, in some embodiments the pathway elements for producing vitamin B12 can include one or more pathway elements are selected from:
In some embodiments of the B12 pathway the
Where the gene(s) encoding the CobG polypeptide, precorrin-3B synthase, together with one or more additional polypeptides that catalyze additional steps in the Cob pathway are transgenes, these are preferably located in the genome of the genetically modified host cell, either integrated into the cell chromosome or on a self-replicating plasmid. The transgene encoding CobG and one or more of the transgenes Cob pathway enzymes may be present in the genome within one or more operons. The promoter driving expression of the transgene encoding CobG and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. Suitable constitutive promoters include apFab family promoters of SEQ ID NO: 138 while a suitable inducible promoter includes pBad (arabinose-inducible) SEQ ID NO: 134 and lac promoter lac p, which is regulated by repressor lacl SEQ ID NO: 135. Suitable terminators include members of the apFAB terminator family including SEQ ID NO: 139. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
B12 cobalamin can be produced using genetically modified host cells such as described in example 5 of WO2020148351 further modified as described herein.
In other embodiments the vitamin B compound is vitamin B5 or pantotheonate, and the host cell disclosed herein further comprises an operative metabolic pathway comprising one or more native or heterologous pathway elements producing vitamin B5. In this context the term “pathway elements” include any step or element relevant for the functioning of the pathway, including proteins, polypeptides, peptides, enzymes, co-factors or the like. Host cells described herein genetically modified to lack or down-regulate CRP-cAMP complex and further modified with a transgene or up-regulated native IlvD gene encoding a dihydroxy-acid dehydratase (EC: 4.2.1.9) and optionally additional transgenes or up-regulated native genes encoding polypeptides PanB, PanE and PanC have an enhanced flux through the B5 pathway, and enhanced production of pantothenic acid.
Accordingly, in some embodiments the pathway elements for producing vitamin B5 can include one or more pathway elements are selected from:
In some embodiments of the B5 pathway
Where the gene encoding the IIvD dihydroxy-acid dehydratase (eg. SEQ ID NO: 194), together with one or more additional polypeptides that catalyze additional steps in the pantothenic acid pathway (encoding PanB, PanE and PanC) are transgenes, they are located in the genome of the genetically modified host cell, either integrated into the cell chromosome or on a self-replicating plasmid. The transgene encoding IlvD and one or more of the transgenes encoding panBEC pathway enzymes may be present in the genome within one or more operons.
The promoter driving expression of the transgene encoding IlvD and one or more additional transgenes is preferably a non-native promoter, which may be a heterologous constitutive-promoter or an inducible-promoter. When the promoter is a heterologous constitutive promoter, then a suitable promoter includes the apFab family SEQ ID NO: 138, while a suitable inducible promoter includes: pBad (arabinose inducible SEQ ID NO: 134 and Lacl SEQ ID NO: 135. Suitable terminators include members of the apFAB terminator family including SEQ ID NO: 139. The selected promoter and terminator may be operably linked to the respective gene, either to provide individual gene regulation or for regulation of an operon.
B5 pantothenoate can be produced using genetically modified host cells such as described in example 6 of WO2020148351 further modified as described herein.
For any of the Vitamin B compound pathways the IscR factor may be a mutant polypeptide having has at least one amino acid substitution selected from the group consisting of L15X, C92X, C98X, C104X, and H107X; wherein X is any amino acid other than the corresponding amino acid residue in the native IscR comprised in SEQ ID NO: 17. The amino acid substitution in the mutant IscR polypeptide can particularly be selected from the group consisting of:
In the host cell one or more genes and/or polypeptides of the pathway for the vitamin B compound may be heterologous to the host cell and may be present in the host cell in more than one copy, such as least 2 copies, such as least 3 copies, such as least 4 copies. The heterologous genes of the pathway for the vitamin B compound may be inserted in a plasmid or integrated in a chromosome. The host cell may in some embodiments comprise a transporter molecule facilitating transport of a precursor for or a product of the pathway for the vitamin B compound and/or the host cell may be further genetically modified to provide an increased amount of a substrate consumed in the pathway for the vitamin B compound. In addition, the host cell may further be genetically modified to exhibit increased tolerance towards one or more substrates, intermediates, or products in the pathway for the vitamin B compound and/or one or more native or endogenous genes of the host cell are deleted, disrupted and/or attenuated. For example, where the vitamin B compound is B3 complex and/or quinolate it is desirable to down-regulate the NAD salvage pathway for example by deleting, disrupting and/or attenuating the nadR and/or pncC genes thereby reducing NR consumption.
In a particular embodiment, the host cell may also be modified to overexpress one or more genes in the pathway for the vitamin B compound.
The host cell of the invention may be any host cell suitable for hosting and expressing the pathway for the vitamin B compound. Such cell may be a prokaryotic or eukaryotic cell. Suitable prokaryotic host cells can be of a genus selected from Escherichia, Bacillus, Brevibacterium, Burkholderia, Campylobacter, Corynebacterium, Serratia, Lactobacillus, Lactococcus, Acinetobacter, Acetobacter or Pseudomonas. Particularly useful prokaryotic host cells are of the genus Escherichia, Corynebacterium, Bacillus, Serratia, or Pseudomonas, such as the species Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Serratia marcescens, Pseudomonas putida and/or Pseudomonas mutabilis. Useful eukaryotic host cells include mammalian, insect, plant, fungal or archaeal cells. Among the eukaryotic host cells fungal cells of the genuses Saccharomyces, Kluveromyces, Candida, Pichia, Debaromyces, Hansenula, Yarrowia, Zygosaccharomyces, Schizosaccharomyces and Ashbya are particularly useful, such as the species Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces bensis, Saccharomyces oviformis, Yarrowia lipolytica, Pichia pastoris or Ashbya gossypii.
The invention also provides mutated CRP, CRR and/or CyaA polypeptides. In one embodiment a mutated CRP polypeptide is provided which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CRP comprised in SEQ ID NO: 39 or 97 and comprises one or more mutations in positions corresponding to positions T12, D138, T146, F69, R82, V139, G57, K58, E59, M60, 161, L62, S63, G72, E73, L74, R83, S84, T128, S129, A136, F137, Q171, E172, 1173, G174, Q175, 1176, V177, G178, C179, S180, R181, E182, T183, V184, G185, R186 of SEQ ID NO: 39 or 97.
In another embodiment a mutated CRR polypeptide is provided which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CRR comprised in SEQ ID NO: 41 or 99 and comprises one or more mutations in positions corresponding to H76, H91 of SEQ ID NO: 41 or 99.
In another embodiment a mutated CyaA polypeptide is provided which is at least 20%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identical to the CyaA comprised in SEQ ID NO: 43 or 101 and comprises one or more mutations in positions corresponding to G60, K59, L63, T65, R188, G195, K196, R192, S103, S113, D114, D116, W118, E185, T189, K260, K264, K332, W200, D300 of SEQ ID NO: 43 or 101.
