Patent Application
20230374445
The contents of the electronic sequence listing (GNBI_001_02WO_SeqList_ST26.xml; Size: 467,880 bytes; and Date of Creation: May 17, 2023) are herein incorporated by reference in its entirety.
The disclosure relates to recombinant bacteria and methods for producing poly(3-hydroxybutyrate-co-3-hydroxyvalerate).
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a polyhydroxyalkanoate-type microbial biopolymer that is biocompatible and biodegradable and could serve as a viable alternative for many petroleum-derived polymers. The many useful features of PHBV, for example, absorption capacity, low cytotoxicity, piezoelectricity, and thermoplasticity, render it a very promising material with broad applications in a wide range of applications, in particular biomaterial applications. Amongst the different biomaterial applications, PHBV may be suited for absorbable surgical sutures, drug release and delivery systems, medical packaging, and tissue engineering such as biodegradable medical implants, biosensors, porous scaffolds, and tissue patches.
The vast array of potential applications of PHBV may be achieved by varying properties such as composition, molecular weight (MW) and crystallinity, which affect the mechanical and thermal characteristics of the biopolymer. These properties are influenced by, for example, the species or strains of microbes, carbon source, and growth parameters. There are inherent difficulties in maintaining consistent polymer properties (i.e. Mw and composition) and in achieving a specific composition (i.e. tailoring 3-hydroxyvalerate (HV) content) when the microbial culture is highly heterogeneous. A recombinant approach that generates specific strains that modulates the expression level or activity of specific enzymes, including heterologous enzymes, involved in metabolic pathways may provide an avenue for controlling production of PHBV with consistent polymer properties (such as, a desired Mw) and specific compositions.
The disclosure provides recombinantly-modified bacterial host cells that exhibit improved production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV from substrates, such as, volatile fatty acids (VFAs) and glycerol. The disclosed recombinant bacterial host cells have been engineered to express catalytic proteins that enhance flux through metabolic pathways, thereby promoting uptake of the substrates and their conversion to PHBV. Notably, the disclosed recombinantly-modified bacterial host cells may be used for the small-scale and large-scale production of PHBV per the methods disclosed herein.
The disclosure provides bacterial host cells, comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway.
In embodiments, the bacterial host cells comprise: a first operon comprising: (a) a nucleic acid molecule encoding a PhaC protein, wherein the PhaC protein is a Cupriavidus sp. S-6 PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, wherein the PhaA protein is a Cupriavidus sp. S-6 PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein, wherein the BktB protein is a Cupriavidus gilardii QJ1 BktB protein, and (ii) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; and a sleeping beauty mutase (Sbm) operon comprising a promoter, wherein each of the first and the second operons comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2). In embodiments, the bacterial host cells are capable of converting glycerol to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV.
The disclosure further provides bacterial host cells comprising: comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, (e) a nucleic acid molecule encoding a LvaE protein, (f) a nucleic acid molecule encoding a propionate-CoA transferase, (g) a nucleic acid molecule encoding a FadE protein, (h) a nucleic acid molecule encoding a FadB protein, and (i) a nucleic acid molecule encoding a AtoB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway. In embodiments, the bacterial host cells are capable of converting one or more volatile fatty acids (VFAs) to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV.
The disclosure also provides methods of producing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using the bacterial host cells disclosed herein, as well as methods of metabolizing glycerol or VFAs using the bacterial host cells disclosed herein.
Throughout the disclosure, a reference may be made using an abbreviation of a gene name or a polypeptide name, and it is understood that such an abbreviated gene or polypeptide name represents the genus of genes or polypeptides, respectively. Such gene names include all genes encoding the same polypeptide and homologous polypeptides having the same physiological function. Polypeptide names include all polypeptides that have the same activity (e.g., that catalyze the same fundamental chemical reaction).
Unless otherwise indicated, the accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A.
EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo.
The term “recombinant”, or a derivative thereof as used herein refers to a cell or a polynucleotide molecule that has been modified by the introduction of a heterologous polynucleotide sequence, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cells, or the recombinant cells express, as a result of deliberate human intervention, native genes that are otherwise abnormally expressed, underexpressed or not expressed at all. The terms “recombination,” “recombining,” and generating a “recombined” polynucleotide molecule refer generally to the assembly of two or more polynucleotide fragments wherein the assembly gives rise to a chimeric polynucleotide made from the assembled parts.
The term “poly(3-hydroxybutyrate-co-3-hydroxyvalerate)”, “PHBV”, or “PHBV polymer”, or a derivative thereof as used herein refers to a polyhydroxyalkanoate-type polymer that can be produced by bacteria through fermentation of a carbon source, for example, sugar, lipids, polyol, or fatty acids. PHBV is a copolymer of 3-hydroxybutyric acid (HB) and 3-hydroxyvaleric acid (HV; also known as 3-hydroxypentanoic acid). PHBV can have varying HB and HV content. PHBV is biocompatible, biodegradable, and non-toxic, and is useful in the production of bioplastics. The many useful features of PHBV include absorption capacity, low cytotoxicity, piezoelectricity, and thermoplasticity. PHBV has a broad range of applications, including biomaterial applications such as production of absorbable surgical sutures, drug release and delivery systems, medical packaging, and tissue engineering, e.g. biodegradable medical implants, biosensors, porous scaffolds, and tissue patches.
The term “acyl-CoA synthetase” as used herein refers to an enzyme which can catalyze the esterification, in some cases concomitant with transport, of fatty acids into metabolically active CoA thioesters for subsequent degradation or incorporation into phospholipids. Acyl-CoA synthetase enzymes can be categorized based on their specificity to short, medium, or long chain fatty acids. For example, short chain acyl-CoA synthetase catalyzes chemical reactions with fatty acid with fewer than 6 carbons. Medium chain acyl-CoA synthetase catalyzes chemical reactions with fatty acids with 6 to 12 carbons. Acyl-CoA synthetase includes, but is not limited to, fatty acid-CoA ligase. In embodiments, an acyl-CoA synthetase comprises an enzyme under the enzyme classification numbers EC 6.2.1.1, EC 6.2.1.2, EC 6.2.1.3, EC 6.2.1.17, or EC 6.2.1.40. Additionally, one of ordinary skill in the art will appreciate that some enzymes classified under a different enzyme class can have acyl-CoA synthetase activity as well. Such non-specific “acyl-CoA synthetase” are, therefore, also included in this definition. Nucleic acid sequences encoding acyl-CoA synthetase are known in the art, and such acyl-CoA synthetase are publicly available.
The term “acetate-CoA transferase” as used herein refers to an enzyme that can act upon a fatty acid substrate and an acetyl-CoA substrate to catalyze a reversible chemical reaction to produce acetate and a corresponding acyl-CoA. The enzyme can also act upon a VFA substrate and an acetyl-CoA substrate to produce a corresponding acyl-CoA and acetate. A person of ordinary skill in the art would readily understand that the enzyme is capable of catalyzing the reversible reaction in both forward and reverse directions. In embodiments, an acetate CoA transferase has broad substrate specificity for short-chain acyl-CoA thioesters with the activity decreasing when the length of the carboxylic acid chain exceeds four carbons. The enzyme includes, but is not limited to, short-chain acyl-CoA:acetate-CoA transferase. In embodiments, an acetate-CoA transferase is an enzyme under the enzyme classification number EC 2.8.3.8. The terms “acetate” and “acetic acid” are used interchangeably herein. Similarly, the use of any term which describes an organic acid likewise includes, and is used interchangeably with, the corresponding salt form of the organic acid. In embodiments, the acetate-CoA transferase comprises a first subunit, optionally a MELS_RS00170 polypeptide or an AtoA polypeptide, and a second subunit, optionally a MELS_RS00175 polypeptide or AtoD polypeptide. In embodiments, the acetate-CoA transferase comprises a MELS_RS00170 polypeptide and a MELS_RS00175 polypeptide. In embodiments, the acetate-CoA transferase comprises an AtoD polypeptide and an AtoA polypeptide.
The term “propionate-CoA transferase” as used herein refers to an enzyme that acts upon substrates acetyl-CoA and propionate. Propionate-CoA transferase catalyzes a chemical reaction with its substrates to produce acetate and propionyl-CoA. The enzyme can also include, but is not limited to, acetyl-CoA:propionate-CoA transferase, propionate-coenzyme A transferase, propionate-CoA:lactoyl-CoA transferase, propionyl-CoA:acetate-CoA transferase, or propionyl-CoA transferase. In embodiments, a propionate-CoA transferase comprises an enzyme under the enzyme classification number EC 2.8.3.1.
The term “β-ketothiolase” as used herein refers to an enzyme that acts upon substrates acetyl-CoA and acyl-CoA. β-ketothiolase catalyzes a chemical reaction to produce 3-oxoacyl-CoA and CoA. The enzyme can also include, but is not limited to, acetyl-CoA synthetase, acetyl-CoA acyltransferase, acyl-CoA ligase, 3-ketoacyl-CoA thiolase, or fatty acid oxidation complex subunit beta. In embodiments, a β-ketothiolase comprises an enzyme under the enzyme classification number EC 2.3.1.16.
The term “polyhydroxyalkanoate synthase” as used herein refers to an enzyme that acts upon substrates hydroxybutyryl-CoA and poly(hydroxybutyrate)n. Polyhydroxyalkanoate synthase catalyzes a chemical reaction to produce poly(hydroxylalkanoate)n+1 and CoA. The chemical reaction can yield hydroxylalkanoate polymers. The enzyme can also include, but is not limited to, poly(3-hydroxyalkanoate) polymerase, poly(3-hydroxybutyrate) polymerase, or polyhydroxyalkanoic acid synthase. In embodiments, a polyhydroxyalkanoate synthase comprises an enzyme under the enzyme classification number EC 2.3.1. In embodiments, a polyhydroxyalkanoate synthase comprises short-chain polyhydroxyalkanoate synthase. In embodiments, a polyhydroxyalkanoate synthase polymerizes (R)-HB-CoA and (R)-HV-CoA to produce PHBV.
The term “methylmalonyl-CoA mutase” as used herein refers to an enzyme that catalyzes interconversion of succinyl-CoA and methylmalonyl-CoA. In embodiments, methylmalonyl-CoA mutase comprises an enzyme under the enzyme classification number EC 5.4.99.2.
The term “methylmalonyl-CoA mutase interacting protein”, or a derivative thereof as used herein refers to a protein that interacts with methylmalonyl-CoA mutase and is a member of the G3E family of P-loop GTPases. In embodiments, a methylmalonyl-CoA mutase interacting protein comprises methylmalonyl-CoA mutase-interacting GTPase. The enzyme can also include, but is not limited to, GTPase ArgK, G-protein chaperone, or YgfD protein. In embodiments, a methylmalonyl-CoA mutase interacting protein comprises an enzyme under the enzyme classification number EC 3.6.5.
The term “methylmalonyl-CoA decarboxylase” as used herein refers to an enzyme that acts upon substrate methylmalonyl-CoA and catalyzes decarboxylation of methylmalonyl-CoA into propionyl-CoA. The enzyme can also include, but is not limited to, transcarboxylase. In embodiments, a methylmalonyl-CoA decarboxylase comprises an enzyme under the enzyme classification number EC 4.1.1.
The term “propionyl-CoA:succinate CoA transferase” as used herein refers to an enzyme that acts upon substrates propionyl-CoA and succinate. The enzyme catalyzes the transfer of CoA from propionyl-CoA to succinate. The enzyme produces the products propionate and succinyl-CoA. In embodiments, a propionyl-CoA:succinate CoA transferase comprises an enzyme under the enzyme classification number EC 2.8.3. In embodiments, the bacterial host cell shows reduced or eliminated expression and/or activity, of propionyl-CoA:succinate CoA transferase.
The expression “at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyryl-CoA to succinate”, or a derivative thereof as used herein refers to an enzymatic pathway that starts with butyryl-CoA as a substrate and through at least one enzyme produces the product succinate. This pathway may involve the production of intermediates such as butyraldehyde and succinate semialdehyde. In embodiments, the pathway for conversion of butyrl-CoA to succinate comprises enzymes CoA-dependent propanal dehydrogenase, optionally PduP, β-alanine transaminase, optionally KES23458, and NADP+-dependent succinate semialdehyde dehydrogenase, optionally GabD.
The term “CoA-dependent propanal dehydrogenase” or “CoA-dependent propionaldehyde dehydrogenase” as used herein refers to an enzyme that reversibly converts 1-propanal (propionaldehyde) to propionyl-CoA (propionyl-CoA). In some instances, CoA-dependent propanal dehydrogenase enzymes, for example PduP, may have preferences for substrates with 2-4 or 2-6 carbons, and are able to reversibly convert butyryl-CoA to butyraldehyde. In some instances, CoA-dependent propanal dehydrogenase enzymes may have specificity for aldehydes containing 4 carbons. In embodiments, a CoA-dependent propanal dehydrogenase comprises an enzyme under the enzyme classification number EC 1.2.1.10.
The term “CoA-acylating aldehyde dehydrogenase” as used herein refers to an enzyme that can convert acetyl-CoA and butyryl-CoA to the corresponding aldehydes. In some instances, CoA-acylating aldehyde dehydrogenase enzymes may have preferences for substrates with 2-4 or 2-6 carbons, and are able to convert butyryl-CoA to butyraldehyde. In embodiments, a CoA-acylating aldehyde dehydrogenase comprises an enzyme under the enzyme classification number EC 1.2.1.27.
The term “β-alanine transaminase” as used herein refers to an enzyme that acts upon substrates β-alanine and pyruvate. β-alanine transaminase catalyzes a chemical reaction to produce 3-oxopropionate and L-alanine. The enzyme can also include, but is not limited to, β-alanine:pyruvate aminotransferase, β-alanine:pyruvate transaminase, Ω-amino acid aminotransferase, or Ω-amino acid:pyruvate aminotransferase. In embodiments, a β-alanine transaminase comprises an enzyme under the enzyme classification number EC 2.6.1.18.
The term “NADP+-dependent succinate semialdehyde dehydrogenase”, or a derivative thereof as used herein refers to an enzyme that acts upon substrates NADP+, H2O, and succinate semialdehyde. NADP+-dependent succinate semialdehyde dehydrogenase catalyzes a chemical reaction to produce succinate, NADPH and two H+ ions. The enzyme can include, but is not limited to, succinic semialdehyde dehydrogenase (NADP+), succinyl semialdehyde dehydrogenase (NADP+), succinate semialdehyde:NADP+ oxidoreductase, or NADP-dependent succinate-semialdehyde dehydrogenase. In embodiments, a NADP+-dependent succinate semialdehyde dehydrogenase is an enzyme under the enzyme classification number EC 1.2.1.79.
The expression “at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyryl-CoA to 3-hydroxybutyryl-CoA”, or a derivative thereof as used herein refers to an enzymatic pathway that starts with butyryl-CoA as a substrate and through at least one enzyme produces the product 3-hydroxybutyryl-CoA. This pathway may involve the production of intermediates such as, for example, crotonyl-CoA. In embodiments, the pathway for conversion of butyryl-CoA to 3-hydroxybutyryl-CoA comprises enzymes acyl-CoA dehydrogenase, optionally a short-chain acyl-CoA dehydrogenase, optionally at least one of PP_2216, BC_5341, MELS_RS10970, and FadE, and an enoyl-CoA hydratase/isomerase, optionally at least one of H16_RS27940, PhaJ, and PaaZ.
The term “acyl-CoA dehydrogenase”, or a derivative thereof as used herein refers to an enzyme that catalyzes the dehydrogenation of acyl-coenzymes A (acyl-CoAs) to 2-enoyl-CoAs. Acyl-CoA dehydrogenase enzymes can be categorized based on their specificity to short, medium, or long chain fatty acids. For example, short-chain acyl-CoA dehydrogenase catalyzes fatty acid oxidation of acyl-CoAs with 4-6 carbons. In embodiments, an acyl-CoA dehydrogenase comprises an enzyme under the enzyme classification number EC 1.3.8.7 or EC 1.3.8.8. Additionally, one of ordinary skill in the art will appreciate that some enzymes classified under a different enzyme class can have acyl-CoA dehydrogenase activity as well. Such non-specific “acyl-CoA dehydrogenase” are, therefore, also included in this definition. Nucleic acid sequences encoding acyl-CoA dehydrogenase are known in the art, and such acyl-CoA dehydrogenase are publicly available.