Also provided for herein are polynucleotide constructs harboring gene(s) encoding native or mutated CRP, CyaA or CRR operably linked to one or more control sequences, said polynucleotide constructs comprising mutations to delete, disrupt, and/or an attenuate the gene transcription or translation or the activity and/or function of the encoded CRP, CyaA or CRR. Alternatively, the polynucleotide constructs provided for herein harbor gene(s) encoding native or mutated CpdA or CadD operably linked to one or more control sequences, said polynucleotide constructs comprising mutations to increase the gene transcription or translation of the cAMP degrading activity of the encoded CpdA or CadD.
The control sequences direct the expression of the encoded CRP, CyaA, CRR, CpdA and/or CadD in the host cell harboring the polynucleotide construct. Conditions for the expression should be compatible with the control sequences. The control sequence may be heterologous or native to the gene(s) encoding the CRP, CyaA, CRR, CpdA or CadD and/or to the host cell. In some embodiments both the control sequence and the gene(s) encoding the CRP, CyaA, CRR, CpdA and/or CadD are heterologous to the host cell and optionally also to each other. In one embodiment the polynucleotide construct is an expression vector, comprising the gene(s) encoding the CRP, CyaA, CRR, CpdA and/or CadD operably linked to the one or more control sequences.
Polynucleotides may be manipulated in a variety of ways to modify expression of the CRP, CyaA, CRR, CpdA or CadD. Manipulation of the polynucleotide prior to its insertion into an expression vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, which is a polynucleotide that is recognized by a host cell for expression of a polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The promoter may also be an inducible promoter. Selecting a suitable promoter for expression in yeast is well-known and is well understood by persons skilled in the art.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3′-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used.
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
The control sequence may also be a leader, a non-translated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5′-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.
The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3′-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
It may also be desirable to add regulatory sequences that regulate expression of the CRP, CyaA CRR, CpdA and/or CadD relative to the growth of the host cell. Examples of regulatory systems are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
Various nucleotide sequences in addition to the polynucleotide construct of the invention may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide sequence encoding the CRP, CyaA, CRR, CpdA and/or CadD at such sites. The recombinant expression vector may be any vector (e.g., a plasmid or virus or chromosomal) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the CRP, CyaA, CRR, CpdA and/or CadD. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid (linear or closed circular plasmid), an extrachromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may, when introduced into the host cell, integrate into the genome, and replicate together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector may contain one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene from which the product provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
The vector may further contain element(s) that permits integration of the vector into genome (being a vector in itself) of the host cell or permits autonomous replication of the vector in the cell independent of the genome. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, such as 400 to 10,000 base pairs, and such as 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.
As mentioned, supra, more than one copy of a gene encoding pathway elements for vitamin B compounds may be inserted into a host cell to increase production of the vitamin B compound. An increase in the gene copy number can be obtained by integrating one or more additional copies of a gene into the host cell genome or by including an amplifiable selectable marker gene with the gene, so that cells containing amplified copies of the selectable marker gene—and thereby additional copies of the polynucleotide—can be selected by cultivating the cells in the presence of the appropriate selectable agent. The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present disclosure are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
In a separate embodiment the host cell comprising the polynucleotide constructs and/or vectors as disclosed herein.
Further provided for herein are cell cultures comprising the genetically modified host cells of the invention and a growth medium. Suitable growth mediums for relevant prokaryotic or eukaryotic host cells are widely known in the art.
The invention also provides a method for producing vitamin B compounds (such as biotin or thiamine) comprising
The cell culture can be cultivated in a nutrient medium and at conditions suitable for production of the vitamin B compounds of the invention and/or for propagating cell count using methods known in the art. For example, the culture may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermenters in a suitable medium and under conditions allowing the host cells to grow and/or propagate, optionally to be recovered and/or isolated.
The cultivation can take place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., from catalogues of the American Type Culture Collection). The selection of the appropriate medium may be based on the choice of host cell and/or based on the regulatory requirements for the host cell. Such media are available in the art. The medium may, if desired, contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms. Accordingly, in an embodiment a suitable nutrient medium can include one or more of (i) trace metals; (ii) vitamins; (iii) salts (such as salts of phosphate, magnesium, potassium, zinc, iron); (iv) nitrogen sources (such as YNB, ammonium sulfate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and/or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and/or acetate); (vi) nucleobases; (vii) aminoglycosides; and/or (viii) antibiotics (such as G418 and hygromycin B).
The cultivation of the host cell may be performed over a period of from about 0.5 to about 30 days. The cultivation process may be a batch process, continuous or fed-batch process, suitably performed at a temperature in the range of 0-100° C. or 0-80° C., for example, from about 0° C. to about 50° C. and/or at a pH, for example, from about 2 to about 10. Preferred fermentation conditions are a temperature in the range of from about 25° C. to about 55° C. and at a pH of from about 3 to about 9. The appropriate conditions are usually selected based on the choice of host cell. Accordingly, in some embodiments the method of the invention comprising one or more elements selected from:
The cell culture of the invention may be recovered and or isolated using methods known in the art. For example, the compound(s) may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, spray-drying, or lyophilization. In a particular embodiment the method includes a recovery and/or isolation step comprising separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the vitamin B compound and subjecting the supernatant to one or more steps selected from:
The yield of vitamin B compound, such as biotin or thiamine provided by the method of the invention is typically higher than when producing Vitamin B compounds by methods employing host cells without reduced CRP-cAMP complex formation and/or increased degradation and/or decreased binding of cAMP, in some embodiments at least 10% higher such as at least 50%, such as at least 100%, such as least 150%, such as at least 200% higher.
The method of the invention may further comprise one or more steps of mixing the vitamin B compound with one or more carriers, agents, adjuvants, additives and/or excipients, optionally pharmaceutical grade carriers, agents, adjuvants, additives and/or excipients.
The method of the invention may further comprise one or more in vitro steps in the process of producing the vitamin B compound. It may also comprise one or more in vivo steps performed in another cell than the host cell of the invention. For example, precursors for and/or intermediates in the pathway for the vitamin B compound may be produced in another cell and isolated therefrom and then fed to a cell culture of the invention for conversion into the vitamin B compound. Accordingly, in one embodiment the method of the invention further comprises feeding one or more exogenous vitamin B precursors to the host cell culture, such as O-methylpimeloyl-acyl carrier protein, pimeloyl-acyl carrier protein, KAPA, DAPA, DTB and/or pimelate for biotin production, whereas precursors such as thiazole, or HMP would be suitable for production of thiamine, TMP or TPP.
The disclosure also describes a fermentation composition comprising the cell culture of the invention and the vitamin B compound-either comprised in the cells or in the medium. In the fermentation composition the genetically modified host cells may be wholly or partially lysed and/or disintegrated. In some embodiments at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the genetically modified host cells in the fermentation composition are lysed and/or disintegrated. Further, in the fermentation composition of the invention at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material may have been separated and/or removed from a liquid phase of the fermentation composition.
The fermentation composition may further comprise one or more compounds of
Suitable supplemental nutrients can include one or more of (i) trace metals; (ii) vitamins; (iii) salts (such as salts of phosphate, magnesium, potassium, zinc, iron); (iv) nitrogen sources (such as YNB, ammonium sulfate, urea, yeast extracts, ammonium nitrate, ammonium chloride, malt extract, peptone and/or amino acids); (v) carbon source (such as dextrose, sucrose, glycerol, glucose, maltose, molasses, starch, cellulose, xylan, pectin, lignocellolytic biomass hydrolysate, and/or acetate); (vi) nucleobases; (vii) aminoglycosides; and/or (viii) antibiotics (such as G418, hygromycin B, spectinomycin and/or Kanamycin).
The invention also provides a composition comprising the fermentation composition of the invention and one or more carriers, agents, adjuvants, additives and/or excipients and at least trace amounts of one or more metabolites of the cell culture, optionally signature metabolites for the genetically modified host cell. Suitable carriers, agents, adjuvants, additives and/or excipients includes formulation additives, stabilising agent, fillers and the like. The composition and the one or more carriers, agents, adjuvants, additives and/or excipients can suitably be formulated into in a dry solid form, e.g., by using methods known in the art, such as spray drying, spray cooling, lyophilization, flash freezing, granulation, microgranulation, encapsulation or microencapsulation. The composition and the one or more carriers, agents, adjuvants, additives and/or excipients can also be formulated into a liquid stabilized form using methods known in the art, such as adding to the fermentation composition one or more stabilizers such as sugars and/or polyols (e.g., sugar alcohols) and/or organic acids (e.g., lactic acid).
The composition of the invention may be further refined into a pharmaceutical preparation, a dietary supplement, a cosmetic, a food/flavor preparation, a feed preparation and/or an analytical or diagnostic reagent optionally using one or more steps of the methods described herein for producing the vitamin B compound including mixing the vitamin B compound with one or more pharmaceutical grade carriers, agents, adjuvants, additives and/or excipients. In one embodiment, the pharmaceutical composition is a pharmaceutical preparation obtainable from the method of the invention. The pharmaceutical preparation may be a dry preparation, optionally in the form of a powder, tablet, capsule, hard chewable and or soft lozenge or a gum. Alternatively, the pharmaceutical preparation may in form of a liquid pharmaceutical solution. Such pharmaceutical preparations may be used as a medicament in a method for treating and/or relieving a disease and/or medical condition, in particular in a mammal, in particular for use in the treatment of a nutritional deficiency. Accordingly, the invention further provides a method for preventing, treating and/or relieving a disease and/or medical condition comprising administering a therapeutically effective amount of the pharmaceutical composition of the invention to a mammal in need of treatment and/or relief. Diseases and/or medical conditions treatable or relievable by the pharmaceutical composition includes but is not limited to diseases and/or medical conditions associated with lacking or insufficient bodily intake of vitamin B compounds. The pharmaceutical preparation can be administered parenterally, such as topically, epicutaneously, sublingually, buccally, nasally, intradermally, intravenously, and/or intramuscularly. The pharmaceutical composition can also be administered enterally via the gastrointestinal tract.
The invention also provides a kit of parts comprising
In some embodiments, the kit comprises a genetically modified cell capable of producing a vitamin B compound, wherein the genetically modified cell expresses pathway enzymes producing the vitamin B compound. In one embodiment the Vitamin B compound is Biotin (B7) and the genetically modified cell expresses Biotin synthase (BioB) and optionally one or more biotin pathway enzymes or factors selected from BioC, BioH, BioF, BioA, Biok, BioD, Biol, BioW and IscR. In other embodiments the genetically modified cell expresses thiamine synthase (ThiC) and optionally one or more thiamine pathway enzymes or factors selected from ThiD, ThiF, This, ThiH, ThiG, ThiM, ThiE, TMP phosphatase, Thik, ThiL, Thil, IscR and ThiO. In some embodiments, the kit comprises a genetically modified cell capable of producing riboflavin (B2). In some embodiments, the kit comprises a genetically modified cell capable of producing niacin (B3). In some embodiments, the kit comprises a genetically modified cell capable of producing pantothenic acid (B5). In some embodiments, the kit comprises a genetically modified cell capable of producing pyridoxine (B6). In some embodiments, the kit comprises a genetically modified cell capable of producing folate (B9). In some embodiments, the kit comprises a genetically modified cell capable of producing cobalamin (B12).
The present application contains a Sequence Listing prepared in PatentIn included below but also submitted electronically in ST26 format which is hereby incorporated by reference in its entirety.
Chemicals used herein, e.g., for buffers, media and substrates are commercial products of at least reagent grade.
mMOPS Medium
The minimal medium (mMOPS) used herein had the following composition in demineralized H2O (dH2O):
pH of the mMOPS medium was adjusted to 7.4±0.1 with NaOH or H2SO4.
The minimal screening medium (S medium) used herein had the following composition in dH2O:
pH of the S medium was adjusted to 7.4±0.1 with NaOH or H2SO4
The LB agar medium used herein had the following composition in dH2O:
mMOPS Agar Medium
The mMOPS agar medium used herein had the following composition in mMOPS medium:
The fermentation batch medium (B medium) used herein had the following composition in dH2O:
The fermentation feed medium (F medium) used herein had the following composition in dH2O:
The fermentation feed medium (F medium) used herein had the following composition in dH2O:
The antibiotics solution used herein had the following composition in dH2O:
Strains of Escherichia coli used herein were the following:
E. coli K-12 BW251131 parent strain: rrnB3 ΔlacZ4787
Escherichia coli BioB
Escherichia coli BioB
Escherichia coli BioB
Escherichia coli BioB
1Commercially available strain e.g. from The Coli Genetic Stock Center; http://cgsc2.biology.yale.edu/KeioList.php
2WO2019012058A1
Further strains of Escherichia coli used herein were the following:
E. coli K-12 BW251131 parent strain: rrnB3 ΔlacZ4787
1Commercially available strain, e.g., from the Coli Genetic Stock Center; http://cgsc2.biology.yale.edu/KeioList.php
2WO2017103221A1
3WO2019012058A1
Plasmids used herein were as follows:
Further plasmids used herein were as follows:
To measure optical densities (OD) of a cell culture as cuvette OD at 600 nm (cOD600), the culture was diluted 10-fold with dH2O to a final volume of 1 mL and transferred to a 1.5 mL transparent cuvette with 10 mm pathlength. The diluted culture was measured at 600 nm and 10 mm pathlength on a mySPEC (VWR). If the diluted culture was measured to cOD600>0.4, the culture was further diluted 10-fold and remeasured.
For quantification of biotin in supernatant samples from small-scale screening and fermentations, a bioassay involving biotin auxotrophic strain BS1093, mMOPS (excluding biotin) supplemented with zeocin and >5 of biotin standards in the dynamic growth range of BS1093 was used following the assay described in section 1.6 of the methods in the examples in WO2019012058. Specifically, the supernatant from each culture was diluted alongside >5 biotin standards in the concentration range of 0 μM (mg/L) to 40 μM (12.9 mg biotin/L) prepared in Milli-Q water. 15 μl of each diluted supernatant and each of the biotin standards was then added to a well of a 96-well microtiter plate. Each well contained prior to addition 135 μl of mMOPS medium without biotin but with supplementation of zeocin and inoculated to an initial OD600 of 0.01 with a biotin-starved overnight culture of BS1093. The plate was sealed with a breathable seal and incubated at 37° C. with 275 rpm shaking for 20 h before OD600 was measured. A biotin bioassay calibration curve was obtained using the dynamic range of growth response of BS1093 to the biotin standards. For sample wells, the calibration curve was used to calculate concentrations of biotin produced based on the growth response of BS1093 in the individual wells.