The term “enoyl-CoA hydratase/isomerase”, or a derivative thereof as used herein refers to an enzyme that acts upon substrates hydroxyacyl-CoA and NAD+. The enzyme catalyzes a chemical reaction to produce 3-oxoacyl-CoA, NADH, and a H+ ion. The enzyme can also include, but is not limited to, fatty acid oxidation complex subunit-α, enoyl-CoA hydratase, delta-(2)-trans-enoyl-CoA isomerase, 2-hydroxybutyryl-CoA epimerase, or 3-hydroxyacyl-CoA dehydrogenase. In embodiments, an enoyl-CoA hydratase/isomerase is an enzyme under the enzyme classification number EC 4.2.1.17, EC 5.1.2.3, EC 5.3.3.8, EC 1.1.1.35, EC 3.3.2.12 or EC 1.12.1.91.
The term “propionyl-CoA synthetase” as used herein refers to an enzyme that catalyzes the synthesis of propionyl-CoA from propionate and CoA, using ATP. Propionyl-CoA synthetase can also include, but is not limited to, propionate-CoA ligase. In embodiments, a propionyl-CoA synthetase is an enzyme under the enzyme classification number EC 6.2.1.17.
The term “glutamate decarboxylase” as used herein refers to an enzyme that catalyzes a chemical reaction to convert L-glutamate into gamma-aminobutyrate (GABA). The chemical reaction consumes an H+ ion and produces CO2. Glutamate decarboxylase can also include, but is not limited to, glutamate decarboxylase-α or glutamate decarboxylase-β. In embodiments, a glutamate decarboxylase comprises an enzyme under the enzyme classification number EC 4.1.1.15.
The term “succinyl-CoA transferase” as used herein refers to an enzyme that acts upon substrates succinate and 3-oxoacyl-CoA. The enzyme catalyzes a chemical reaction to produce succinyl-CoA and 3-oxo acid. Succinyl-CoA transferase can include, but is not limited to, 3-oxoacid coenzyme A-transferase, 3-ketoacid CoA-transferase, 3-ketoacid coenzyme A transferase, 3-oxo-CoA transferase, 3-oxoacid CoA dehydrogenase, acetoacetate succinyl-CoA transferase, acetoacetyl coenzyme A-succinic thiophorase, succinyl coenzyme A-acetoacetyl coenzyme A-transferase, or succinyl-CoA transferase. In embodiments, a succinyl-CoA transferase comprises an enzyme under the enzyme classification number EC 2.8.3.5.
The term “succinyl-CoA synthetase” as used herein refers to an enzyme that acts upon substrates succinate and CoA. The enzyme catalyzes a chemical reaction which consumes ATP to produce succinyl-CoA and ADP. The enzyme can also include, but is not limited to, a succinate-CoA ligase. In embodiments, succinyl-CoA synthetase comprises an enzyme under the enzyme classification number EC 6.2.1.5. In embodiments, the succinyl-CoA synthetase comprises a first subunit, optionally a SucC polypeptide, and a second subunit optionally a SucD polypeptide. In embodiments, the succinyl-CoA synthetase comprises a SucC polypeptide and a SucD polypeptide.
The term “glutamate dehydrogenase” as used herein refers to an enzyme that catalyzes the reversible conversion of ketoglutarate to glutamate, such as L-glutamate. In embodiments, the glutamate dehydrogenase comprises an enzyme under the enzyme classification number EC 1.4.1.4. In embodiments, the glutamate dehydrogenase is GdhA.
The term “attenuate”, or a derivative thereof as used here means to weaken, reduce or diminish. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is reduced such that the enzyme activity is not impacted by the presence of a compound. In a particular example, an enzyme that has been modified to be less active can be referred to as attenuated. A functional modification of the sequence encoding an enzyme can be used to attenuate expression of an enzyme. Sequence modifications may include, for example, a mutation, deletion, or insertion of one or more nucleotides in a gene sequence or a sequence controlling the transcription or translation of a gene sequence, which modification results in a reduction or inhibition of production of the gene product, or renders the gene product non-functional. In some examples, a functional deletion is described as a knock-out mutation. Other methods are available for attenuating expression of an enzyme. For example, attenuation can be accomplished by modifying the sequence encoding any gene described herein, e.g. by mutation, placing the gene under the control of a less active promoter, expressing interfering RNAs, ribozymes, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional interference, or antisense sequences that target the gene of interest, and/or by changing the physical or chemical environment, such as temperature, pH, or solute concentration, such that the optimal activity of the gene or gene product is not realized. The skill person will appreciate that such attenuation effects can be achieved through any other techniques known in the art.
The term “homologous genes”, or a derivative thereof as used herein refers to a pair of genes from different but related species, which correspond to each other and which are identical or similar to each other. The term encompasses genes that are separated by the speciation process during the development of new species (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes). Homologous polypeptides are polypeptides that are encoded by these homologous genes, and/or polypeptides having the same physiological function. The term “homolog”, or a derivative thereof as used herein refers to a homologous protein and to the gene encoding it.
The term “operably linked”, or a derivative thereof as used herein in the context of a polynucleotide sequence, refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner, for instance, the placement of one polynucleotide sequence into a functional relationship with another polynucleotide sequence. For example, a transcriptional regulatory sequence or a promoter is operably linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. A ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Aspects of the transcription process include, but not limited to, initiation, elongation, attenuation and termination. In general, an operably linked transcriptional regulatory sequence joined in cis with the coding sequence, but it is not necessarily directly adjacent to it, and the polynucleotide sequences being linked are contiguous and in the same reading frame.
The term “operon region” as used herein refers to a group of contiguous genes that are transcribed as a single transcription unit from a common promoter, and are thereby subject to co-regulation. In other words, an operon comprises a common promoter is operably linked to the group of contiguous genes in the operon. In embodiments, the operon comprises a regulator segment.
The term “orthologs” or “orthologous genes”, or a derivative thereof as used herein refers to genes in different species that have evolved from a common ancestral gene by speciation. Typically, orthologs retain the same function during the course of evolution. Identification of orthologs finds use in the reliable prediction of gene function in genomes of different species.
A “promoter” as used herein refers to a polynucleotide sequence that functions to direct transcription of a downstream gene. In embodiments, the promoter is appropriate to a host cell, such as a bacterial cell, in which the target gene is being expressed. The promoter, together with other transcriptional and translational regulatory polynucleotide sequences (also termed “control sequences”) is necessary to express a given gene. In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
The term “regulatory segment”, “regulatory sequence”, or “expression control sequence”, or a derivative thereof as used herein refers to a polynucleotide sequence that is operatively linked with another polynucleotide sequence that encodes the amino acid sequence of a polypeptide chain to effect the expression of that encoded amino acid sequence. The regulatory sequence can inhibit, repress, promote, or drive the expression of the operably linked polynucleotide sequence encoding the amino acid sequence.
The terms “proportional yield” and “percentage yield” are used interchangeably herein referring to the amount of a desired product in relation to other products that are within the same mixture produced by a recombinant bacterial cell of the present disclosure. For example, the proportional yield of a desired product can be improved such that it is more predominant over the other components in the product mixture to reduce the burden of purification. In another example, the proportional yield of an undesired product (i.e. a component that will need to be removed from the desired product) can be reduced such that it is less predominant over the desired component in the product mixture to achieve the same end.
The term “substitution”, or a derivative thereof as used herein means replacing an amino acid in the sequence of a precursor polypeptide with another amino acid at a particular position, resulting in a mutant of the precursor polypeptide. The amino acid used as a substitute can be a naturally-occurring amino acid, or can be a synthetic or non naturally-occurring amino acid.
The term “surfactants” as used herein refers to substances that are capable of reducing the surface tension of a liquid in which they are dissolved. Surfactants are typically composed of a water-soluble head and a hydrocarbon chain or tail. The water-soluble head is hydrophilic and can be either ionic or nonionic, whereas the hydrocarbon chain is hydrophobic. Surfactants are used in a variety of products, including detergents and cleaners, and in chemical processes. Surfactants can be used to aid in the extraction and isolation of biopolymers such as those described herein. There are four types of surfactants: anionic surfactants, cationic surfactants, amphoteric surfactants, and non-ionic surfactants, any of which may be used for extraction and isolation of biopolymers, and/or treatment of biopolymers.
The term “wild-type” as used herein means, in the context of gene or protein, a polynucleotide or protein sequence that occurs in nature. In embodiments, the wild-type sequence refers to a sequence of interest that is a starting point for recombinant protein engineering.
The term “volatile fatty acid” or “VFA”, or a derivative thereof as used herein refers to fatty acids with less than six carbon atoms. For example, VFA includes, but not limited to formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. The VFA and salt thereof described herein are useful energy and carbon source, and as source materials to be converted to PHBV by bacteria. In embodiments, the carbon or energy source comprises at least one VFA. In embodiments, the at least one VFA comprises at least one of acetic acid, propionic acid, and butyric acid.
The term “biomass” refers to an organic or biological material that can be converted into an energy source. One exemplary source of biomass is plant matter. For example, corn, sugar cane, and switchgrass can be used as biomass. Another non-limiting example of biomass is animal matter, for example cow manure. Biomass also includes waste products from industry, agriculture, forestry, food, perennial grasses, and households. Examples of such waste products which can be used as biomass are fermentation waste, straw, lumber, sewage, garbage and food leftovers. Biomass also includes sources of carbon, such as carbohydrates (e.g., sugars). In embodiments, the biomass comprises pretreated biomass. Biomass may be pretreated by methods including, but not limited to, mechanical chipping, shredding, grinding. Methods of pretreating biomass can also include methods of biological degradation of lignin, hemicellulose, and polyphenols via fungi or chemical treatments with acids, alkali, organic solvents, and ionic liquids to increase internal surface area, and decrease degree of polymerization and crystallinity. In embodiments, physiochemical methods such as steam and other forms of heat can also be used to pretreat biomass. Methods of pretreating biomass produces pretreated biomass.
The term “carbon source” refers to a nutrient (such as sugar) that provides carbon needed for cellular respiration, cellular combustion, and/or synthesis of new organic molecules. A volatile fatty acid is useful as a carbon source for a recombinant bacterial cell described herein. In embodiments, at least one carbon source comprises at least one volatile fatty acid.
The term “granule”, or a derivative thereof as used herein relating to PHBV refers to the form of PHBV accumulated inside bacteria. PHBV is stored inside bacteria as discrete water-insoluble intracellular granules. PHBV granules can be extracted from bacteria by the methods described herein.
The term “mmol/L”, or a derivative thereof as used herein refers to a measure of the concentration of a solute in a solution in the unit of mmol of the solute per litre solution.
The term “Cmmol/L”, or a derivative thereof as used herein refers to a measure of the concentration of a solute in a solution in the unit of mmol of carbon per litre solution.
The term “VFA mmol/L”, or a derivative thereof as used herein refers to a measure of the concentration of total VFA in a solution in the unit of mmol of VFA per litre solution.
The term “mol %”, or a derivative thereof as used herein when relating to HV content in PHBV refers to a measure of molar percentage of HV in PHBV. For example, PHBV can have a HV content of 0-5 mol %, 5-10 mol %, 10-20 mol %, 20-50 mol %, 1-20 mol %, 1-30 mol %, 1-40 mol %, or 1-50 mol %, 1-60 mol %, 1-70 mol %, or 1-80 mol %.
The phrase “substantially free”, or a derivative thereof as used herein is used to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result. For example, a medium or a composition that is “substantially free of” glycerol would either completely lack glycerol, or so nearly completely lack glycerol that the effect would be the same as if it completely lacked glycerol. In other words, a composition that is “substantially free of” an element may still actually contain such item as long as there is no measurable effect thereof. For example, a medium or a composition that is substantially free of an ingredient or element comprises less than about 1% by wt or less than about 1% vol/vol of the ingredient or element in the composition.
The term (w/v), or a derivative thereof as used herein refers to a measure of the concentration of a solution or mixture obtained by dividing the mass or weight of the solute by the volume of the solution or mixture.
The term (w/w), or a derivative thereof as used herein refers to a measure of the concentration of a solution or mixture obtained by dividing the mass or weight of the solute by the weight of the solution or mixture.
In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The term “consisting essentially of”, or a derivative thereof as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps. Finally, terms of degree such as “substantially”, “about” and “approximately”, or a derivative thereof as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes for example 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about”.
As used herein, the term “polypeptide” as used herein encompasses both peptides and proteins, unless indicated otherwise. The 3-letter code as well as the 1-letter code for amino acid residues as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. It is also understood that a polypeptide may be coded for by more than one polynucleotide sequence due to the degeneracy of the genetic code. An enzyme is a protein that is also a biocatalyst, which accelerate chemical reactions. It is understood that the enzymes described herein, unless otherwise stated, have substrate specificities and enzymatic activity (e.g. catalytic rate) with respect to their substrates. For example, an acyl-CoA synthetase polypeptide has acyl-CoA synthetase activity.
The term “nucleic acid molecule” or its derivatives thereof as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, the nucleic acid molecules of the disclosure can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically double-stranded or a mixture of single- and double-stranded regions. In addition, the nucleic acid molecules can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The nucleic acid molecules of the disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule” embraces chemically, enzymatically, or metabolically modified forms. The term “polynucleotide” shall have a corresponding meaning.
As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. The extent of identity (homology) between two sequences can be ascertained using a computer program and mathematical algorithm. Percentage identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk/Tools/msa/clustalo using default parameters. See, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.” (2011 October 11) Molecular systems biology 7:539. For the purposes of calculating identity to a sequence, extensions such as tags are not included.
The term “plasmid”, “vector”, or “construct” as used herein refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in some microorganism such as bacteria, or integrates into the host chromosome. The plasmid can be part of an expression system. The plasmid is useful for creating a recombinant bacterial cell, for example, that produces polypeptides which catalyze the synthesis of a biopolymer, including PHBV described herein.
The terms “expression” or “express” refers to the production of mRNA from the polynucleotide sequence of a gene or portion of a gene. The production of any polypeptide which is encoded by the mRNA, gene, or portion of the gene is also included within the scope of the terms.
The term “encoding” refers to the property of polynucleotide sequences to behave as templates for the production of other macromolecules such as mRNA, polypeptides, and cDNA.
The term “host strain” or “host cell” refers to a suitable host for an expression vector or genomically-integrated expression cassette comprising polynucleotide of the present disclosure.
A “segment” of a nucleotide sequence is a sequence of contiguous nucleotides. A segment can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 85, 100, 110, 120, 130, 145, 150, 160, 175, 200, 250, 300, 350, 400, 450, 500 or more contiguous nucleotides.
The definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art.
The disclosure provides bacterial host cells, comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaB protein, (c) a nucleic acid molecule encoding a PhaA protein, and (d) a nucleic acid molecule encoding a BktB protein. In embodiments, the bacterial host cells disclosed herein comprise more than one copy (for example, two copies, three copies, 4 hours copies, or 5 or more copies) of the nucleic acid molecule encoding a PhaC protein.
In embodiments, the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway. Further details are provided in Miscevic D et al., Applied Microbiology and Biotechnology 2021, 105:1435-1446, and Srirangan K et al., Scientific Reports 2016, 6:36470, the contents of each of which are incorporated herein by reference in their entireties for all purposes. In embodiments, the bacterial host cell comprises a sleeping beauty mutase (Sbm) operon comprising a promoter. In embodiments, the bacterial host cell comprises a sleeping beauty mutase (Sbm) operon comprising a Ptrc promoter. In embodiments, the Ptrc promoter comprises a nucleic acid sequence having at least 95% (for example, about 96%, about 97%, about 98%, about 99% or about 100%) identity to SEQ ID NO: 254. In embodiments, the Ptrc promoter comprises the nucleic acid sequence of SEQ ID NO: 254. In embodiments, the Ptrc promoter consists of the nucleic acid sequence of SEQ ID NO: 254.
In embodiments, one or more of the PhaA protein, the PhaB protein, the PhaC protein and the BktB protein are catalytically active at a temperature in the range of about 30° C. to about 50° C. In embodiments, each of the PhaA protein, the PhaB protein, the PhaC protein and the BktB protein are catalytically active at a temperature in the range of about 30° C. to about 50° C. In embodiments, each of the PhaA protein, the PhaB protein, the PhaC protein and the BktB protein are catalytically active at a temperature in the range of about 37° C. to about 50° C.