For quantification of thiamine in supernatant samples from small-scale cultivations and fermentations, a bioassay involving thiamine auxotrophic strain BS04501 cultivated in mMOPS supplemented with zeocin and >5 thiamine standards in the dynamic growth range of BS04501 was used following the assay described in section 1.6 of the methods in the examples in WO2019012058. Specifically, the supernatant from each culture was diluted alongside >5 thiamine standards in the concentration range of 0 μM (mg/L) to 60 μM (15.9 mg thiamine/L) prepared in Milli-Q water. 15 μL of each diluted supernatant and each of the biotin standards was then added to a well of a 96-well microtiter plate. Each well contained prior to addition 135 μL of mMOPS medium without thiamine but with supplementation of zeocin and inoculated to an initial OD600 of 0.01 with a thiamine-starved overnight culture of BS04501. The plate was sealed with a breathable seal and incubated at 37° C. with 275 rpm shaking for 20 hours before OD600 was measured. A thiamine bioassay calibration curve was obtained using the dynamic range of growth response of BS04501 to the thiamine standards. For sample wells, the calibration curve was used to calculate concentrations of thiamine produced based on the growth response of BS04501 in the individual wells.
For quantification of pantothenic acid, similar procedures were followed but using other standards and strains are reported elsewhere:
(https://www.sciencedirect.com/science/article/abs/pii/0076687979622188).
For the quantification of DCW in culture samples harvested from fermentations a known volume of culture was added to a centrifuge tube. Prior to addition this centrifuge tube was dried at 80° C. for 24 h and weighed with the weight recorded. The culture was then spun at 4000 g for 20 min at 4° C., the supernatant carefully removed, and the pellet resuspended in Milli-Q water, then spun at 4000 g for 20 min at 4° C. and again the supernatant carefully removed. This washing step was repeated and then the remaining wet cell mass was dried at 80° C. The dried cell mass was regularly weighed until no further weight loss was recorded. The dried centrifuge tube weight was subtracted from the dried cell mass weight and g/L DCW calculated based on the known volume of culture transferred to the centrifuge tube. This procedure was done in duplicate or triplicate for each culture sample with the final DCW g/L representing an average of measurements.
Commercial standards for the analytes of interest were purchased. Biotin (BTN) was acquired from Sigma Aldrich, biotin sulfoxide (BX), biotin sulfone (BSN), 7-keto-8-aminopelargomic acid (KAPA) and 7,8-diaminopelargonic acid (DAPA) from Santa Cruz Biotechnology and d-desthiobiotin (DTB) from Biosynth Carbosynth. The internal standards d4-biotin (d4-BTN) and 13C5-biotin sulfoxide (13C5—BX) were purchased from Sigma Aldrich. Water (H2O) and acetonitrile (ACN) were purchased from Honeywell and dimethyl sulfoxide (DMSO) and acetic acid (CH3COOH) were purchased from Carl Roth. Stock solutions of the analytes and internal standards were prepared in DMSO to a concentration of 1 mg mL−1. Working standard solutions of the stock solutions were then prepared in H2O. Calibration curves in the concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 and 1000 ng mL−1 were prepared in H2O containing 50 ng mL−1 of 13C5—BX and 5 ng mL−1 of d4-BTN. An internal standard mixture (ISTD MIX) containing 5 μg mL−1 of 13C5—BX and 0.5 μg mL−1 of d4-BTN was prepared in H2O.
Before analyses, the samples from the bioreactors are diluted and a mixture of internal standards (ISTD MIX) is added to correct for possible technical variation. The dilution factor differs depending on which of the analytes are to be quantified. For the quantification of BX, BSN, KAPA and DAPA a 1:10 dilution is prepared. First 890 μL of H2O is pipetted into a glass vial. Then 10 μL of the ISTD MIX and 100 μL of the original sample are added, and the solution is vortex mixed. For the quantification of BTN and DTB a two-step dilution pattern is followed to achieve a 1:1000 dilution of the original sample. First a 1:10 dilution is created by pipetting 900 μL of H2O and 100 μL of the original sample into a glass vial. The solution is vortex mixed. Finally, 980 μL of H2O, 10 μL of the ISTD MIX and 10 μL of the 1:10 diluted sample are pipetted into a vial and the solution is vortex mixed.
The samples are randomized after sample preparation and analyzed by ultra-high performance liquid chromatography (Infinity II, Agilent Technologies) coupled to tandem mass spectrometry (6470 Triple Quadrupole, Agilent Technologies) using electrospray ionization in positive ion mode. Selected reaction monitoring is used for quantifying the analytes. Fragmentor voltages, collision energies and cell accelerator voltages are optimized for each ion transition.
The analytes are separated chromatographically before they enter the mass spectrometer. This is done using a ACQUITY UPLC HSS T3 Column (2.1 mm×100 mm, particle size 1.8 μm, Waters Corporation) and H2O+0.1% (v/v) CH3COOH as eluent A and ACN+0.1% (v/v) CH3COOH as eluent B with a flow rate of 0.4 mL min−1. The elution gradient is as follows: 0-0.5 min 0% B, 0.5-1.5 min 0% to 15% B, 1.5-3 min 15% B, 3-5 min 15% to 100% B, 5-7 min 100% B. After each run, the column is re-equilibrated at 0% B for 2 min. The injection volume for each sample is 5 μL. All data is acquired using the MassHunter Acquisition software (Version 10.0, Build 10.0.142).
All data is processed using the MassHunter Quantitative Analysis software (Version B.09.00, Build 9.0.647.0). The peak areas for the analytes of interest are normalized against the peak areas of the corresponding internal standards (BSN, BX, DAPA and KAPA are normalized against 13C5—BX and BTN and DTB are normalized against d4-BTN). Data quality is ensured by evaluating technical replicates of a specific fermentation sample as well as commercial standards. The quantified concentrations of BTN and DTB in these samples are required to remain within ±10% for the data set to be approved.
Standard techniques were used for DNA isolation, amplification, purification and cloning (restriction digestion, ligation), transformation and the like. Such techniques are well known in the art and standard protocols can be found in: Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview NY. And M. Green, J. Sambrook (2012) Molecular Cloning: a laboratory manual. 4th Edition, Cold Spring Harbor Laboratory Press, CSH, NY., which are both hereby incorporated by reference in their entireties.
Commercial standards for the analytes of interest were purchased. thiamine was acquired from Sigma Aldrich, thiamine sulfoxide (BX), thiamine sulfone (BSN), 7-keto-8-aminopelargomic acid (KAPA) and 7,8-diaminopelargonic acid (DAPA) from Santa Cruz Biotechnology and d-desthiothiamine (DTB) from Biosynth Carbosynth. The internal standards d4-thiamine (d4-BTN) and 13C5-thiamine sulfoxide (13C5—BX) were purchased from Sigma Aldrich. Water (H2O) and acetonitrile (ACN) were purchased from Honeywell and dimethyl sulfoxide (DMSO) and acetic acid (CH3COOH) were purchased from Carl Roth.
Stock solutions of the analytes and internal standards were prepared in DMSO to a concentration of 1 mg/mL. Working standard solutions of the stock solutions were then prepared in H2O. Calibration curves in the concentrations of 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500 and 1000 ng/ml were prepared in H2O containing 50 ng/ml of 13C5—BX and 5 ng/ml of d4-BTN. An internal standard mixture (ISTD MIX) containing 5 μg/mL of 13C5—BX and 0.5 μg/mL of d4-BTN was prepared in H2O.
Before analyses, the samples from the bioreactors were diluted and a mixture of internal standards (ISTD MIX) is added to correct for possible technical variation. The dilution factor differs depending on which of the analytes were to be quantified. For the quantification of BX, BSN, KAPA and DAPA a 1:10 dilution is prepared. First 890 μL of H2O is pipetted into a glass vial. Then 10 μL of the ISTD MIX and 100 μL of the original sample were added, and the solution was vortex mixed. For the quantification of BTN and DTB a two-step dilution pattern was followed to achieve a 1:1000 dilution of the original sample. First a 1:10 dilution was created by pipetting 900 μL of H2O and 100 μL of the original sample into a glass vial. The solution was vortex mixed. Finally, 980 μL of H2O, 10 μL of the ISTD MIX and 10 μl of the 1:10 diluted sample were pipetted into a vial and the solution is vortex mixed.
The samples were randomized after sample preparation and analyzed by ultra-high performance liquid chromatography (Infinity II, Agilent Technologies) coupled to tandem mass spectrometry (6470 Triple Quadrupole, Agilent Technologies) using electrospray ionization in positive ion mode. Selected reaction monitoring was used for quantifying the analytes. Fragmentor voltages, collision energies and cell accelerator voltages were optimized for each ion transition.
The analytes were separated chromatographically before they enter the mass spectrometer. This was done using a ACQUITY UPLC HSS T3 Column (2.1 mm×100 mm, particle size 1.8 μm, Waters Corporation) and H2O+0.1% (v/v) CH3COOH as eluent A and ACN+0.1% (v/v) CH3COOH as eluent B with a flow rate of 0.4 mL min−1. The elution gradient was as follows: 0-0.5 min 0% B, 0.5-1.5 min 0% to 15% B, 1.5-3 min 15% B, 3-5 min 15% to 100% B, 5-7 min 100% B. After each run, the column was re-equilibrated at 0% B for 2 min. The injection volume for each sample was 5 μL. All data is acquired using the MassHunter Acquisition software (Version 10.0, Build 10.0.142).
All data was processed using the MassHunter Quantitative Analysis software (Version B.09.00, Build 9.0.647.0). The peak areas for the analytes of interest were normalized against the peak areas of the corresponding internal standards (BSN, BX, DAPA and KAPA were normalized against 13C5—BX and BTN and DTB were normalized against d4-BTN). Data quality was ensured by evaluating technical replicates of a specific fermentation sample as well as commercial standards. The quantified concentrations of BTN and DTB in these samples were required to remain within +10% for the data set to be approved.
Standard techniques were used for DNA isolation, amplification, purification, and cloning (restriction digestion, ligation), transformation and the like. Such techniques were well known in the art and standard protocols can be found in for example: Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview NY. And M. Green, J. Sambrook (2012) Molecular Cloning: a laboratory manual. 4th Edition, Cold Spring Harbor Laboratory Press, CSH, NY., which were both hereby incorporated by reference in their entireties.
CRP is activated as a transcriptional regulator by binding to its allosteric activator cyclic AMP (cAMP) which is produced by the enzyme adenylate cyclase (encoded by CyaA). Under low-glucose conditions adenylate cyclase is activated by the phosphorylated state of glucose-specific enzyme IIA or EIIA (encoded by CRR). Mutations to effectively knock out CyaA, CRR and CRP were introduced into parent such as BS1575 by multiplex automated genome engineering (MAGE) as described in Methods in Enzymology, 498, 409-426, 2011 using the DNA oligos shown in the Table 1 to introduce the desired mutations. Translational knockouts were generated by introducing 3 stop codons immediately following the start codon of the gene to eliminate any translation of the relevant polypeptide and separately a frameshift mutation was introduced into CyaA (at L169 of the translated polypeptide) using oligo moBS506 to eliminate production of a functional protein.
Successful introduction of the desired mutations was verified by PCR amplification of the region followed by Sanger sequencing.
An alternative to translational knockouts is the complete removal of a gene from the chromosome by methods known in the art. To construct strain BS4755 the gene CyaA was completely removed from the chromosome of strain BS1575 using methods known in the art and replaced with only a short FRT sequence. One method to achieve this removal of the CyaA gene is by generating a DNA fragment carrying a Kanamycin resistance gene flanked by homologous regions of CyaA and transforming this DNA into strain BS1575 carrying the λRed recombinase genes expressed from an inducible promoter. One protocol for such a method can be found in Datsenko, K. A. and Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 97 (12): 6640-5, DOI: 10.1073/pnas.120163297.
cAMP phosphodiesterase (CpdA) hydrolyzes cAMP, thereby regulating levels in E. coli. Overexpression of CpdA increases hydrolysis of cAMP and therefore decreases the levels of functional CRP-cAMP complex.
cAMP deaminase (CadD) is an enzyme found in, e.g., Leptospira interrogans that deaminates cAMP to cyclic-3′,5′-inosine monophosphate (ACS Chem. Biol. 2013, 8, 12, 2622-2629). Heterologous expression of CadD in E. coli leads to degradation of cAMP and therefore decreases the levels of functional CRP-cAMP complex.
To construct a strain overexpressing CpdA (BSBS6275) or CadD (BSBS6276) the respective genes are expressed using a constitutive or inducible promoter from a plasmid or from the chromosome. To generate said expression plasmids the genes are amplified using Phusion U polymerase (Thermo Fischer Scientific) following manufacturer's protocol and using primers containing uracil for recognition by USER restriction enzymes. Similarly, plasmid backbones are amplified. DNA fragments are digested and ligated using USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) following the manufacturers' protocols. These mixtures are transformed by electroporation into BS1575, and the transformed cells are grown on selective LB agar overnight at 37° C. To express the genes from the chromosome of E. coli they are introduced as DNA fragments carrying a promoter, the gene, an antibiotic resistance cassette and flanking regions homologous to the integration site into the chromosome using methods known in the art such as by transforming the respective DNA into strain carrying the λRed recombinase genes expressed from an inducible promoter and selecting for antibiotic resistance.
pBS679: An IPTG-inducible (Promoter SEQ ID NO: 49) transgene encoding BioB (SEQ ID NO: 38) was cloned on plasmid pBS679 by amplification of the gene and a ribosome binding site using Phusion U polymerase (Thermo Fischer Scientific) following manufacturer's protocol and using primers containing uracil for recognition by USER restriction enzymes. Similarly, a plasmid backbone carrying origin pSC101 and an Ampicillin resistance cassette as well as the promoter sequence was amplified. DNA fragments were digested and ligated using USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) following the manufacturers' protocols. These mixtures were transformed by electroporation into BS1575 and transformed cells were grown on selective LB agar supplemented with ampicillin overnight at 37° C.