In embodiments, the PhaA protein is a Cupriavidus sp. S-6 PhaA protein, a Cupriavidus gilardii QJ1 PhaA protein, or a Cupriavidus necator PhaA protein. In embodiments, the PhaA protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 241. In embodiments, the PhaA protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 241. In embodiments, the PhaA protein comprises or consists of the amino acid sequence of SEQ ID NO: 241. Further details are provided in Sheu D-S et al., Journal of bacteriology 2012, 194:2620-2629, the contents of which are incorporated herein by reference in its entirety for all purposes.
In embodiments, the nucleic acid molecule encoding a PhaA protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 248. In embodiments, the nucleic acid molecule encoding a PhaA protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 248. In embodiments, the nucleic acid molecule encoding a PhaA protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 248.
In embodiments, the PhaB protein is a Cupriavidus sp. S-6 PhaB protein, a Cupriavidus gilardii QJ1 PhaB protein, or a Cupriavidus necator PhaB protein. In embodiments, the PhaB protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 242. In embodiments, the PhaB protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 242. In embodiments, the PhaB protein comprises or consists of the amino acid sequence of SEQ ID NO: 242.
In embodiments, the nucleic acid molecule encoding a PhaB protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 249. In embodiments, the nucleic acid molecule encoding a PhaB protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 249. In embodiments, the nucleic acid molecule encoding a PhaB protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 249.
In embodiments, the PhaC protein is a Cupriavidus sp. S-6 PhaC protein, a Cupriavidus gilardii QJ1 PhaC protein, or a Cupriavidus necator PhaC protein. In embodiments, the PhaC protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 243. In embodiments, the PhaC protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 243. In embodiments, the PhaC protein comprises or consists of the amino acid sequence of SEQ ID NO: 243.
In embodiments, the nucleic acid molecule encoding a PhaC protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 250. In embodiments, the nucleic acid molecule encoding a PhaC protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 250. In embodiments, the nucleic acid molecule encoding a PhaC protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 250.
In embodiments, the BtkB protein is a Cupriavidus sp. S-6 BtkB protein, a Cupriavidus gilardii QJ1 BtkB protein, or a Cupriavidus necator BtkB protein. In embodiments, the BtkB protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 245. In embodiments, the BtkB protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 245. In embodiments, the BtkB protein comprises or consists of the amino acid sequence of SEQ ID NO: 245.
In embodiments, the nucleic acid molecule encoding a BtkB protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 251. In embodiments, the nucleic acid molecule encoding a BtkB protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 251. In embodiments, the nucleic acid molecule encoding a BtkB protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 251.
In embodiments, the bacterial host cell comprises: a first operon, comprising: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaB protein, and (c) a nucleic acid molecule encoding a PhaA protein. In embodiments, the bacterial host cell comprises: a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein and (ii) a nucleic acid molecule encoding a PhaB protein. In embodiments, the bacterial host cell comprises: a first operon, comprising: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaB protein, (c) a nucleic acid molecule encoding a PhaA protein; and a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein and (ii) a nucleic acid molecule encoding a PhaB protein.
In embodiments, the first and/or second operons comprise a promoter operably linked to the genes in the first and/or the second operons. In embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2) or the nucleic acid sequence of SEQ ID NO: 254 (Ptrc). In embodiments of the first operon, the nucleic acid molecule encoding the PhaC protein is operably linked to a promoter. In embodiments, the first operon comprises the following nucleic acid molecules in the order (i) through (iii): (i) a nucleic acid molecule encoding a PhaC protein, (ii) a nucleic acid molecule encoding a PhaA protein, and (iii) a nucleic acid molecule encoding a PhaB protein.
The disclosure further provides bacterial host cells, comprising: a first operon comprising: (a) a nucleic acid molecule encoding a PhaC protein, wherein the PhaC protein is a Cupriavidus sp. S-6 PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, wherein the PhaA protein is a Cupriavidus sp. S-6 PhaA protein, and (c) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein, wherein the BktB protein is a Cupriavidus gilardii QJ1 BktB protein, and (ii) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; and a sleeping beauty mutase (Sbm) operon comprising a promoter. In embodiments, each of the first and the second operons comprises the promoter comprising the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2).
The disclosure further provides bacterial host cells, comprising: a first operon comprising (a) a nucleic acid molecule encoding a PhaC protein, wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 250, (b) a nucleic acid molecule encoding a PhaA protein, wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 248, (c) a nucleic acid molecule encoding a PhaB protein, wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 249, and; a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein, wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 251, and (ii) a nucleic acid molecule encoding a PhaB protein, wherein the nucleic acid molecule comprises a sequence having at least 90% identity to SEQ ID NO: 249; and a sleeping beauty mutase (Sbm) operon comprises a promoter that is operably linked to the genes in the Sbm operon. In embodiments, each of the first and the second operons comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2).
In embodiments, the bacterial host cells disclosed herein are capable of converting glycerol to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV. In embodiments, the bacterial host cell is capable of converting glycerol into poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV at a temperature in the range of about 30° C. to about 50° C. In embodiments, the bacterial host cells disclosed herein are capable of converting glycerol to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV with a weight average molecular weight (Mw) of about 0.5 MDa to about 2.0 MDa, for example, about 0.6 MDa, about 0.7 MDa, about 0.8 MDa, about 0.9 MDa, about 1 MDa, about 1.1 MDa, about 1.2 MDa, about 1.3 MDa, about 1.4 MDa, about 1.5 MDa, about 1.6 MDa, about 1.7 MDa, about 1.8 MDa, about 1.9 MDa or about 2 MDa, including all subranges and values that lie therebetween. In embodiments, the bacterial host cells disclosed herein are capable of converting glycerol to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV with a weight average molecular weight (Mw) of about 1 MDa to about 1.5 MDa.
In embodiments, the bacterial host cell exhibits reduced or eliminated succinate dehydrogenase (sdhA) function. In embodiments, the bacterial host cell comprises a nucleic acid molecule encoding a fusion protein, comprising sdhA and a protease degradation tag, wherein the expression of the fusion protein is regulated by a EsaR quorum sensing system. Further details are provided in Gupta A et al., Nature biotechnology 2017, 35:273-279 and Shong J et al., ACS chemical biology 2013, 8:789-795, the contents of each of which are incorporated herein by reference in their entireties for all purposes.
In embodiments, the bacterial host cell comprises a nucleic acid molecule encoding sulA, wherein the nucleic acid molecule is operably linked to an inducible promoter. In embodiments, the inducible promoter is a temperature-inducible promoter. Further details are provided in Zhang X-C et al., Metabolic Engineering 2018, 45:32-42, and Jechlinger W, et al., Journal of biotechnology 2005, 116:11-20, the contents of each of which are incorporated herein by reference in its entirety for all purposes.
In embodiments, the bacterial host cell comprises one or more of the following: (a) a nucleic acid molecule encoding a LvaE protein, (b) a nucleic acid molecule encoding a propionate-CoA transferase, (c) a nucleic acid molecule encoding a FadE protein, (d) a nucleic acid molecule encoding a FadB protein, and (e) a nucleic acid molecule encoding a AtoB protein. In embodiments, the bacterial host cell comprises: a third operon, comprising: (a) a nucleic acid molecule encoding a FadE protein, and (b) a nucleic acid molecule encoding a FadB protein.
In embodiments, the bacterial host cell comprises: a third operon, comprising: (a) a nucleic acid molecule encoding a FadE protein, (b) a nucleic acid molecule encoding a FadB protein, and (c) a nucleic acid molecule encoding a AtoB protein. In embodiments, the bacterial host cell comprises: a fourth operon, comprising: (a) a nucleic acid molecule encoding a LvaE protein, and (b) a nucleic acid molecule encoding a propionate-CoA transferase. In embodiments, the FadE protein, the FadB protein and/or the AtoB protein are expressed in Escherichia coli str. K-12 substr. MG1655.
In some embodiments, the bacterial host cell has reduced or eliminated activity of the AtoB protein. In some embodiments, the heterologous and/or the endogenous nucleic acid sequences that encode the AtoB protein in the bacterial host cell are inactivated and/or deleted.
In embodiments, the bacterial host cell comprises: a third operon, comprising: (a) a nucleic acid molecule encoding a FadE protein, and (b) a nucleic acid molecule encoding a FadB protein; and a fourth operon, comprising: (a) a nucleic acid molecule encoding a LvaE protein, and (b) a nucleic acid molecule encoding a propionate-CoA transferase. In embodiments, the bacterial host cell comprises: a third operon, comprising: (a) a nucleic acid molecule encoding a FadE protein, (b) a nucleic acid molecule encoding a FadB protein, and (c) a nucleic acid molecule encoding a AtoB protein; and a fourth operon, comprising: (a) a nucleic acid molecule encoding a LvaE protein, and (b) a nucleic acid molecule encoding a propionate-CoA transferase.
In embodiments, the propionate CoA-transferase is a Clostridium propionicum propionate CoA-transferase (Pct(Cp)) or a Megasphaera elsdenii propionate CoA-transferase (Pct(Me)). In embodiments, the propionate CoA-transferase is a Clostridium propionicum (Pct(Cp)). Further details are provided in Zhuang Q et al. Microb Cell Fact 18, 135 (2019), the contents of which are incorporated herein by reference in its entirety for all purposes. In embodiments, the Pct(Cp) protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 30. In embodiments, the Pct(Cp) protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 30. In embodiments, the Pct(Cp) protein comprises or consists of the amino acid sequence of SEQ ID NO: 30.
In embodiments, the nucleic acid molecule encoding a Pct(Cp) protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 89. In embodiments, the nucleic acid molecule encoding a Pct(Cp) protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 89. In embodiments, the nucleic acid molecule encoding a Pct(Cp) protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 89.
In embodiments, the LvaE protein is a Pseudomonas putida LvaE protein. Further details are provided in Rand J M et al., Nature microbiology 2017, 2:1624-1634, the contents of which are incorporated herein by reference in its entirety for all purposes. In embodiments, the LvaE protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 247. In embodiments, the LvaE protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 247. In embodiments, the LvaE protein comprises or consists of the amino acid sequence of SEQ ID NO: 247.
In embodiments, the nucleic acid molecule encoding a LvaE protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 253. In embodiments, the nucleic acid molecule encoding a LvaE protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 253. In embodiments, the nucleic acid molecule encoding a LvaE protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 253.
In embodiments, the FadE protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 13. In embodiments, the FadE protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 13. In embodiments, the FadE protein comprises or consists of the amino acid sequence of SEQ ID NO: 13. In embodiments, the nucleic acid molecule encoding a FadE protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 72. In embodiments, the nucleic acid molecule encoding a FadE protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 72. In embodiments, the nucleic acid molecule encoding a FadE protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 72.
In embodiments, the FadB protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 12. In embodiments, the FadB protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 12. In embodiments, the FadB protein comprises or consists of the amino acid sequence of SEQ ID NO: 12. In embodiments, the nucleic acid molecule encoding a FadB protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 71. In embodiments, the nucleic acid molecule encoding a FadB protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 71. In embodiments, the nucleic acid molecule encoding a FadB protein comprises or consists of the the nucleic acid sequence of SEQ ID NO: 71.
In embodiments, the AtoB protein comprises an amino acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 182. In embodiments, the AtoB protein comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 182. In embodiments, the AtoB protein comprises or consists of the amino acid sequence of SEQ ID NO: 182. In embodiments, the nucleic acid molecule encoding a AtoB protein comprises a nucleic acid sequence having at least 80% (for example, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%) identity to SEQ ID NO: 191. In embodiments, the nucleic acid molecule encoding a AtoB protein comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 191. In embodiments, the nucleic acid molecule encoding a AtoB protein comprises or consists of the nucleic acid sequence of SEQ ID NO: 191.
In embodiments, each of the first, second, third and fourth operons comprises a promoter operably linked to the genes in the first, second, third and fourth operons. In embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2) or the nucleic acid sequence of SEQ ID NO: 254 (Ptrc). In embodiments, each of the first, second, third and fourth operons comprises an inducible or a constitutive promoter. In embodiments, each of the first, second and fourth operons comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 233 (Pgracmax2), and the third operon comprises a promoter comprising the nucleic acid sequence of SEQ ID NO: 254 (Ptrc).
In embodiments, the promoter comprising a Ptrc promoter. In embodiments, the promoter comprises a Pgracmax2 promoter. In embodiments, the Pgracmax2 promoter comprises a nucleic acid sequence having at least 95% (for example, about 96%, about 97%, about 98%, about 99% or about 100%) identity to SEQ ID NO: 233. In embodiments, the Pgracmax2 promoter comprises the nucleic acid sequence of SEQ ID NO: 233. In embodiments, the Pgracmax2 promoter consists of the nucleic acid sequence of SEQ ID NO: 233.
The disclosure provides bacterial host cells, comprising: a first operon comprising (a) a nucleic acid molecule encoding a PhaC protein, wherein the PhaC protein is a Cupriavidus sp. S-6 PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, wherein the PhaA protein is a Cupriavidus sp. S-6 PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; a second operon comprising: (i) a nucleic acid molecule encoding a BktB protein, wherein the BktB protein is a Cupriavidus gilardii QJ1 BktB protein, and (ii) a nucleic acid molecule encoding a PhaB protein, wherein the PhaB protein is a Cupriavidus sp. S-6 PhaB protein; a third operon, comprising: (a) a nucleic acid molecule encoding a FadE protein, (b) a nucleic acid molecule encoding a FadB protein, and (c) a nucleic acid molecule encoding a AtoB protein; a fourth operon, comprising: (a) a nucleic acid molecule encoding a LvaE protein, wherein the LvaE protein is a Pseudomonas putida LvaE protein, and (b) a nucleic acid molecule encoding a propionate-CoA transferase, wherein the propionate CoA-transferase is a Clostridium propionicum propionate CoA-transferase (Pct(Cp)), and a sleeping beauty mutase (Sbm) operon comprises an inducible promoter,
The disclosure further provides bacterial host cells, comprising:
In embodiments, the bacterial host cell exhibits reduced or eliminated function of an endogenous lacI repressor. In embodiments, the bacterial host cell comprises a deletion of the nucleic acid sequence encoding an endogenous lacI repressor. In embodiments, the bacterial host cell comprises a nucleic acid molecule encoding an enoyl-CoA hydratase/isomerase PhaJ. In embodiments, the nucleic acid molecule encoding an enoyl-CoA hydratase/isomerase PhaJ is derived from Aeromonas caviae, or a homolog thereof.
In embodiments, the bacterial host cell comprises one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding an CoA-acylating aldehyde dehydrogenase (Ald); (b) a nucleic acid molecule encoding an glutamate decarboxylase GadB; and (c) β-alanine transaminase KES23458. In embodiments, the CoA-acylating aldehyde dehydrogenase (Ald) is derived from Clostridium beijerinckii, or a homolog thereof. In embodiments, the nucleic acid molecule encoding an glutamate decarboxylase GadB is derived from E. coli or Lactobacillus senmaizukei. In embodiments, the nucleic acid molecule encoding the β-alanine transaminase KES23458 is derived from Pseudomonas sp. strain AAC.
In embodiments, the bacterial host cell is capable of converting one or more volatile fatty acids (VFAs) to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV. In embodiments, the bacterial host cell is capable of growing in a medium containing more than 100 mM VFAs. In embodiments, the bacterial host cell has a doubling time of at least about 0.1 hour−1 (1/hour) in a medium containing more than 100 mM VFAs, for example, about 0.1 hour−1 (1/hour), 0.2 hour−1, 0.3 hour−1, 0.4 hour−1, 0.5 hour−1, 0.6 hour−1, 0.7 hour−1, 0.8 hour−1, 0.9 hour−1, 1 hour−1, 2 hour−1, 3 hour−1, 4 hour−1, 5 hour−1, or about 6 hour−1 in a medium containing more than 100 mM VFAs. In embodiments, the bacterial host cell is capable of growing in a medium containing more than 225 mM VFAs. In embodiments, the bacterial host cell has a doubling time of at least about 0.1 hour−1 (1/hour) in a medium containing more than 225 mM VFAs. In embodiments, the bacterial host cell has a doubling time of at least about 0.1 hour−1 (1/hour) in a medium containing more than 225 mM VFAs, for example, about 0.1 hour−1 (1/hour), 0.2 hour−1, 0.3 hour−1, 0.4 hour−1, 0.5 hour−1, 0.6 hour−1, 0.7 hour−1, 0.8 hour−1, 0.9 hour−1, 1 hour−1, 2 hour−1, 3 hour−1, 4 hour−1, 5 hour−1, or about 6 hour−1 in a medium containing more than 225 mM VFAs.
In embodiments, the bacterial host cell is capable of growing in a medium containing a concentration of VFAs in the range of about 100 mM to about 1000 mM. In embodiments, the bacterial host cell has a doubling time of at least about 0.1 hour−1 (1/hour) in a medium containing a concentration of VFAs in the range of about 100 mM to about 1000 mM, for example, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, or about 1000 mM, including all values and subranges that lie therebetween.
In embodiments, the one or more volatile fatty acids comprises a mixture of acetate, propionate, and butyrate. In embodiments, the mixture of acetate, propionate, and butyrate comprises 50 mol % acetate, 20 mol % propionate, and 30 mol % butyrate. In embodiments, the bacterial host cell is Escherichia coli. In embodiments, at least one of the one or more nucleic acid molecules is integrated into the bacterial host cell genome. In embodiments, all of the one or more nucleic acid molecules are integrated into the bacterial host cell genome. In embodiments, the bacterial host cell comprises at least one plasmid, wherein the at least one plasmid comprises at least one of the one or more nucleic acid molecules.
In embodiments, the bacterial host cells disclosed herein may be engineered to improve glycerol uptake. For instance, In embodiments, the bacterial host cells disclosed herein may express a mutant glycerol kinase GlpK that is not inhibited by fructose bisphosphate. The mutant glycerol kinase GlpK may be expressed from constitutive or inducible promoters. Further details are provided in Kim K et al., Metabolic Engineering 2022, 69:59-72, Herring C D et al., Nature genetics 2006, 38:1406-1412, and Kang M, et al., Frontiers in microbiology 2019, 10:1845, the contents of which are incorporated herein by reference in its entirety for all purposes.
In embodiments, the bacterial host cells disclosed herein are engineered to express one or more copies of a polyhydroxyalkanoate (PHA) depolymerase.
Exemplary recombinant bacteria host cells disclosed herein are listed below in Table 10:
The disclosure provides methods of metabolizing glycerol using a bacterial host cell, the method comprising: growing bacterial host cells, comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway in a medium containing glycerol, wherein the method results in the conversion of glycerol to one or more metabolic products by the bacterial host cell. In embodiments, the medium is a liquid medium.
The disclosure provides methods of producing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the method comprising: growing bacterial host cells, comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway in a medium containing glycerol, wherein the method results in the conversion of glycerol to PHBV by the bacterial host cell.
The disclosure provides methods of producing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the method comprising: (a) growing bacterial host cells, comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway in a medium containing glycerol at a first temperature for a first period to form a bacterial culture, and (b) incubating the bacterial culture at a second temperature for a second period. In embodiments, the method results in the conversion of glycerol to PHBV by the bacterial host cell.
In embodiments, the first temperature is in a range of about 30° C. to about 37° C., for example, about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., or about 37° C., including all values and subranges that lie therebetween. In embodiments, the first temperature is about 37° C. In embodiments, the second temperature is in a range of about 37° C. to about 50° C., for example, about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., or about 50° C., including all values and subranges that lie therebetween. In embodiments, the second temperature is in a range of about 37° C. to about 45° C.
In embodiments, the first period is in the range of about 1 hour to about 24 hours. In embodiments, the first period is in the range of about 1 hour to about 16 hours. In embodiments, the first period lasts for about 16 hours to about 36 hours—for example, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours. In embodiments, the first period lasts for about 16 hours to about 24 hours. In embodiments, optical density, dissolved oxygen, or base consumption are used as metrics for determining when the growth phase is complete. Maximum optical density during growth phase may depend on a number of factors, such as, for example, inoculation density, fermentation conditions, type of spectrophotometer used for measurements, and media composition.
In embodiments, the second period is in the range of about 24 hours to about 44 hours. In embodiments, the second period is in the range of about 12 hours to about 60 hours, for example, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, or about 69 hours, including all values and subranges that lie therebetween.
In embodiments of the methods disclosed herein, the bacterial host cells are grown at a first temperature in a range of about 30° C. to about 37° C. until about the 16 hour-timepoint to about the 24 hour-timepoint to form a bacterial culture, and thereafter, incubating the bacterial culture at a second temperature until about the 48 hour-timepoint to about the 60 hour-timepoint.
In embodiments, the methods disclosed herein comprise producing PHBV from glycerol with a weight average molecular weight (Mw) of about 0.5 MDa to about 2.0 MDa, for example, about 0.6 MDa, about 0.7 MDa, about 0.8 MDa, about 0.9 MDa, about 1 MDa, about 1.1 MDa, about 1.2 MDa, about 1.3 MDa, about 1.4 MDa, about 1.5 MDa, about 1.6 MDa, about 1.7 MDa, about 1.8 MDa, about 1.9 MDa or about 2 MDa, including all subranges and values that lie therebetween. In embodiments, the methods disclosed herein comprise producing PHBV from glycerol with a weight average molecular weight (Mw) of about 1 MDa to about 1.5 MDa. In embodiments, the weight average molecular weight (Mw) is determined using gel permeation chromatography. In specific embodiments, the Mw is determined using conventional gel permeation chromatography with a single refractive index detector, against a polystyrene standard for Mw calibration. In embodiments, the medium contains more than about 0.7 g/g glycerol.
The disclosure provides methods of metabolizing volatile fatty acids (VFAs) in a bacterial medium, the method comprising: growing bacterial host cells comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, (e) a nucleic acid molecule encoding a LvaE protein, (f) a nucleic acid molecule encoding a propionate-CoA transferase, (g) a nucleic acid molecule encoding a FadE protein, (h) a nucleic acid molecule encoding a FadB protein, and (i) a nucleic acid molecule encoding a AtoB protein in a medium containing one or more volatile fatty acids (VFAs). In embodiments, the methods disclosed herein result in the conversion of VFAs to one or more metabolic products by the bacterial host cell.
The disclosure provides methods of producing poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the method comprising: growing bacterial host cells comprising one or more of the following nucleic acid molecules: (a) a nucleic acid molecule encoding a PhaC protein, (b) a nucleic acid molecule encoding a PhaA protein, (c) a nucleic acid molecule encoding a PhaB protein, and (d) a nucleic acid molecule encoding a BktB protein, (e) a nucleic acid molecule encoding a LvaE protein, (f) a nucleic acid molecule encoding a propionate-CoA transferase, (g) a nucleic acid molecule encoding a FadE protein, (h) a nucleic acid molecule encoding a FadB protein, and (i) a nucleic acid molecule encoding a AtoB protein, wherein the bacterial host cell comprises an activated sleeping beauty mutase (Sbm) pathway in a medium containing one or more volatile fatty acids (VFAs). In embodiments, the methods disclosed herein result in the conversion of VFAs to PHBV by the bacterial host cell. In embodiments, the methods disclosed herein comprise producing PHBV from VFAs with a weight average molecular weight (Mw) of about 3 MDa.
E. coli has a natural capacity to dissimilate acetate as sole carbon source, and acetate can be converted to (R)-HB-CoA. The pathway to dissimilate acetate can be manipulated, without wishing to be bound by theory, and begins with the conversion of acetate to acetyl-CoA via an acetate kinase polypeptide and a phosphate acetyltransferase AckA-Pta polypeptide (encoded by ackA-pta), an acetyl-CoA synthetase Acs or AcsA polypeptide (encoded by acs and acsA from Bacillus subtilis, respectively), and/or a propionyl-CoA synthetase PrpE polypeptide (encoded by prpE and can be derived from Salmonella enterica, Cupriavidus necator, or E. coli) followed by the fusion of two acetyl-CoA moieties to yield acetoacetyl-CoA via a β-ketothiolase BktB polypeptide or PhaA polypeptide (encoded by bktB and phaA, respectively, from C. necator). Acetoacetyl-CoA is then reduced to (R)-HB-CoA by a NADPH-dependent acetoacetyl-CoA reductase PhaB polypeptide (encoded by phaB from C. necator) or by a NADH-dependent acetoacetyl-CoA reductase PhaB(Hb) polypeptide (encoded by phaB(Hb) from Halomonas bluephagenesis TD01). Alternatively, acetate can be converted to succinate via the glyoxylate shunt, and succinate can be converted to succinyl-CoA by blocking its conversion to fumarate by knocking out or down sdhA (encoding succinate:quinone oxidoreductase, FAD binding protein SdhA).
This disclosure provides conversion of succinate to succinyl-CoA by expression of a succinyl-CoA transferase CKL_RS14680 polypeptide (encoded by CKL_RS14680 from Clostridium kluyveri), succinyl-CoA synthetase polypeptides (encoded by sucC and sucD), or a propionyl-CoA transferase YgfH polypeptide (encoded by ygfH). Without wishing to be bound by theory, the Sbm pathway is a dormant pathway in E. coli for the production of various chemicals derived from propionyl-CoA (including PHBV) using glycerol as carbon source. This disclosure also provides coupling of the Sbm pathway with pathways for VFA dissimilation to provide control over HV content, i.e. by diverting succinate produced from acetate and butyrate toward (R)-HV-CoA production. In this pathway, succinyl-CoA is converted to L-methylmalonyl-CoA by a methylmalonyl-CoA mutase Sbm polypeptide (encoded by sbm), which is subsequently converted to propionyl-CoA via a methylmalonyl-CoA decarboxylase YgfG polypeptide (encoded by ygfG). Propionyl-CoA is fused with acetyl-CoA via a PhaA polypeptide or a BktB polypeptide to yield 3-ketovaleryl-CoA, which is subsequently converted to (R)-HV-CoA via a PhaB polypeptide or a PhaB(Hb) polypeptide. On the other hand, propionate is converted directly to propionyl-CoA by a PrpE polypeptide or a propionate-CoA transferase Pct polypeptide (derived from Clostridium propionicum or Megasphaera elsdenii, i.e. Pct(Cp) or Pct (Me)), following propionate uptake into the cell by passive diffusion, or via a proline:Na+ symporter PutP polypeptide or a short-chain fatty acid transporter AtoE polypeptide (encoded by putP and atoE, respectively).
This disclosure provides conversion of butyrate to HB-CoA or succinate through distinct engineered pathways. Without wishing to be bound by theory, the first pathway may exist in natural PHA producers and begins with the uptake of butyrate into the cell by passive diffusion or a short-chain fatty acid transporter AtoE polypeptide (encoded by atoE), followed by conversion of butyrate to butyryl-CoA via a short/medium chain acyl-CoA synthetase LvaE polypeptide (encoded by lvaE from Pseudomonas putida), propionate-CoA transferase Pct polypeptide, or an acetate CoA-transferase AtoD polypeptide and an AtoA polypeptide or an acetate CoA-transferase MELS_RS00170 polypeptide and a MELS_RS00175 polypeptide (encoded by atoD and atoA, and MELS_RS00170 and MELS_RS00175 from M. elsdenii, respectively).
Butyryl-CoA is then converted to crotonyl-CoA via a short-chain acyl-CoA dehydrogenase PP_2216 polypeptide, a BC_5341 polypeptide, a MELS_RS10970 polypeptide, or a FadE polypeptide (encoded by PP_2216 from P. putida, BC_5341 from Bacillus cereus, MELS_RS10970 from M. elsdenii, and fadE, respectively), which is subsequently converted to (R)-HB-CoA via an enoyl-CoA hydratase/isomerase H16_RS27940 polypeptide, an enoyl-CoA hydratase/isomerase PhaJ polypeptide, or bifunctional protein PaaZ polypeptide (encoded by H16_RS27940 from C. necator, phaJ from Aeromonas caviae (Ac) or Aromatoleum aromaticum (Aa), and paaZ, respectively). Further details are provided in Wang X et al., Journal of biotechnology 2018, 280:62-69, the contents of which are incorporated herein by reference in its entirety for all purposes.
The bifunctional protein PaaZ polypeptide has enoyl-CoA hydratase activity that converts crotonyl-CoA to (R)-HB-CoA. Crotonyl-CoA can also be sequentially converted to (S)-HB-CoA and acetoacetyl-CoA by native multifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA epimerase/Δ3-cis-Δ2-trans-enoyl-CoA isomerase/L-3-hydroxyacyl-CoA dehydrogenase polypeptides FadB and FadJ. This disclosure provides conversion of butyrate to succinate which occurs through a synthetic pathway in which butyrate is converted to butyryl-CoA, which is then converted to butyraldehyde via a CoA-dependent propanal dehydrogenase PduP polypeptide (encoded by pduP from S. enterica, Klebsiella pneumoniae, or Listeria monocytogenes) or a CoA-acylating aldehyde dehydrogenase Ald polypeptide (encoded by ald from Clostridium beijerinckii). In parallel, without wishing to be bound by theory, L-glutamate is converted to 4-aminobutyrate by an engineered glutamate decarboxylase GadAe polypeptide, an engineered glutamate decarboxylase GadBe(Ec) polypeptide (with the same modifications as GadAe), an engineered glutamate decarboxylase GadBe(Lb) polypeptide with amino acid substitutions K17I, D294G, E312S, and Q346H (further details provided in Shi et al., Enzyme and Microbial Technology 2014, 61:35-43, the contents of which are incorporated herein by reference in its entirety for all purposes), a glutamate decarboxylase GadB(Lp) polypeptide, a glutamate decarboxylase Gad(Ls) polypeptide, or a glutamate decarboxylase Gad polypeptide (encoded by gadAe, gadBe(Ec), gadBe(Lb) from Lactobacillus brevis, gadB(Lp) from Lactobacillus plantarum, gad(Ls) from Lactobacillus senmaizukei, and gad from Arabidopsis thaliana, respectively). L-glutamate production can be enhanced by expressing a glutamate dehydrogenase GdhA polypeptide (encoded by gdhA), that converts ketoglutarate to L-glutamate, for increased 4-aminobutyrate production (further details are provided in Soma Y et al., Metabolic Engineering 2017, 43:54-63, the contents of which are incorporated herein by reference in its entirety for all purposes). This disclosure provides conversion of butyraldehyde and 4-aminobutyrate to succinate semialdehyde via a β-alanine transaminase KES23458 polypeptide (encoded by FG99_15380 from Pseudomonas sp. strain AAC). Succinate semialdehyde is oxidized to succinate by a NADP+-dependent succinate semialdehyde dehydrogenase GabD polypeptide (encoded by gabD). (R)-HB-CoA and (R)-HV-CoA are polymerized by a short-chain polyhydroxyalkanoate synthase PhaC polypeptide (encoded by phaC from C. necator) to yield PHBV. PhaC mutants are also useful for polymerizing (R)-HB-CoA and (R)-HV-CoA. For example, PhaC(F420S) (SEQ ID NO: 226) can dimerize at a faster rate relative to wild-type PhaC [25], and the PhaC(G4D) mutation (SEQ ID NO: 230) increases soluble expression relative to wild-type PhaC [26]. These are beneficial attributes for increasing PHBV biosynthesis and molecular weight.
Further details are provided in Tang C-D, et al., International Journal of Biological Macromolecules 2020, 160:372-379; and Ho NAT, et al., Journal of Bioscience and Bioengineering 2013, 115:154-158, Yin J, et al., Applied microbiology and biotechnology 2015, 99:5523-5534, Phan T T P, et al., Journal of biotechnology 2012, 157:167-172, Olins P O, et al., Journal of Biological Chemistry 1989, 264:16973-16976, Arab B, et al., Fermentation 2023, 9:14, Puigbo P et al., Nucleic acids research 2007, 36:D524-D527, Agus J, et al., Polymer degradation and stability 2006, 91:1138-1146; Normi Y M, et al., Macromolecular bioscience 2005, 5:197-206, Chinese Patent Application CN105063790A, International Patent Application WO1990000067A1, the contents of each which are incorporated herein by reference in its entirety for all purposes.
In embodiments, the Pct polypeptide comprises a Pct(Cp) polypeptide or a Pct(Me) polypeptide. In embodiments, the PduP polypeptide comprises a PduP(Kp) polypeptide or a PduP(Se) polypeptide. In embodiments, the recombinant bacterial cell further comprises a proline:Na+ symporter, optionally a PutP polypeptide, or a short-chain fatty acid transporter, optionally an AtoE polypeptide.