PBS1565: A plasmid encoding genes BioFADCH (SEQ ID Nos. 6, 8, 12, 2, 4, respectively) driven by constitutive promoter apFAB346 (SEQ ID No: 50) was cloned on a plasmid backbone carrying a Kanamycin resistance cassette and pBR322 origin of replication using USER cloning methods as described above for pBS679 construction. Each gene was preceded by a ribosome binding site.
Transformation: Plasmids pBS679 and/or pBS1565 were transformed into background strains of interest using electroporation transformation protocols well known in the art and transformant strains were selected on selective Agar plates contains Ampicillin (pBS679), Kanamycin (pBS1565) or both.
E. coli strains require a functional CRP-cAMP complex to express pathways allowing utilization of alternative carbon sources such as succinate. A strain that cannot form a functional CRP-cAMP complex cannot grow on succinate as a sole carbon source. To confirm that CyaA, CRR and CRP knockout strains were all defective in the formation of CRP-cAMP, growth of the CyaA, CRR and CRP knockout strains of example 1 were tested on medium containing glucose or succinate as the sole carbon source.
To show visually that strains defective in CyaA are unable to grow on succinate a colony of strain BS1575 (wt) and a colony of strain BS4264 (CyaA mutant) were inoculated in 2 mL of mMOPS medium containing 2 g/L succinate as the sole carbon source. The cultures were incubated at 37° C. for 24 h while shaking. The culture of BS1575 was grown to high density after this time while the culture of BS4264 showed no detectable growth (
Similarly strains BS4260, BS4261, BS4262 and control strain BS1575 were inoculated from a single colony each in 200 μL of mMOPS medium containing either 2 g/L succinate or 2 g/L glucose in a microtiter plate and incubated at 37° C. for 24 h while shaking. The resulting cultures were measured for growth by measuring absorbance at 600 nm in a spectrophotometer. As shown in table 2 below all strains grew to high density on glucose medium while only the control strain BS1575 was able to grow on succinate medium.
E. coli strains require a functional CRP-cAMP complex to express pathways allowing utilization of alternative carbon sources such as succinate. Strains overexpressing CpdA or CadD (two enzymes that degrade cAMP) have lower levels of functional CRP-cAMP complex and therefore cannot grow on succinate as a sole carbon source. To show that CRP-cAMP complex formation is decreased in strains overexpressing CpdA or CadD their growth is tested using succinate as a sole carbon source.
Strains BS6275, BS6276 and control strain BS1575 are inoculated from a single colony each in 200 μL of mMOPS medium containing either 2 g/L succinate or 2 g/L glucose in a microtiter plate and incubated at 37° C. for 24 h while shaking. The resulting cultures are measured for growth by measuring absorbance at 600 nm in a spectrophotometer. As shown in table 3 all strains grow to high density on glucose medium while only the control strain BS1575 grow on succinate medium.
To show that biotin synthases (BioB) activity is greatly increased in E. coli strains defective in CRP-cAMP complex formation such strains overexpressing BioB were cultivated in the presence of biotin precursor desthiobiotin (DTB) and the production of biotin in each culture was measured compared to a control culture.
The strains tested all expressed BioB from a plasmid. Control parent strain (BS2658) and KO mutants of CRR, CyaA and CRP (BS4378, BS4118 and BS4377, respectively) are described above in the section: Strains and plasmids. BioB expression was IPTG inducible and the IPTG concentration that gives maximal activity was chosen for each strain. All strains were cultured, in triplicate, in 400 μL mMOPS medium containing 100 μg/mL ampicillin under optimal IPTG concentrations for induction of BioB and in the presence of 0.1 g/L biotin synthase substrate (DTB). The strains were grown in deep well plates for 24 h at 37° C. shaken at 275 rpm, after which biotin production was evaluated using a growth-based bioassay according to procedure II. The resulting biotin titers are shown in
It has been reported that strains defective in CRP-cAMP formation increase the yield of cell biomass produced from glucose (Perrenoud & Sauer (2005)). To test if the biotin titer increases seen in example 6 were simply due to increases in biomass formation per gram of glucose both biotin titers and biomass levels were carefully measured in a fed-batch cultivation of two strains expressing BioB with added DTB: a wildtype strain (BS2154) and a strain with a frameshift mutation in CyaA (BS3079). A 1 mL glycerol stock of BS3079 (BioB overexpressing strain with a CyaA knockout genotype) and BS2154 (BioB overexpressing strain with a CyaA positive genotype) were inoculated into two separate 250 ml shake flasks containing 50 mL mMOPS medium with ampicillin. The shake flasks were incubated at 37° C. and 250 rpm shake for 17 h resulting in an optical density of cOD600=2.59 and COD600=2.62 respectively. 200 mL B medium supplemented with ampicillin, 0.4 g/L desthiobiotin and 1 ml of a 1% (v/v) antifoam solution was added to four Applikon 500 mL MiniBio Reactors with temperature set to 37° C., pH controlled to pH=7 by addition of 5 M NH4OH and dissolved oxygen (DO) set-point to DO=15% by agitation speed, sparging was fixed at 1 VVM. 10 ml of each strain preculture was used to inoculate the B medium in duplicate reactors resulting in an initial cOD600=0.13 for all reactors and strains. Once the CO2 in the outlet gas reached greater than 0.4% each fermentation was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.0191 g/L for BS3079 (4.5 h) and 0.006 g/L for BS2154 (3.8 h), the concentrations represented previously determined induction optimums for biotin production. Following the depletion of glucose in the medium a fed batch phase was initiated by the addition of F medium to each fermentation at a constant feed rate of 1.6 ml/h. This transition took place between 8.5-11 h. The DO control continued to operate at DO=15% with the agitation increasing in all reactors to between 1250-1800 rpm. Additional 1 mL 1% (v/v) antifoam solution was added at 7.00 h and additional 0.4 g/L desthiobiotin was added at 24 h. The fermentations were terminated at 48 h. Culture samples were taken from the fermenter at, 4.5, 6.5, 24, 29 and 48 h after inoculation. From these samples optical density (cOD600) was measured according to analytical procedure I and DCW (g/L) measured according to analytical procedure III. Supernatants were obtained by spinning biomass down in a microcentrifuge at 17,000 G for 1 min. Supernatants were screened for biotin using LC-MS method described in analytical procedure IV. From the data the biotin yield per g DCW was calculated (mg biotin produced/g DCW produced) this calculation can be seen over the course of the fermentation in
This clearly showed that the increase in biomass does not explain the increased BioB activity. Increase in biotin production by mutating CyaA is much higher than increase in biomass. The CyaA frameshift mutation and thereby the elimination of CRP-cAMP complex in the cell indeed increases the activity of BioB per cell.
To determine if mutations in CRP-cAMP complex formation improve biotin production directly from glucose (de novo) biotin production was measured in fed-batch fermentation in representative CyaA wildtype and knockout strains each carrying two plasmids: pBS1565 (DTB production genes) and pBS679 (BioB).