In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyrate to butyryl-CoA. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyryl-CoA to butyraldehyde. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyraldehyde and optionally 4-aminobutyrate to succinate semialdehyde. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of succinate semialdehyde to succinate. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of L-glutamate to 4-aminobutyrate. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of butyryl-CoA to crotonyl-CoA. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of crotonyl-CoA to 3-hydroxybutyryl-CoA. In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding a polypeptide that catalyzes the conversion of succinate to succinyl-CoA.
In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding at least one, at least two, at least three, at least four, or at least five of a polypeptide that catalyzes the conversion of butyrate to butyryl-CoA, a polypeptide that catalyzes the conversion of butyryl-CoA to butyraldehyde, a polypeptide that catalyzes the conversion of butyraldehyde and 4-aminobutyrate to succinate semialdehyde, a polypeptide that catalyzes the conversion of succinate semialdehyde to succinate, and a polypeptide that catalyzes the conversion of L-glutamate to 4-aminobutyrate.
In embodiments, the recombinant bacterial cell comprises at least one recombinant nucleic acid molecule encoding at least one, at least two, or at least three of a polypeptide that catalyzes the conversion of butyrate to butyryl-CoA, a polypeptide that catalyzes the conversion of butryryl-CoA to crotonyl-CoA, and a polypeptide that catalyzes the conversion of crotonyl-CoA to 3-hydroxybutyryl-CoA.
In a specific embodiment, the recombinant bacterial cell for producing PHBV comprises:
In embodiments, the recombinant bacterial cell further comprises a glutamate decarboxylase polypeptide, optionally a GadAe polypeptide, a GadBe(Ec) polypeptide, a GadBe(Lb) polypeptide, a GadB(Lp) polypeptide, a Gad(Ls) polypeptide, or a Gad polypeptide. In embodiments, the recombinant bacterial cell further comprises a second β-ketothiolase polypeptide, optionally a PhaA polypeptide. In embodiments, the recombinant bacterial cell further comprises a succinyl-CoA transferase polypeptide, optionally a CKL_RS14680 polypeptide, or succinyl-CoA synthetase polypeptides, optionally a SucC polypeptide and a SucD polypeptide.
In embodiments, the recombinant bacterial cell comprises a Pct(Cp) polypeptide, an LvaE polypeptide, a PhaJ(Ac) polypeptide, a FadE polypeptide, a GadAe polypeptide, a FG99_15380 polypeptide, a PduP(Se) polypeptide, a GabD polypeptide, a CKL_RS14680 polypeptide, and an AtoC(Con) polypeptide comprising a serine at the position corresponding to position 129 of SEQ ID NO: 203. In some embodiment, the recombinant bacterial cell further comprises a PhaC polypeptide, a PhaB polypeptide, a BktB polypeptide, and a PhaA polypeptide.
In embodiments, the nucleic acid molecule described herein is optionally a heterologous nucleic acid molecule having a nucleic acid sequence encoding a recombinant polypeptide described herein. In embodiments, the recombinant bacterial cell comprises stably incorporated into the genome a heterologous nucleic acid molecule having a nucleic acid sequence encoding a recombinant polypeptide described herein.
The bacterial strain described herein can include heterologous nucleic acid that contains transcriptional and translational regulatory elements. For example, transcriptional regulatory elements can include promoter such as Pgracmax2 and transcriptional terminator, and translational regulatory elements can include ribosomal binding site (RBS) such as RBS from gene 10 of Phage T7 (T7.RBS) that can significantly enhance translation efficiency relative to the consensus RBS of E. coli. Translation efficiency may also be enhanced by combining other RBSs, e.g. the consensus Gram-positive RBS (i.e. AAGGAGG), with a nine bp sequence derived from T7.RBS (i.e. TTAACTTTA) to facilitate base-pairing with the 16S rRNA of E. coli (e.g. RBS1). In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having promoter Pgracmax2. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having translational regulatory element T7.RBS. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having promoter Pgracmax2 and at least one translational regulatory element. In embodiments, the at least one translational regulatory element is T7.RBS, Gram-positive RBS, or RBS1. In embodiments, the at least one translational regulatory element is combined T7.RBS and Gram-positive RBS. In embodiments, the at least one translational regulatory element is combined T7.RBS and Gram-positive RBS, and RBS1. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 232. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 233. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 234. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 235. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 236. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 233, 234, and 236. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 232 and 236. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 237. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NOs: 233, 234, 236, and 237. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NOs: 232, 236, and 237. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having a transcriptional terminator. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 238. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having Pgracmax2, combined T7.RBS and Gram-positive RBS, RBS1, and transcriptional terminator. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NOs: 233, 234, 236, and 238. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NOs: 232, 236, and 238. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 239. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NO: 240. In embodiments, the recombinant bacterial cell comprises a nucleic acid molecule having the sequence of SEQ ID NOs: 239 and 240. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 239 is integrated into a nonessential gene locus. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 239 is integrated into the bcsA locus. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 240 is integrated into a nonessential gene locus. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 240 is integrated into the intF locus. In embodiments, the nucleic acid molecule is integrated into one or more loci of bacterial strain CPC-Sbm. In embodiments, the nucleic acid molecule is integrated into one or more loci of K-12 derived bacterial strain. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 239 is integrated into the bcsA locus of strain CPC-Sbm and the nucleic acid molecule having the sequence of SEQ ID NO: 240 is integrated into the intF locus of strain CPC-Sbm. In embodiments, the nucleic acid molecule having the sequence of SEQ ID NO: 236 is integrated into the bcsA locus of K-12 derived strain and the nucleic acid molecule having the sequence of SEQ ID NO: 240 is integrated into the intF locus of K-12 derived strain. In embodiments, the nucleic acid molecule comprises Pgracmax2::(T7.RBS)bktB:(RBS1)phaB.
In embodiments, the nucleic acid molecule comprises Pgracmax2::(T7.RBS)phaC:(RBS1)phaA. In embodiments, the nucleic acid molecule comprises Pgracmax2::(T7.RBS)bktB:(RBS1)phaB) and (Pgracmax2::(T7.RBS)phaC:(RBS1)phaA. In embodiments, the recombinant bacterial strain is CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB), intF::(Pgracmax2::(T7.RBS)phaC:(RBS1)phaA).
The expression of recombinant polypeptide in a particular bacteria species can be improved by codon optimization. In some examples described herein, codon optimization was completed by first optimizing a gene sequence for expression in E. coli K12 using the Codon Optimization Tool provided by Integrated DNA Technologies (USA), followed by further optimization of the optimized sequence via the OPTIMIZER web server using the “guided random” method that is based on a Monte Carlo algorithm (further details are provided in Puigbo P et al., Nucleic acids research 2007, 36:D524-D527, and Puigbo P et al., Nucleic acids research 2007, 35:W126-W131, the contents of which are incorporated herein by reference in its entirety for all purposes). Finally, manual adjustments were made to the sequence resulting from the second optimization procedure using the codon frequency table for E. coli K12 from the Codon Usage Database (as provided at Nakamura Y, et al., Nucleic acids research 2000, 28:292-292) as a reference and the manual optimization option found in the Codon Optimization Tool provided by Integrated DNA Technologies. In embodiments, the heterologous nucleic acid molecule has an optimized nucleic acid sequence for encoding a recombinant polypeptide described herein for expression in a bacterial cell described herein.
Amino acid sequences described herein are set out in Table 1.
E.coli with the
In embodiments, the recombinant bacterial cell for producing PHBV comprises at least one polypeptide having an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to any one of SEQ ID NO: 1-26, 28-38, 40-59, 172-173, 176-184, 194-203, 215-217, 224-226, and 230, or a polypeptide having an accession no. shown in Table 6. In embodiments, the polypeptide is a recombinant polypeptide. In embodiments, the acyl-CoA synthetase has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 26, the acetate CoA-transferase polypeptides having an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 4 and 5 or 172 and 173, or a polypeptide having an accession no. WP_053001645.1, QGU62017.1, WP_155555734.1, WP_038355059.1, MLY49728.1, WP_105269001.1, WP_105284960.1, WP_149476985.1, WP_108188772.1, WP_000850520.1, WP_138957179.1, WP_123267594.1, WP_114680602.1, WP_047500919.1, or WP_004184954.1, and the propionate-CoA transferase polypeptide has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 30 or 31 or a polypeptide having an accession no. WP_066087637.1, NCC15629.1, WP_054329786.1, WP_072853413.1, CDC28613.1, WP_016408311.1, WP_088107724.1, WP_160302233.1, WP_004038625.1, WP_054336166.1, WP_036203125.1, WP_044502862.1, WP_065360594.1, KXA66894.1, WP_095629974.1, WP_087478516.1, WP_107195767.1, WP_048515067.1, WP_101912966.1, WP_156208970.1, KXB92430.1, WP_023053187.1, WP_039891686.1, or KXB92214.1. In embodiments, the PutP polypeptide has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 195. In embodiments, the AtoE polypeptide has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 6. In embodiments, the first β-ketothiolase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 8, or a polypeptide having an accession no. WP_013956457.1, WP_035820088.1, WP_092317205.1, WP_115013782.1, WP_116382528.1, WP_018311404.1, WP_063238655.1, WP_116321050.1, AGW89814.1, WP_062798985.1, WP_133094381.1, AGW95651.1, WP_140952189.1, WP_144195740.1, or WP_011516125.1. In embodiments, the NADPH-dependent acetoacetyl-CoA reductase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% 99.4% 99.5% 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 35, or a polypeptide having an accession no. RWA53825.1, WP_042885115.1, WP_039016191.1, WP_116336746.1, WP_112777371.1, WP_006577377.1, WP_135705030.1, WP_133096842.1, WP_124684436.1, WP_116321053.1, WP_006155939.1, WP_045241722.1, WP_011297519.1, WP_144195744.1, or ODV43053.1. In embodiments, the NADH-dependent acetoacetyl-CoA reductase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 225, or a polypeptide having an accession no. WP_162219671.1, WP_126946472.1, WP_120385833.1, WP_030074446.1, WP_188637499.1, WP_058579713.1, WP_083023226.1, WP_039183428.1, WP_159340906.1, or WP_096653461.1. In embodiments, the short-chain polyhydroxyalkanoate synthase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 36, 226, or 230, or a polypeptide having an accession no. ACZ57807.1, WP_010810133.1, WP_013956451.1, AAW65074.1, WP_018311399.1, AGW89808.1, WP_115678329.1, WP_062798976.1, WP_115013788.1, or WP_115680054.1, WP_112777370.1. In embodiments, the CoA-dependent propanal dehydrogenase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 32 or 33, or a polypeptide having an accession no. WP_109231734.1, WP_109848747.1, WP_136028274.1, WP_100680758.1, WP_100631313.1, WP_049157539.1, WP_029884370.1, MXH33721.1, WP_144232363.1, WP_153679752.1, WP_148849915.1, EBS2830838.1, WP_112213940.1, WP_064370270.1, WP_001097684.1, WP_001528442.1, WP_080203692.1, WP_108450871.1, WP_009652778.1, WP_142983670.1, WP_105274032.1, WP_070556870.1, WP_142502560.1, WP_012131760.1, WP_012906342.1, WP_006683971.1, WP_103775053.1, WP_060570657.1, or WP_135321437.1, the β-alanine transaminase polypeptide has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 15 or 16, or a polypeptide having an accession no. WP_116425784.1, WP_069862932.1, WP_043315988.1, WP_009614288.1, WP_089392503.1, WP_109934365.1, WP_090268322.1, WP_138519936.1, WP_138213347.1, WP_015474919.1, WP_043256620.1, WP_084311461.1, WP_053816481.1, WP_070656248.1, or WP_077524299.1, or the NADP+-dependent succinate semialdehyde dehydrogenase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 17 or a polypeptide having an accession no. WP_105285925.1, WP_135494970.1, WP_094315749.1, WP_161983589.1, WP_000772895.1, WP_078167276.1, WP_016249103.1, WP_105267583.1, WP_149461599.1, WP_128880059.1, WP_149461599.1, WP_060773285.1, WP_153257801.1, or WP_108418849.1, WP_045446520.1. In embodiments, the short-chain acyl-CoA dehydrogenase polypeptide has an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 38, 7, 28, or 13, or a polypeptide having accession no. WP_003250094.1, WP_104887321.1, WP_039614175.1, WP_023662689.1, WP_085706434.1, WP_070087269.1, WP_060512757.1, WP_144171976.1, WP_054884005.1, WP_051100719.1, WP_099814118.1, WP_125859423.1, WP_125464833.1, WP_090345830.1, WP_110994568.1, WP_088022147.1, WP_098448816.1, WP_149216716.1, WP_101167410.1, WP_143881711.1, WP_085450733.1, WP_144504985.1, BCA34359.1, WP_098299175.1, WP_071710801.1, CKE48212.1, WP_163095898.1, WP_071725959.1, WP_136445333.1, WP_128975345.1, WP_020723925.1, WP_048514244.1, WP_074501184.1, KXB91325.1, WP_154877386.1, WP_107195291.1, WP_087477538.1, WP_095630133.1, WP_091647756.1, WP_023053225.1, WP_101912630.1, WP_075572446.1, WP_006790232.1, WP_006942404.1, WP_094316844.1, WP_130224094.1, WP_135404353.1, WP_046076114.1, WP_011069257.1, WP_135489829.1, WP_085448671.1, WP_124782953.1, WP_153879457.1, EDR1571704.1, WP_103776898.1, WP_008783785.1, WP_087053141.1, WP_079225425.1, or WP_137366593.1, WP_000973041.1, and the enoyl-CoA hydratase/isomerase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 22, 37, or 196, or a polypeptide having accession no. WP_051591491.1, WP_114130480.1, WP_078200706.1, EON20731.1, PKO64515.1, WP_092007571.1, WP_162566377.1, WP_137921632.1, WP_162591754.1, WP_103260220.1, WP_104454254.1, OJW67134.1, WP_041998622.1, WP_043760202.1, WP_043129860.1, WP_042076944.1, WP_100860962.1, WP_163157368.1, WP_042638062.1, WP_106886672.1, WP_033131291.1, WP_025327110.1, WP_040094291.1, WP_139745378.1, WP_169200570.1, WP_053422493.1, WP_169118971.1, WP_169202263.1, AUL99438.1, WP_136349851.1, WP_136385326.1, WP_187719679.1, or WP_107493682.1, WP_169262136.1. In embodiments, the propionyl-CoA synthetase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 43, 44, or 45, or a polypeptide having an accession no. WP_081623799.1, WP_115213214.1, WP_082818978.1, WP_116324638.1, WP_092309442.1, AMR79067.1, WP_151072146.1, WP_029046365.1, AGW91162.1, WP_116321975.1, WP_039006728.1, WP_092134378.1, WP_109580644.1, WP_035882297.1, WP_149135646.1, WP_024249411.1, WP_130258507.1, WP_000010307.1, WP_138159881.1, WP_105281240.1, WP_000010239.1, WP_000010244.1, WP_160524152.1, WP_105270931.1, WP_160530253.1, WP_016235155.1, WP_061090735.1, WP_103014998.1, WP_094761423.1, ATX90159.1, WP_127836169.1, WP_103776706.1, WP_044259075.1, WP_012904755.1, WP_043015332.1, WP_008783866.1, WP_153690685.1, WP_058587683.1, WP_101700584.1, WP_042324663.1, WP_123268908.1, WP_137351112.1, WP_048219548.1, or WP_160955604.1, WP_012133646.1. In embodiments, the glutamate decarboxylase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 19, 20, 194, 216, 217, or 224, or a polypeptide having an accession no. XP_002871761.1, KFK41557.1, VVB14898.1, RID41892.1, XP_013661825.1, VDC86651.1, XP_006400267.1, XP_010420446.1, XP_010453919.1, CAA7061503.1, XP_006400266.1, ESQ41721.1, XP_013627326.1, XP_031273023.1, WP_134806912.1, WP_052942456.1, WP_128881419.1, WP_135383171.1, WP_054518524.1, WP_138158972.1, WP_103194808.1, WP_000358851.1, WP_107164449.1, WP_000358937.1, WP_135385956.1, WP_113623060.1, EAB0955940.1, WP_134806912.1, WP_052942456.1, WP_128881419.1, WP_135383171.1, WP_054518524.1, WP_138158972.1, WP_103194808.1, WP_000358851.1, WP_107164449.1, WP_000358937.1, WP_135385956.1, WP_113623060.1, EAB0955940.1, WP_125641322.1, WP_226457942.1, BAN05709.1, MBL3537851.1, WP_039105805.1, WP_052957185.1, KIR08754.1, WP_125574762.1, WP_063488771.1, or WP_017262688.1. In embodiments, the glutamate dehydrogenase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% 99.4% 99.5% 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 215. In embodiments, the second β-ketothiolase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 34, or a polypeptide having an accession no. WP_013956452.1, SCU96900.1, WP_035820078.1, 4O9C_A, WP_116382525.1, WP_092317196.1, WP_062798979.1, WP_116321054.1, AGW89809.1, WP_039016192.1, WP_063238652.1, WP_029049660.1, WP_011297518.1, WP_124684437.1, or WP_109580845.1. In embodiments, the succinyl-CoA transferase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 10 or a polypeptide having an accession no. WP_073539834.1, or WP_010236491.1, or the succinyl-CoA synthetase polypeptides having an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 50 and 51 or a polypeptide having an accession no. WP_111780024.1, WP_105268114.1, WP_149508492.1, EBH0782533.1, WP_079789068.1, EAA0703253.1, WP_001048612.1, WP_103776364.1, HAC6539881.1, WP_139538723.1, WP_040076526.1, WP_152308781.1, WP_061708388.1, WP_159152251.1, WP_159754306.1, WP_148048643.1, WP_161983406.1, WP_128882005.1, SEK68167.1, WP_064567804.1, WP_090133347.1, EDS6037479.1, WP_015965312.1, WP_154777294.1, WP_108473875.1, WP_162082208.1, or WP_154158334.1. In embodiments, the CoA-acylating aldehyde dehydrogenase polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 184 or a polypeptide having an accession no. WP_077830381.1, WP_065419149.1, WP_017211959.1, WP_077844109.1, AAD31841.1, WP_087702529.1, WP_077868466.1, WP_077366605.1, WP_026888070.1, WP_077860531.1, WP_022747467.1, WP_077863550.1, WP_009171375.1, WP_128214949.1, WP_160679606.1, WP_012059995.1, WP_041898834.1, or WP_015395720.1. In embodiments, the bifunctional protein polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 29 or a polypeptide having an accession no. WP_160599600.1, WP_152066042.1, WP_094316530.1, WP_032252644.1, WP_001186464.1, WP_125401136.1, WP_001186494.1, WP_119163289.1, WP_095281943.1, WP_045888522.1, WP_058840681.1, WP_095440732.1, WP_162382197.1, WP_059385322.1, or WP_045286529.1.
In embodiments, the recombinant bacterial cell for producing PHBV comprises a recombinant nucleic acid molecule having at least 75% sequence identity to at least one, two, three, four, five, six, seven, eight, or nine of SEQ ID NO: 89, 85, 97, 96, 79, 93, 94, 95, 67, 228, 229, and 231, optionally wherein the recombinant bacterial cell comprises inactivation of iclR, optionally inactivation of SdhA, optionally wherein the recombinant bacterial cell comprises inactivation of at least one nonessential gene.
In embodiments, the recombinant bacterial cell for producing PHBV comprises a recombinant nucleic acid molecule having at least 75% sequence identity to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve of SEQ ID NO: 89, 85, 97, 96, 79, 74, 92, 76, 93, 94, 95, 67, 228, 229, and 231, optionally wherein the recombinant bacterial cell comprises inactivation of iclR, optionally inactivation of SdhA, optionally wherein the recombinant bacterial cell comprises inactivation of at least one nonessential gene. In embodiments, the at least one nonessential gene is a nucleic acid molecule encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 21, 18, 47, 12, 14, 13, 53, 58, 52, 54, 176, 177, 178, 179, 180, 181, 182, 183, 40, 41, 42, 197, 198, 199, 200, 201, and 202.
For example, fadR is a nonessential gene that can be inactivated without significantly affecting cell viability, said inactivation of fadR would derepress expression of fadE, and the derepression of fadE facilitates the conversion of butyryl-CoA to crotonyl-CoA. Further details are provided in Jenkins L S et al., Journal of Bacteriology 1987, 169:42-52, the contents of which are incorporated herein by reference in its entirety for all purposes. Cell viability can be measured, for example, by BacTiter-Glo™, LIVE/DEAD™ BacLight™ Bacterial Viability assay, or LIVE BacLight™ Bacterial Gram Stain, where cells with inactivated genes having +/−25% viability on a quantifiable index as compared to parental and/or wildtype are considered to be not significantly affected. In embodiments, the recombinant bacterial cell comprises inactivation of FadR. In embodiments, the FadR comprises a nucleic acid molecule encoding a polypeptide having an amino acid sequence of SEQ ID NO: 201. In embodiments, the FadR comprises a nucleic acid molecule having a nucleic acid sequence of SEQ ID NO: 211.
In embodiments, the recombinant bacterial cell for producing PHBV comprises a recombinant nucleic acid molecule having at least 75% sequence identity to at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of SEQ ID NO: 89, 85, 97, 96, 79, 74, 92, 76, 69, 93, 94, 95, 67, 228, 229, and 231, optionally wherein the recombinant bacterial cell comprises inactivation of iclR.
In addition, AtoC(Con) which is a DNA-binding transcriptional activator/ornithine decarboxylase inhibitor that activates transcription of the atoDAEB operon for enhanced VFA uptake and conversion to acyl-CoAs, can be mutated at position 129 from isoleucine to serine to confer constitutive expression of the atoDAEB operon. Accordingly, In embodiments, the recombinant bacterial cell for producing PHBV comprises a DNA-binding transcriptional activator/ornithine decarboxylase inhibitor, optionally an AtoC polypeptide. Further details are provided in Pauli G et al. European Journal of Biochemistry 1972, 29:553-562, the contents of which are incorporated herein by reference in its entirety for all purposes. In embodiments, the AtoC polypeptide has an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 203, wherein the AtoC(Con) polypeptide comprises a serine at the position corresponding to position 129 of SEQ ID NO: 203.
The presence of acetate or butyrate can affect bacterial cell viability. Expression of small noncoding RNAs, such as DsrA, RprA and ArcZ, can increase the tolerance of bacterial cells to the presence of acetate and/or butyrate. In embodiments, the recombinant bacterial cell for producing PHBV comprises noncoding RNAs, optionally DsrA, RprA, or ArcZ. In embodiments, the recombinant bacterial cell for producing PHBV comprises noncoding RNA DsrA, noncoding RNA RprA, and noncoding RNA ArcZ. In embodiments, the recombinant bacterial cell for producing PHBV comprises a DNA nucleic acid molecule having nucleic acid sequence encoding for noncoding RNA DsrA, RprA, or ArcZ. In embodiments, the recombinant bacterial cell for producing PHBV comprises a DNA nucleic acid molecule having nucleic acid sequence encoding for noncoding RNA DsrA, RprA, and ArcZ. In embodiments, the recombinant bacterial cell for producing PHBV comprises a nucleic acid molecule having nucleic acid sequence of SEQ ID NO: 27, 39, or 214. In embodiments, the recombinant bacterial cell for producing PHBV comprises a nucleic acid molecule having nucleic acid sequence of SEQ ID NO: 27, 39, and 214. In embodiments, the recombinant bacterial cell for producing PHBV comprises a nucleic acid molecule having nucleic acid sequence of SEQ ID NO: 221, a nucleic acid molecule having nucleic acid sequence of SEQ ID NO: 222, and a nucleic acid molecule having nucleic acid sequence of SEQ ID NO: 223.
Exemplary nucleic acid sequences described herein are set out in Table 2, Table 3A, Table 3B, Table 3C, Table 3D, and Table 4.
E.coli
E.coli
E.coli
In embodiments, the recombinant bacterial cell for producing PHBV comprises at least one nucleic acid molecule having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3% 99.4% 99.5% 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to any one of SEQ ID NO: 60-118, 174-175, 185-193, 204-213, 218-220, 227-229, and 231, or a complementary sequence thereof, or a segment thereof. In embodiments, the at least one nucleic acid molecule described herein is optionally a heterologous nucleic acid molecule having a nucleic acid sequence encoding a recombinant polypeptide described herein. In embodiments, the acyl-CoA synthetase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 85 or 86, the acetate CoA-transferase polypeptides are encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 63 and 64 or 174 and 175, the propionate-CoA transferase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 89 or 90. In embodiments, the PutP polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 205. In embodiments, the AtoE polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 65. In embodiments, the first β-ketothiolase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 67. In embodiments, the NADPH-dependent acetoacetyl-CoA reductase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 94. In embodiments, the NADH-dependent acetoacetyl-CoA reductase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 228. In embodiments, the short-chain polyhydroxyalkanoate synthase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 95, 229, or 231. In embodiments, the CoA-dependent propanal dehydrogenase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 91 or 92, the β-alanine transaminase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 74 or 75, or the NADP+-dependent succinate semialdehyde dehydrogenase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 76. In embodiments, the short-chain acyl-CoA dehydrogenase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 97, 98, 66, 87, or 72, and the enoyl-CoA hydratase/isomerase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 81, 96, or 206. In embodiments, the propionyl-CoA synthetase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 102, 103, or 104. In embodiments, the glutamate decarboxylase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 78, 79, 204, 219, 220, or 227. In embodiments, the glutamate dehydrogenase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 218. In embodiments, the second p-ketothiolase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 93. In embodiments, the succinyl-CoA transferase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 69. In embodiments, the succinyl-CoA synthetase polypeptides are encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 109 and 110. In embodiments, the CoA-acylating aldehyde dehydrogenase polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 193. In embodiments, the bifunctional protein polypeptide is encoded by a nucleic acid molecule, optionally a heterologous nucleic acid molecule, having a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to SEQ ID NO: 88. In embodiments, the at least one heterologous nucleic acid molecule encoding a polypeptide is operably linked to a promoter capable of expressing a heterologous nucleic acid sequence encoding the recombinant polypeptide in a bacterial cell.
Also provided is a plasmid comprising nucleic acid sequence described herein. In embodiments, the plasmid comprises a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to any one of SEQ ID NO: 162-171.
In an aspect, the heterologous nucleic acid molecule or plasmid is codon-optimized for expression in a bacterial cell described herein. In embodiments, the bacterial cell is selected from the group consisting of Escherichia coli, optionally strain K-12 or a derivative thereof, optionally CPC-Sbm or a derivative thereof, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Lactococcus lactis, Pseudomonas putida, Cupriavidus necator, Cupriavidus gilardii, Cupriavidus sp. S-6, and Lactobacillus reuteri.
In embodiments, the nucleic acid molecule comprises an isolated and/or purified nucleic acid molecule. In embodiments, a nucleic acid molecule, a plasmid, or an expression system comprising these isolated and/or purified nucleic acid molecules, may be used to create a recombinant bacterial cell that produces polypeptides which catalyze the synthesis of PHBV. Therefore, some embodiments relate to a recombinant bacterial cell comprising a nucleic acid molecule, a plasmid, or an expression system having at least one of SEQ ID NO: 60-118, 162-170, 185-193, 204-213, 218-220, 227-229, and 231, or having at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9% sequence identity to at least one of SEQ ID NO: 60-118, 162-170, 185-193, 204-213, 218-220, 227-229, and 231.