A 1 ml glycerol stock of BS4759 (de novo biotin producing strain with a CyaA knockout genotype) and BS3304 (de novo biotin producing strain with a CyaA positive genotype) were inoculated into two separate 250 mL shake flasks containing 50 mL mMOPS medium with ampicillin and kanamycin. The shake flasks were incubated at 37° C. and 250 rpm shake for 20 h resulting in an optical density of COD600 2.5-3.5. 0.9 mL B medium supplemented with ampicillin and kanamycin was added to a BioLector 32 well microfluidic plate equipped with pH, DO and biomass fluorescence (m2p-labs). 5% (v/v) of each strain culture was used to inoculate the B medium in triplicate fermentation wells resulting in an initial cOD600 0.125-0.175 for each well and strain. The microfluidic plate was then sealed with gas-permeable sealing foil with evaporation reduction and loaded into the BioLector Pro (m2p-labs, Baesweiler, Germany). The temperature set to 37° C., pH controlled in each well to pH=7 by addition of 5 M NH4OH, the agitation speed of the plate set to 1300 rpm (3 mm orbit) and the relative humidity in the chamber controlled at 85%. Four hours after inoculation the plate fermentations were paused, and each culture well was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 0.0048 g/L. The fermentation was resumed and following the depletion of glucose in the batch medium a fed batch phase was initiated by the addition of G medium to each culture well. This addition was controlled to an unlimited 1 μL pulse provided the DO % in each well registered greater than 70%. This transition took place between 6.7-11 h. The fermentations were terminated at 24 h. An endpoint culture sample was taken from each fermentation well at 24 h after inoculation. From this sample optical density (cOD600) was measured according to analytical procedure I. Supernatants were obtained by spinning biomass down in a centrifuge at 4000 g, 5 min at 4° C. Supernatants were screened for biotin using LC-MS method described in analytical procedure IV. The final biotin yield was calculated by normalizing the mg biotin produced by the total g glucose fed to each well. The biotin titre and yield for each strain can be seen in
Strains overexpressing CpdA and CadD (BS6275 and BS6276) as described in Example 2 are transformed with pBS679 plasmid carrying BioB. Strain BS2658 is used as a control. BioB expression is IPTG inducible and the IPTG concentration that gives maximal activity is chosen for each strain. All strains are cultured, in triplicate, in 400 μL mMOPS medium containing 100 μg/mL ampicillin under optimal IPTG concentrations for induction of BioB and in the presence of 0.1 g/L biotin synthase substrate (DTB). The strains are grown in deep well plates for 24 h at 37° C. shaken at 275 rpm, after which biotin production is evaluated using a growth-based bioassay according to procedure II. The results in table 4 below show significantly higher biotin titers in the strains overexpressing CpdA and CadD than the control strain indicating that overexpression of cAMP degrading enzymes also improves biotin production.
Using the ΔthiP mutant BS00734 as starting strain, a PCR cassette of the Arabidopsis thaliana phosphatase was introduced into the chromosome at location KO-176 via the “clonetegration” method generally known in the art. The resulting strain BS04565 was used as the starting point for mutagenesis of IscR. This transcription factor regulates the expression of dozens of genes involved in the biosynthesis of FeS clusters. By mutating IscR to favor the apoprotein formation, with the aim to improve thiamine production. Accordingly, IscR was mutated in BS04565 by multiplex automated genome engineering (MAGE) as described in Methods in Enzymology, 498, 409-426, 2011 using the DNA oligo shown in Table 5, yielding strain BS04701.
CRP was activated as a transcriptional regulator by binding to its allosteric activator cyclic AMP (cAMP) which was produced by the enzyme adenylate cyclase (encoded by cyaA). Under low-glucose conditions adenylate cyclase was activated by the phosphorylated state of glucose-specific enzyme IIA or EIIA (encoded by CRR). Mutations to effectively knock out CRP were introduced into parent strain BS04701 by MAGE. Translational knockouts were generated by introducing 3 stop codons immediately following the start codon of the gene to eliminate any translation of the relevant polypeptide with the DNA oligo shown in Table 5. An alternative to translational knockouts was the complete removal of a gene from the chromosome by methods known in the art. To construct strain BS04782, the gene cyaA was completely removed from the chromosome of strain BS04701 using methods known in the art and replaced with only a short FRT sequence. One method to achieve this removal of the cyaA gene was by generating a DNA fragment carrying a Kanamycin resistance gene flanked by homologous regions of cyaA and transforming strain BS04701 (carrying the λRed recombinase genes expressed from an inducible promoter) with this DNA cassette. One protocol for such a method can be found in Datsenko, K. A. and Wanner, B. L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, PNAS, 2000, 97 (12): 6640-5, DOI: 10.1073/pnas.120163297.
Successful introduction of the desired mutations was verified by PCR amplification of the region followed by Sanger sequencing using the oligos in Table 5.
pBS2180: An IPTG-inducible (Promoter SEQ ID NO: 107) transgene encoding ThiC (SEQ ID NO: 52) was cloned on plasmid pBS2180 by amplification of the gene from E. coli 's chromosome and a ribosome binding site using Phusion U polymerase (Thermo Fischer Scientific) following manufacturer's protocol and using primers containing Uracils for recognition by USER restriction enzymesly, a plasmid backbone carrying origin pSC101 and an Ampicillin resistance cassette as well as the promoter sequence was amplified. DNA fragments were digested and ligated using USER enzyme (New England Biolabs) and T4 ligase (Thermo Fischer Scientific) following the manufacturers' protocols. These mixtures were introduced by electroporation into BS04608 and transformed cells were grown on selective LB agar supplemented with ampicillin overnight at 37° C.
pBS2184: A plasmid encoding genes ThiMDE (SEQ ID Nos. 64, 4, 72, respectively) driven by constitutive promoter apFAB71 (SEQ ID No: 108) was cloned on a plasmid backbone carrying a Kanamycin resistance cassette and pBR322 origin of replication using USER cloning methods as described above for pBS2180 construction. Each gene, except ThiD that is located downstream of ThiM, was preceded by a ribosome binding site.
Transformation: Plasmids pBS2180 and/or pBS2184 were introduced into background strains of interest using electroporation transformation protocols that were well known in the art and transformant strains were selected on selective agar plates contains ampicillin (pBS2180), kanamycin (pBS2184) or both.
To show that phosphomethylpyrimidine synthase (ThiC) activity is greatly increased in E. coli strains defective in CRP-cAMP complex formation such strains overexpressing key thiamine genes (ThiMDE) were cultivated in mMOPS with thiazole supplementation (THZ) and the production of thiamine in each culture was measured compared to a control culture.
The strains tested all expressed ThiC from a first plasmid (pBS2180) and ThiMDE (pBS2184) from a second plasmid. Control parent strains (BS04608) and IscR mutant (BS04726) as well as KO mutants of CRP (BS04739) and CyaA (BS04786) were described above in the section: Strains and plasmids. ThiC expression was IPTG inducible and the IPTG concentration that gave maximal activity was chosen for each strain, whereas ThiMDE expression was constitutive. All strains were cultured, in 4 replicates in 400 μL mMOPS medium containing 100 μg/mL ampicillin under optimal IPTG concentrations for induction of ThiC and 50 μg/mL kanamycin for plasmid expressing ThiMDE.