A person of ordinary skill in the art would readily understand that the disclosed polypeptide amino acid and nucleic acid sequences may be used interchangeably with any of their corresponding homologs. For example, In embodiments, the recombinant bacterial cell for producing PHBV comprises at least one nucleic acid molecule encoding a polypeptide corresponding to any of the homologs listed in Table 6. In embodiments, a homolog of AckA comprises a polypeptide having an accession no. WP_151250307.1, WP_025758333.1, WP_000095714.1, WP_094316684.1, WP_000095699.1, WP_059270696.1, WP_160523843.1, WP_108188758.1, WP_000095694.1, WP_079781741.1, WP_000095691.1, WP_162383091.1, WP_110248734.1, WP_016529145.1, or WP_064543869.1. In embodiments, a homolog of Acs comprises a polypeptide having an accession no. WP_094321046.1, WP_134796521.1, WP_000078234.1, WP_000078255.1, WP_160523940.1, WP_130258462.1, WP_135490640.1, WP_000078187.1, WP_000078188.1, WP_105283185.1, WP_079225661.1, WP_151218054.1, EAX3726079.1, WP_061075561.1, or WP_087051807.1. In embodiments, a homolog of Ald comprises a polypeptide having an accession no. WP_077830381.1, WP_065419149.1, WP_017211959.1, WP_077844109.1, AAD31841.1, WP_087702529.1, WP_077868466.1, WP_077366605.1, WP_026888070.1, WP_077860531.1, WP_022747467.1, WP_077863550.1, WP_009171375.1, WP_128214949.1, WP_160679606.1, WP_012059995.1, WP_041898834.1, or WP_015395720.1. In embodiments, a homolog of AcsA comprises a polypeptide having an accession no. WP_047183033.1, WP_144459203.1, WP_071577026.1, WP_061186774.1, WP_075747112.1, WP_010329597.1, WP_024714615.1, WP_162101126.1, WP_105990205.1, WP_061572550.1, WP_109567131.1, WP_061523123.1, or WP_103526694.1. In embodiments, a homolog of AtoA comprises a polypeptide having an accession no. WP_103053735.1, WP_137325583.1, WP_050899668.1, WP_000339071.1, WP_128880225.1, WP_047462387.1, WP_135321227.1, WP_090049661.1, WP_004184955.1, WP_151219893.1, WP_100682748.1, WP_013365500.1, WP_000339048.1, or WP_087857377.1. In embodiments, a homolog of AtoD comprises a polypeptide having an accession no. WP_053001645.1, QGU62017.1, WP_155555734.1, WP_038355059.1, MLY49728.1, WP_105269001.1, WP_105284960.1, WP_149476985.1, WP_108188772.1, WP_000850520.1, WP_138957179.1, WP_123267594.1, WP_114680602.1, WP_047500919.1, or WP_004184954.1. In embodiments, a homolog of BC_5341 comprises a polypeptide having an accession no. WP_088022147.1, WP_098448816.1, WP_149216716.1, WP_101167410.1, WP_143881711.1, WP_085450733.1, WP_144504985.1, BCA34359.1, WP_098299175.1, WP_071710801.1, CKE48212.1, WP_163095898.1, WP_071725959.1, WP_136445333.1, or WP_128975345.1. In embodiments, a homolog of BktB comprises a polypeptide having an accession no. WP_013956457.1, WP_035820088.1, WP_092317205.1, WP_115013782.1, WP_116382528.1, WP_018311404.1, WP_063238655.1, WP_116321050.1, AGW89814.1, WP_062798985.1, WP_133094381.1, AGW95651.1, WP_140952189.1, WP_144195740.1, or WP_011516125.1. In embodiments, a homolog of PhaC comprises a polypeptide having an accession no. ACZ57807.1, WP_010810133.1, WP_013956451.1, AAW65074.1, WP_018311399.1, AGW89808.1, WP_115678329.1, WP_062798976.1, WP_115013788.1, WP_115680054.1, or WP_112777370.1. In embodiments, a homolog of CKL_RS14680 comprises a polypeptide having an accession no. WP_073539834.1 or WP_010236491.1. In embodiments, a homolog of FadE comprises a polypeptide having an accession no. WP_094316844.1, WP_130224094.1, WP_135404353.1, WP_046076114.1, WP_011069257.1, WP_135489829.1, WP_085448671.1, WP_124782953.1, WP_153879457.1, EDR1571704.1, WP_103776898.1, WP_008783785.1, WP_087053141.1, WP_079225425.1, WP_137366593.1, or WP_000973041.1. In embodiments, a homolog of PhaJ(Aa) comprises a polypeptide having an accession no. WP_169200570.1, WP_053422493.1, WP_169118971.1, WP_169202263.1, AUL99438.1, WP_136349851.1, WP_136385326.1, WP_187719679.1, WP_107493682.1, or WP_169262136.1. In embodiments, a homolog of GabD comprises a polypeptide having an accession no. WP_105285925.1, WP_135494970.1, WP_094315749.1, WP_161983589.1, WP_000772895.1, WP_078167276.1, WP_016249103.1, WP_105267583.1, WP_149461599.1, WP_128880059.1, WP_149461599.1, WP_060773285.1, WP_153257801.1, WP_108418849.1, or WP_045446520.1. In embodiments, a homolog of Gad comprises a polypeptide having an accession no. XP_002871761.1, KFK41557.1, VVB14898.1, RID41892.1, XP_013661825.1, VDC86651.1, XP_006400267.1, XP_010420446.1, XP_010453919.1, CAA7061503.1, XP_006400266.1, ESQ41721.1, XP_013627326.1, or XP_031273023.1. In embodiments, a homolog of GadAe comprises a polypeptide having an accession no. WP_134806912.1, WP_052942456.1, WP_128881419.1, WP_135383171.1, WP_054518524.1, WP_138158972.1, WP_103194808.1, WP_000358851.1, WP_107164449.1, WP_000358937.1, WP_135385956.1, WP_113623060.1, or EAB0955940.1. In embodiments, a homolog of GadBe(Ec) comprises a polypeptide having an accession no. WP_134806912.1, WP_052942456.1, WP_128881419.1, WP_135383171.1, WP_054518524.1, WP_138158972.1, WP_103194808.1, WP_000358851.1, WP_107164449.1, WP_000358937.1, WP_135385956.1, WP_113623060.1, or EAB0955940.1. In embodiments, a homolog of GadBe(Lb) polypeptide comprises a polypeptide having an accession no. STX19016.1, QBY21422.1, ANN49747.1, KIO99344.1, ERK41051.1, KRN34776.1, KRL97822.1, WP_057717368.1, VDG20388.1, WP_165444417.1, or AHX56280.1. In embodiments, a homolog of GadB(Lp) polypeptide comprises a polypeptide having an accession no. BBA26472.1, SPD93437.1, KTF01778.1, RDF95564.1, AQY71158.1, KRL97822.1, AHX56280.1, TBX37968.1, AHX56282.1, AHX56281.1, AHX56283.1, or WP_048001054.1. In embodiments, a homolog of Gad(Ls) polypeptide comprises a polypeptide having an accession no. WP_125641322.1, WP_226457942.1, BAN05709.1, MBL3537851.1, WP_039105805.1, WP_052957185.1, KIR08754.1, WP_125574762.1, WP_063488771.1, or WP_017262688.1. In embodiments, a homolog of GdhA polypeptide comprises a polypeptide having an accession no. WP_077135411.1, EFY1585775.1, EFW0012466.1, WP_135489199.1, WP_105291250.1, EEW3328042.1, WP_105274563.1, AGB78530.1, WP_113858645.1, WP_181668454.1, or WP_203398179.1. In embodiments, a homolog of H16_RS27940 comprises a polypeptide having an accession no. WP_051591491.1, WP_114130480.1, WP_078200706.1, EON20731.1, PKO64515.1, WP_092007571.1, WP_162566377.1, WP_137921632.1, or WP_162591754.1. In embodiments, a homolog of KES23458 comprises a polypeptide having an accession no. WP_116425784.1, WP_069862932.1, WP_043315988.1, WP_009614288.1, WP_089392503.1, WP_109934365.1, WP_090268322.1, WP_138519936.1, WP_138213347.1, WP_015474919.1, WP_043256620.1, WP_084311461.1, WP_053816481.1, WP_070656248.1, or WP_077524299.1. In embodiments, a homolog of LvaE comprises a polypeptide having an accession no. WP_051095536.1, AGA73676.1, WP_054905284.1, OFQ86312.1, OFQ81524.1, WP_102880076.1, WP_092297027.1, WP_160291004.1, WP_081520035.1, WP_104443972.1, WP_046855848.1, WP_134690622.1, WP_103303932.1, WP_042129240.1, or BAV75244.1. In embodiments, a homolog of MELS_RS10970 comprises a polypeptide having an accession no. WP_020723925.1, WP_048514244.1, WP_074501184.1, KXB91325.1, WP_154877386.1, WP_107195291.1, WP_087477538.1, WP_095630133.1, WP_091647756.1, WP_023053225.1, WP_101912630.1, WP_075572446.1, WP_006790232.1, or WP_006942404.1. In embodiments, a homolog of PaaZ comprises a polypeptide having an accession no. WP_160599600.1, WP_152066042.1, WP_094316530.1, WP_032252644.1, WP_001186464.1, WP_125401136.1, WP_001186494.1, WP_119163289.1, WP_095281943.1, WP_045888522.1, WP_058840681.1, WP_095440732.1, WP_162382197.1, WP_059385322.1, or WP_045286529.1. In embodiments, a homolog of Pct(Cp) comprises a polypeptide having an accession no. WP_066087637.1, NCC15629.1, WP_054329786.1, WP_072853413.1, CDC28613.1, WP_016408311.1, WP_088107724.1, WP_160302233.1, or WP_004038625.1. In embodiments, a homolog of Pct(Me) comprises a polypeptide having an accession no. WP_054336166.1, WP_036203125.1, WP_044502862.1, WP_065360594.1, KXA66894.1, WP_095629974.1, WP_087478516.1, WP_107195767.1, WP_048515067.1, WP_101912966.1, WP_156208970.1, KXB92430.1, WP_023053187.1, WP_039891686.1, or KXB92214.1. In embodiments, a homolog of PduP(Kp) comprises a polypeptide having an accession no. WP_109231734.1, WP_109848747.1, WP_136028274.1, WP_100680758.1, WP_100631313.1, WP_049157539.1, WP_029884370.1, MXH33721.1, WP_144232363.1, WP_153679752.1, WP_148849915.1, EBS2830838.1, WP_112213940.1, or WP_064370270.1.
In embodiments, a homolog of PduP(Se) comprises a polypeptide having an accession no. WP_001097684.1, WP_001528442.1, WP_080203692.1, WP_108450871.1, WP_009652778.1, WP_142983670.1, WP_105274032.1, WP_070556870.1, WP_142502560.1, WP_012131760.1, WP_012906342.1, WP_006683971.1, WP_103775053.1, WP_060570657.1, or WP_135321437.1. In embodiments, a homolog of PhaA comprises a polypeptide having an accession no. WP_013956452.1, SCU96900.1, WP_035820078.1, 4O9C_A, WP_116382525.1, WP_092317196.1, WP_062798979.1, WP_116321054.1, AGW89809.1, WP_039016192.1, WP_063238652.1, WP_029049660.1, WP_011297518.1, WP_124684437.1, or WP_109580845.1. In embodiments, a homolog of PhaB comprises a polypeptide having an accession no. RWA53825.1, WP_042885115.1, WP_039016191.1, WP_116336746.1, WP_112777371.1, WP_006577377.1, WP_135705030.1, WP_133096842.1, WP_124684436.1, WP_116321053.1, WP_006155939.1, WP_045241722.1, WP_011297519.1, WP_144195744.1, or ODV43053.1. In embodiments, a homolog of PhaB(Hb) comprises a polypeptide having an accession no. WP_162219671.1, WP_126946472.1, WP_120385833.1, WP_030074446.1, WP_188637499.1, WP_058579713.1, WP_083023226.1, WP_039183428.1, WP_159340906.1, or WP_096653461.1. In embodiments, a homolog of PhaJ(Ac) comprises a polypeptide having an accession no. WP_103260220.1, WP_104454254.1, OJW67134.1, WP_041998622.1, WP_043760202.1, WP_043129860.1, WP_042076944.1, WP_100860962.1, WP_163157368.1, WP_042638062.1, WP_106886672.1, WP_033131291.1, WP_025327110.1, WP_040094291.1, or WP_139745378.1. In embodiments, a homolog of PP_2216 comprises a polypeptide having an accession no. WP_003250094.1, WP_104887321.1, WP_039614175.1, WP_023662689.1, WP_085706434.1, WP_070087269.1, WP_060512757.1, WP_144171976.1, WP_054884005.1, WP_051100719.1, WP_099814118.1, WP_125859423.1, WP_125464833.1, WP_090345830.1, or WP_110994568.1. In embodiments, a homolog of PrpE(Cn) comprises a polypeptide having an accession no. WP_081623799.1, WP_115213214.1, WP_082818978.1, WP_116324638.1, WP_092309442.1, AMR79067.1, WP_151072146.1, WP_029046365.1, AGW91162.1, WP_116321975.1, WP_039006728.1, WP_092134378.1, WP_109580644.1, WP_035882297.1, or WP_149135646.1. In embodiments, a homolog of PrpE(Ec) comprises a polypeptide having an accession no. WP_024249411.1, WP_130258507.1, WP_000010307.1, WP_138159881.1, WP_105281240.1, WP_000010239.1, WP_000010244.1, WP_160524152.1, WP_105270931.1, WP_160530253.1, WP_016235155.1, WP_061090735.1, WP_103014998.1, WP_094761423.1, or ATX90159.1. In embodiments, a homolog of PrpE(Se) comprises a polypeptide having an accession no. WP_127836169.1, WP_103776706.1, WP_044259075.1, WP_012904755.1, WP_043015332.1, WP_008783866.1, WP_153690685.1, WP_058587683.1, WP_101700584.1, WP_042324663.1, WP_123268908.1, WP_137351112.1, WP_048219548.1, WP_160955604.1, or WP_012133646.1. In embodiments, a homolog of Pta comprises a polypeptide having an accession no. WP_119174868.1, WP_114414934.1, WP_112484304.1, WP_000086724.1, WP_135520103.1, WP_113650156.1, WP_105273752.1, WP_079788930.1, WP_000086702.1, WP_135520103.1, WP_038354606.1, WP_025714133.1, WP_071260224.1, WP_046483030.1, or WP_080924257.1. In embodiments, a homolog of Sbm comprises a polypeptide having an accession no. CDW60403.1, WP_096098300.1, QGU68683.1, WP_000073215.1, WP_024250007.1, WP_105273911.1, EBT2497755.1, WP_064198903.1, WP_105271628.1, CDZ86651.1, WP_130258050.1, WP_038355443.1, WP_142462060.1, WP_103769047.1, or WP_137649991.1. In embodiments, a homolog of SucC comprises a polypeptide having an accession no. WP_111780024.1, WP_105268114.1, WP_149508492.1, EBH0782533.1, WP_079789068.1, EAA0703253.1, WP_001048612.1, WP_103776364.1, HAC6539881.1, WP_139538723.1, WP_040076526.1, WP_152308781.1, WP_061708388.1, WP_159152251.1, or WP_159754306.1
In embodiments, a homolog of SucD comprises a polypeptide having an accession no. WP_148048643.1, WP_161983406.1, WP_128882005.1, SEK68167.1, WP_064567804.1, WP_090133347.1, EDS6037479.1, WP_015965312.1, WP_154777294.1, WP_108473875.1, WP_162082208.1, or WP_154158334.1. In embodiments, a homolog of YgfD comprises a polypeptide having an accession no. HBV28035.1, WP_094338169.1, EBT2497754.1, WP_105273912.1, WP_105271629.1, MJD64661.1, MVY25917.1, WP_152060700.1, CDZ86650.1, CDK74861.1, WP_138183055.1, WP_138158389.1, WP_138158874.1, WP_137651359.1, or WP_038355444.1. In embodiments, a homolog of YgfG comprises a polypeptide having an accession no. WP_105273913.1, WP_011069498.1, WP_095785007.1, KAE9894204.1, WP_128881119.1, WP_105287397.1, EBT2497753.1, WP_112366200.1, CDZ86649.1, WP_137653935.1, WP_103750818.1, WP_135521100.1, EFE06586.1, WP_080626129.1, or WP_079226013.1. In embodiments, a homolog of YgfH comprises a polypeptide having an accession no. WP_094321963.1, WP_075331646.1, WP_105271630.1, WP_128881120.1, WP_075328602.1, WP_128861696.1, ECA1898152.1, WP_105273914.1, CDZ86648.1, WP_130221450.1, WP_135519865.1, WP_001027665.1, WP_135407775.1, WP_130221450.1, or WP_135492970.1.
Strains were maintained as glycerol stocks at −80° C., and were revived on non-selective lysogeny broth (LB) agar containing 5 g/L NaCl, 5 g/L yeast extract, 10 g/L tryptone, 15 g/L agar, and antibiotics as required, and incubated overnight at 30-37° C. LB also served as the medium for starter and seed cultures and was supplemented with antibiotics as required. The performance of E. coli strains was evaluated in shake flask cultures in a base medium of the following composition: M9 salts (12.8 g/L Na2HPO4·H2O, 3 g/L KH2PO4, 0.5 g/L NaCl, and 1 g/L NH4Cl), yeast extract (5 g/L), NaHCO3 (10 mM), trace elements (2.86 g/L H3BO3, 1.81 g/L MnCl2·4H2O, 0.22 g/L ZnSO4·7H2O, 0.39 g/L Na2MoO4·2H2O, 79 μg/L CuSO4·5H2O, and 49.4 μg/L Co(NO3)2·6H2O) as a 1000× concentrate), MgSO4 (1 mM), and isopropyl beta-D-1-thiogalactopyranoside (IPTG), with antibiotics added as required. Cultures can be supplemented with sodium acetate, sodium propionate, and/or sodium butyrate at respective concentrations of up to 20 g/L, 10 g/L, and 8 g/L, or a VFA feedstock at up to 75% by volume to facilitate (R)-HB-CoA and (R)-HV-CoA production (to produce PHBV). Additional carbon sources, for example, but not limited to, glucose, glycerol, pretreated biomass, and cheese whey can be used to augment PHBV production and growth. Additionally, nitrogen sources, for example, but not limited to, ammonium salts and corn steep liquor can be used in place of yeast extract. Inducer (i.e. IPTG) concentration may vary between 0 mM and 1 mM to tune expression of pathway enzymes. Cyanocobalamin (vitamin B12) is added to the medium at a concentration of 0.1-2 μM to facilitate the functional expression of Sbm as required. Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, can be added to the medium at a concentration of 0.1-2 mM to facilitate the conversion of L-glutamate to 4-aminobutyrate via a glutamate decarboxylase polypeptide. The same range of medium compositions can be used for bioreactor cultures.
In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising at least one carbon source. In embodiments, the carbon source comprises at least one of VFA, optionally sodium acetate, sodium propionate, sodium butyrate, and glucose, glycerol, biomass, optionally pretreated biomass, and cheese whey. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising at least one of about 0.01 to 20 g/L sodium acetate, about 0.01 to 10 g/L sodium propionate, about 0.01 to 8 g/L sodium butyrate, about 1-10 g/L butyraldehyde, about 1-10 g/L L-glutamate, about 1-10 g/L 4-aminobutyrate, and about 1-10 g/L succinate. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising at least one of about 0.01 to 20 g/L sodium acetate, about 0.01 to 10 g/L sodium propionate, and about 0.01 to 8 g/L sodium butyrate. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium further comprising at least one of about 1-10 g/L butyraldehyde, about 1-10 g/L L-glutamate, about 1-10 g/L 4-aminobutyrate, and about 1-10 g/L succinate. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising between about 20 VFA mmol/L and about 5 VFA mol/L, optionally between about 20 VFA mmol/L and about 90 VFA mmol/L, optionally between about 90 VFA mmol/L land about 180 mmol/L, optionally about or at least 400, 450, 500, 550, 600, 650, 700, 750, or 800 VFA mmol/L, optionally about or up to 1 VFA mol/L. In embodiments, the VFA comprises at least one of about 10-70 mol % acetic acid, about 10-80 mol % propionic acid, and about 10-70 mol % butyric acid. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium containing VFA comprising of at least one of about 20-60 mol % acetic acid, about 5-30 mol % propionic acid, and about 20-60 mol % butyric acid. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising about at least one of about 0.1-20% (w/v) glucose, optionally about 0.1%-15% (w/v) glucose, optionally about 0.1%-10% glucose, about 0.1-20% (w/v) glycerol, optionally about 0.1%-10% (w/v) glycerol, optionally about 0.1%-5% glycerol, about 0.1-50% (w/v) biomass, optionally about 0.1%-25% (w/v) biomass, optionally about 0.1%-10% biomass, optionally about 50% (w/v) pretreated biomass, optionally about 0.1%-25% (w/v) pretreated biomass, optionally about 0.1%-10% pretreated biomass and about 0.1-50% (w/v) cheese whey, optionally about 0.1%-25% (w/v) cheese whey, optionally about 0.1%-10% cheese whey.