The strains were grown in deep well plates for 24 hours at 37° C. shaken at 275 rpm, after which thiamine production was evaluated using a growth-based bioassay according to procedure II. The resulting thiamine titers were shown in
To determine if mutations in CRP-cAMP complex formation improve thiamine production, fed-batch fermentations were performed using all strains.
A 1 ml glycerol stock of BS04608, BS04726, BS04739 and BS04786 were inoculated into four separate 250 ml shakeflasks containing 50 ml mMOPS medium with ampicillin and kanamycin. The shake flasks were incubated at 37° C. at 250 rpm shaking for 20 hours resulting in an optical density of COD600 2.5-3.5. 0.9 ml B medium supplemented with ampicillin and kanamycin was added to a BioLector 32 well microfluidic plate equipped with pH, DO and biomass fluorescence (m2p-labs). 5% (v/v) of each strain culture was used to inoculate the B medium in four fermentation wells resulting in an initial cOD600 0.125-0.175 for each well and strain. The microfluidic plate was then sealed with gas-permeable sealing foil with evaporation reduction and loaded into the Biolector Pro (m2p-labs, Baesweiler, Germany). The temperature set to 37° C., pH controlled in each well to pH=7 by addition of 5 M NH4OH, the agitation speed of the plate set to 1300 rpm (3 mm orbit) and the relative humidity in the chamber controlled at 85%. Four hours after inoculation the plate fermentations were paused, and each culture well was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a concentration of 50 μM. The fermentation was resumed and following the depletion of glucose in the batch medium a fed batch phase was initiated by the addition of G medium to each culture well. This addition was controlled to an unlimited 1 μL pulse provided the DO % in each well registered greater than 70%. This transition took place between 6.7-11 h. The fermentations were terminated at 24 hours. An endpoint culture sample was taken from each fermentation well at 24 hours after inoculation. From this sample optical density (cOD600) was measured according to analytical procedure I. Supernatants were obtained by spinning biomass down in a centrifuge at 4000 g, 5 min at 4° C. Supernatants were screened for thiamine using the bioassay described in analytical procedure II. The thiamine titre is shown in
The following strains of Escherichia coli from example 4 WO2020148351 are used.
The following plasmids are used.
Enhanced Nicotinamide Riboside (NR) Production in a CRP-cAMP Deficient Strain Overexpressing nadABC, nadE* and aphA Genes
The parent E. coli strain BS1575, and a mutant thereof lacking CRP (BS4260), are transformed with a plasmid (pBS_NR) comprising the genes nadABCE*aphA operatively linked an IPTG inducible promoter resulting in strains BS1575_NR and BS4260_NR. The genes expressed in plasmid pBS_NAM include:
Cells of each strain are grown in 50 ml mMOPS medium, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin, at 37° C. and 250 rpm until the OD600 nm reaches 0.6. IPTG is then added to each culture to a final concentration of 0.064 mM that are then incubated for a further 6 h, and subsequently lysed.
NR, present in the recovered lysed cell supernatant, is measured by LC-MS using a 1290 Infinity series UHPLC coupled to a 6470 triple quadrupole from Agilent Technologies (Santa Clara, USA) (Ollagnier-de Choudens et al., 2005). NR production by an E. coli strain expressing the genes of the NR pathway (i.e. nadABCE*aphA), is enhanced when the host strain lacks the CRP-cAMP complex as compared to a strain expressing a gene encoding a native, CRP-cAMP complex.
Enhanced Nicotinamide (NAM) Production in a CRP-cAMP Deficient Strain Overexpressing nadABC, nadE* and NMN Nucleosidase (Chi) Genes.
For nicotinamide NAM production E. coli strain BS1575, and a mutant thereof lacking CRP (BS4260), are transformed with a plasmid (pBS_NAM) comprising the genes nadABCE* and chi operatively linked an IPTG inducible promoter, resulting in strains BS1575_NAM and BS4260_NAM. The genes expressed in plasmid pBS_NAM include:
NAM production by an E. coli strain expressing the genes of the NAM pathway is enhanced when the host strain lacks the CRP-cAMP complex as compared to a strain expressing a gene encoding a native, CRP-cAMP complex.
The following strains of Escherichia coli from example 5 WO2020148351 are used.
The following plasmids used in the example are listed below.
Cobalamin is produced by E. coli cells expressing an IPTG inducible transgene encoding CobG and a constitutively expressing transgenes encoding transgenes encoding CobHIJLFK, CobMNST, CobCDTPduX, CobROQBtuR and CobUSCbiB (see table 9); and where the host E. coli cells further comprise the transgenes cbiNQOM inserted into their genome. E. coli strains BS1575_B12x3 and BS4260_B12x3 are cultured as described in WO2020148351. Cobalamin produced by the cultures is measured as follows: 2.5 mL of NaNO2 8% (w/v) and 2.5 mL of glacial acetic acid are added to 25 ml samples of each culture; which are then boiled for 30 min, and the resulting mixture filtered. Then 20 μL NaCN 10% (w/v) is added to 1 ml of aqueous phase; and 20 L of resulting upper aqueous phase is injected into an HP1100 HPLC system (Agilent). NH2 column (4.6×250 mm2, 5 um) is employed for HPLC analysis with a flow rate of 1.7 mL/min and a wavelength of 360 nm, using a mobile phase of 250 mM phosphoric acid/acetonitrile (30/70, v/v). The production of cobalamin is enhanced when said host E. coli cells comprise the CRP-cAMP deficient strain (BS4260_B12x3) as compared to host E. coli cells comprising WT CRP-cAMP complex (BS1575_B12x3).
The following strains of Escherichia coli are used.
The following plasmids used in the example are listed below.
Enhanced Pantothenate Production in a Strain Lacking CRP-cAMP Complex Overexpressing ilvD, panB, panE and panC Genes
The parent E. coli strain BS1575, and a mutant thereof lacking CRP (BS4260), are transformed with a plasmid (pBS_PAN) comprising the genes ilvD, panB, panE and panC operably linked a constitutive promoter. The E. coli panB gene encodes a) 3-methyl-2-oxobutanoate hydroxymethyl-transferase; E. coli panE encodes 2-dehydropantoate 2-reductase and E. coli panC encodes pantothenate synthetase.
Cells of each strain are cultivated aerobically in 50 ml of NM1 Medium (NM1 composition: glucose, 20 g; (NH4) 2SO4, 20 g; KH2PO4, 2.0 g; MgSO4, 7H2O,0.4 g; NaCl, 1.6 g; yeast extract, 2 g; trace metal solution, supplemented with 1 nmol/L biotin, 100 μg/mL ampicillin at 31° C. and 250 rpm for 24 h. Pantothenate produced in the culture medium by each strain, following normalization for cell density, is measured as described in the methods section and the reference provided therein.
The production of pantothenate is enhanced in the CRP-cAMP deficient strain, BS1575_B5, when co-expressing the transgene encoding ilvD and the transgenes encoding panBEC, as compared to their co-expression in the parent host E. coli strain expressing wild type CRP-cAMP complex (BS4260_B5).
| Number | Date | Country | Kind |
|---|---|---|---|
| 21186217.2 | Jul 2021 | EP | regional |
| 22167864.2 | Apr 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/069711 | 7/14/2022 | WO |