In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising at least one nitrogen source. In embodiments, the nitrogen source comprises at least one of yeast extract, an ammonium salt, and corn steep liquor. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture media comprising at least one of about 0.1-20% (w/v) yeast extract, about 0.1-20% (w/v) ammonium salt, about 0.1-20% (w/v) casamino acids, and about 0.1-20% (w/v) corn steep liquors.
In embodiments, the method comprises culturing a recombinant bacterial cell in a culture media comprising about 0-2 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG), optionally about 0.3 mM IPTG. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture media comprising about 0.1-2 μM cyanocobalamin, optionally about 0.2 μM cyanocobalamin. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture media comprising about 0.1-2 mM pyridoxal 5′-phosphate (PLP), optionally about 0.5 mM PLP.
In a specific embodiment, the method comprises culturing a recombinant bacterial cell in a culture medium comprising about 30 g/L glycerol, about 10 g/L yeast extract, about 10 mM NaHCO3, about 0.4 μM vitamin B12, trace elements, about 0.1 mM IPTG, about 0.23 g/L K2HPO4, about 0.51 g/L NH4Cl, about 49.8 mg/L MgCl2, about 48.1 mg/L K2SO4, about 2.78 mg/L FeSO4·7H2O, about 0.055 mg/L CaCl2, about 2.93 g/L NaCl, and about 0.72 g/L tricine. In embodiments, the trace elements comprises H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, Co(NO3)2·6H2O. In embodiments, the culture medium comprises trace elements at about 2.86 mg/L H3BO3, about 1.81 mg/L MnCl2·4H2O, about 0.222 mg/L ZnSO4·7H2O, about 0.39 mg/L Na2MoO4·2H2O, about 79 ng/L CuSO4·5H2O, about 49.4 ng/L Co(NO3)2·6H2O). In embodiments, the volumetric mass transfer coefficient (kLa) is between 50 and 500 h−1.
Shake flask and bioreactor cultures can be performed at temperatures between 25° C. and 42° C. The starting pH in shake flask cultures can be adjusted to pH 5-9, which is the same pH range that can be maintained in bioreactor cultures. The agitation rate in shake flask cultures may range between 50 and 400 revolutions per min (rpm) and can be adjusted between 100 and 1200 rpm in bioreactor cultures. The dissolved oxygen (DO) concentration will be maintained between 1% and 50% of saturation in bioreactor cultures. Various surfactants and perfluorocarbon- and hydrocarbon-based oxygen carriers can be used to improve PHBV production and growth via improved oxygen mass transfer and altered membrane fluidity.
Growth and PHBV production can be improved, for example, by repeated culturing to acclimate E. coli strains to higher concentrations of VFA. Such repeated culturing involves, for example, culturing the recombinant E. coli cells in a medium containing increasing concentrations of VFA. Culturing can begin in a medium such as a semi-defined medium containing VFA at 1-50 mmol/L, and one or more of, but not limited to, M9 salts, yeast extract, glycerol, MgSO4, MgCl2, K2SO4, tricine, thiamine, (NH4)2HPO4, sodium citrate, CaCl2, FeSO4, K2HPO4, and trace elements such as H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, and Co(NO3)2·6H2O (i.e. the starting medium). The strains can be cultured for 1-7 days in the starting medium, after which time 5-100% of the culture is centrifuged and the resulting cell pellet is resuspended into a fresh medium containing VFA at a concentration of 101-200% of the starting medium. For example, if the starting medium contains 40 mmol/L VFA, the subsequent (second) round of culturing can occur in a medium containing 40.4-80 mmol/L VFA. Similarly, the second round of culturing can occur for 1-7 days, after which time 5-100% of the culture is centrifuged and the resulting cell pellet is resuspended into a fresh medium containing VFA at a concentration of 101-200% of the medium from the second round of culturing. For example, if the second round of culturing occurred in a medium containing 60 mmol/L VFA, the fresh medium can contain 60.6-120 mmol/L VFA. This process can be repeated until the strains can consume all VFA in cultures supplemented with up to 300 mmol/L VFA, with PHBV yields reaching at least 30% of dry cell weight, assuming that VFA that has not been converted to PHBV can be converted to biomass at a concentration of up to 100 g dry cell weight/L.
In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising maintaining a temperature of about 20-42° C., optionally about 25-42° C., optionally about 25-37° C. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising maintaining a pH of about 4-10, optionally about 5-9, optionally about 6-8. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising maintaining an agitation rate of about 50-1200 rpm, optionally about 50-600 rpm, optionally about 100-1200 rpm, optionally about 100-600 rpm. In embodiments, the method comprises culturing a recombinant bacterial cell in a culture medium comprising maintaining dissolved oxygen of about 1-100% of saturation, optionally about 1-5% of saturation, optionally about 6-10% of saturation, optionally about 11-15% of saturation, optionally about 16-20% of saturation, optionally about 21-25% of saturation, optionally about 26-30% of saturation, optionally about 31-35% of saturation, optionally about 36-40% of saturation, optionally about 41-45% of saturation, optionally about 46-50% of saturation, optionally about 51-55% of saturation, optionally about 56-60% of saturation, optionally about 61-65% of saturation, optionally about 66-70% of saturation, optionally about 71-75% of saturation, optionally about 76-80% of saturation, optionally about 81-85% of saturation, optionally about 86-90% of saturation, optionally about 91-95% of saturation, optionally about 96-100% of saturation.
In embodiments, the method comprises culturing a recombinant bacterial cell in a culture media comprising at least one of a surfactant, optionally an anionic surfactant, a cationic surfactant, an amphoteric surfactants, or a non-ionic surfactant, a perfluorocarbon-based oxygen carrier, optionally n-perfluorooctane, perfluorodecalin, perfluoromethyldecalin, or perfluoro-1,3-dimethylcyclohexane) and a hydrocarbon-based oxygen carrier, optionally n-heptane, n-hexadecane, and n-dodecane.
In embodiments, the method described herein comprises producing PHBV in about 1-10 days, optionally about 1-9 days, optionally about 1-8 days, optionally about 1-7 days, optionally about 1-6 days, optionally about 1-5 days, optionally about 1-4 days, optionally about 1-3 days, optionally about 1-2 days, optionally less than 10, 9, 8, 7, 6, 5, 4, 3, or 2, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
In embodiments, the feedstock comprises VFA composition of about: 20-60 mol % acetic acid, 5-30 mol % propionic acid, and 20-60 mol % butyric acid.
In embodiments, the culturing condition for producing intracellular PHBV granules by the recombinant bacterial cell is under pH conditions of 6-9, optionally 6-7 or 7-8, or 8-9, temperature conditions of 20-40° C., optionally 20-25° C., or 25-30° C., or 30-35° C., or 35-40° C. and incubation times of 1 hour to 2 weeks, optionally 1 h to 1 week, optionally 1 h to 5 days, optionally 1 h to 4 days, optionally 1 h to 3 days, optionally 1 h to 2 days, optionally 1-24 h, optionally 1-3 h, or 3-6 h, or 6-9 h, or 9-12 h, or 12-18 h, or 18-24 h. Culturing of the recombinant bacterial cell for producing PHBV may use bubble column reactors, stirred tank reactors, airlift reactors, preferably airlift reactors, flasks such as polycarbonate flasks. PHBV production is done under aerobic condition, for example, when a flask for incubation is vented, or under microaerobic condition, when a flask for incubation is capped.
In embodiments, the method of culturing a recombinant bacterial cell for producing PHBV comprises,
In embodiments, the method comprises culturing a recombinant bacterial cell by repeated culturing in a medium containing increasing concentrations of VFA. In embodiments, the repeated culturing comprises i) culturing in a medium comprising VFA at 1-50 mmol/L, and one or more of M9 salts, yeast extract, glycerol, trace elements, and MgSO4, for 1-7 days; ii) centrifuging 5-100% of the culture and resuspending the resulting cell pellet into a fresh medium comprising VFA at a concentration of 101-200% of the medium of step i), and one or more of M9 salts, yeast extract, glycerol, trace elements, and MgSO4, for 1-7 days; and iii) repeating step ii) until the recombinant bacterial cell is capable of consuming all VFA up to 300 mmol/L VFA in the medium, and the recombinant bacterial cell produces PHBV at a minimum of 30% (w/w) of dry cell weight. In embodiments, the trace elements comprises H3BO3, MnCl2·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, and Co(NO3)2·6H2O.
The PHBV accumulates in the form of granules. The PHBV polymers are stored inside of the cells as discrete granules that are water-insoluble. In embodiments, the accumulation of PHBV granules is monitored, optionally by fluorescence spectroscopy analysis of the PHBV producing culture. In embodiments, the cells are fixed by heating a smear of the PHBV producing culture, which is the liquid mixture that contains the PHBV producing bacteria, on a glass slide. The heat-fixed cells can then be stained with 1% (v/v) aqueous Nile Blue A solution, or another appropriate staining solution and washed with sequences of water, acetic acid and water again. Afterward, the fixed culture can be analyzed using fluorescence microscopy as PHBV granules will fluoresce under these conditions. Optionally, a high throughput Nile Red assay may be used to monitor and quantify the intracellular PHBV granules in a liquid culture using fluorescence spectroscopy.
In an aspect, PHBV polymers are extracted with sequential washes for up to 3 times and lyophilized with a lyophilizer. In embodiments, the PHBV polymers are extracted with sequential washes for up to 3 times and lyophilized with a lyophilizer for about 48 h at temperatures of −20 to −80° C., optionally −30 to −35° C., −35 to −40° C., −40 to −45° C., or −45 to −50° C. Centrifugation or microfiltration with an appropriate centrifuge and microfilter for purification, may also be used during PHBV granule extraction. The skilled person can readily recognize the appropriate centrifuge and microfilter.
In embodiments, the method for producing PHBV from a recombinant bacterial cell comprises:
In embodiments. the culture medium comprises cyanocobalamin, optionally at a concentration of 0.1-2 μM.
In embodiments, the conditions comprise maintaining a temperature of about 20-42° C., optionally about 25-42° C., optionally about 25-37° C. In embodiments, the conditions comprise maintaining a pH of about 4-10, optionally about 5-9, optionally about 6-8.
In embodiments, the culture medium comprises at least one carbon source. In embodiments, the carbon source comprises at least one of VFA, optionally sodium acetate, sodium propionate, sodium butyrate, and glucose, glycerol, biomass, optionally pretreated biomass, and cheese whey. In embodiments, the culture media comprises at least one of about 0.01 to 20 g/L sodium acetate, about 0.01 to 10 g/L sodium propionate, and about 0.01 to 8 g/L sodium butyrate. In embodiments, the VFA comprises at least one of about 10-70 mol % acetic acid, about 10-80 mol % propionic acid, and about 10-70 mol % butyric acid.
In embodiments, the culture medium comprises at least one nitrogen source. In embodiments, the at least one nitrogen source is at least one of an ammonium salt, corn steep liquor, casamino acids, and yeast extract.
In embodiments, PHBV has a hydroxyvaleric acid (HV) content of about 1-20 mol %, about 1-30 mol %, about 1-40 mol %, or about 1-50 mol %.
In embodiments, the method further comprising extracting the PHBV from the bacterial cell and/or isolating PHBV from the culture medium.
List of strains and corresponding labels used in
PHBV can be recovered by any methods known in the art. The method can be an extraction method recovering PHBV from within bacterial cells, or a method recovering PHBV from culture media. A range of parameters (i.e. temperature, treatment time, pH and concentrations) for surfactant (for example SDS or non-ionic surfactant Triton X-100) and hypochlorite can be used to extract PHBV. The purity of PHBV can be determined by methods known in the art, for example, by gas chromatography mass spectroscopy (GC-MS). The recombinant bacterial cells and methods described herein produce PHBV with a mass yield of 5-80% of dry cell weight. The HV content of PHBV can also be determined by methods known in the art, for example, PHBV can be treated in a reflux at 100° C. for 150 min in the presence of chloroform, methanol, and sulfuric acid, and the PHBV is then converted into methyl esters which facilitates the separation of different hydroxyalkanoates present in the copolymer structure for further analysis, for example, by GC-MS. The monomer composition of PHBV can also be determined via proton-nuclear magnetic resonance (1H-NMR). The polymer sample can be solubilized in an appropriate deuterated solvent such as deuterated methylene chloride (CDCl2) at a concentration of 1-10 mg/mL. The analysis can be conducted in a spectrometer operating at 300-600 MHz, and the molar ratio of HB and HV monomers can be taken as the ratio of integrals of the chemical shifts at 1.25 ppm (corresponding to the CH3- group of HB) and at 0.85 ppm (corresponding to the CH3-CH2- group of HV). Dry cell weight (DCW) can be determined by centrifuging culture samples at 2000-6000×g for 10-30 min, followed by at least one wash step using distilled water, and subsequent lyophilization of the cell paste overnight. In embodiments, PHBV composition is analyzed by GC-MS and/or 1H-NMR.
Applications of PHBV with Varying HV Content
The PHBV produced by the recombinant bacterial cell described herein has a defined HV content, which affects properties such as melting point, water permeability, glass transition temperature, and tensile strength of the biopolymer. PHBV with different HV contents thus has different applications.
For example, PHBV with 0-5 mol % HV has properties that are comparable to polylactic acid (PLA) or polystyrene (PS), and it is useful as, for example, 3D printing filament, golf tees, writing utensils, cutlery, and coffee cup lids, which can be manufactured by injection moulding or extrusion of the PHBV with this amount of HV content.
For example, PHBV with 5-10 mol % HV has properties that are comparable to acrylonitrile butadiene styrene (ABS), and it is useful as, for example, building blocks (in toys) and clamshells, which can be manufactured by injection moulding or extrusion of the PHBV with this amount of HV content.
For example, PHBV with 10-20 mol % HV has properties that are comparable to polypropylene (PP) or polyethylene terephthalate (PET), and it is useful as, for example, bioplastic bottles, clothing, straws, electrical insulation, baby wipes, bottle caps, sanitary applicators, yogurt containers, which can be manufactured by blow moulding, injection moulding, profile, extrusion, or textile spinning of the PHBV with this amount of HV content.
For example, PHBV with at least 20 mol % HV has properties that are comparable to polyethylene (PE), and it is useful as, for example, shopping bags, agricultural wrap, paper cup liners, plastic wrap, banners, labels, cigarette filters, which can be manufactured by blow moulding or spray coating of the PHBV with this amount of HV content.
Further, the PHBV produced by the recombinant bacterial cell described herein has applications in the field of biomaterials.
For example, PHBV with at least 20 mol % HV is useful as a flexible porous sheet, for example, for tissue separation to enable healing of pericardiac defect in sheep (see WO1990000067A1, herein incorporated by reference in its entirety).
For example, PHBV with at least 8.25 mol % HV is useful as a film, for example, to immobilize antimicrobial peptide tachyplesin I tagged with PHA-granule-associated protein (PhaP).
For example, PHBV with at least 5 mol % HV, optionally at least 8 mol % HV, is useful as a scaffold, for example, for tissue engineering, such as neural tissue engineering.
For example, PHBV is useful as nanoparticles, for example, PHBV with at least 12 wt % HV is useful to encapsulate photosensitizer 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H, 23H-porphine, for example, for photodynamic therapy for cancer treatment, and PHBV with at least 15% mol % is useful to encapsulate drug, for example, anticancer drug such as Ellipticine.
For example, PHBV with at least 11.3 mol % HV is useful as carrier rods for local antibiotic delivery.
Further details are provided in Xue Q et al., Biomaterials 2018, 178:351-362, Rathbone S, et al., Journal of biomedical materials research Part A 2010, 93:1391-1403, Chen W, et al., Acta biomaterialia 2012, 8:540-548, Pramual S, Journal of Materials Science: Materials in Medicine 2016, 27:40-40, Masood F, Materials science & engineering C, Materials for biological applications 2013, 33:1054-1060, and Türesin F, et al., Journal of Biomaterials Science, Polymer Edition 2001, 12:195-207, the contents of which are incorporated herein by reference in its entirety for all purposes.
For example, 10-30 wt % PHBV, where the PHBV has at least 5-25% wt % HV is useful as a PHBV/polylactic acid absorbable suture, for example, for nerve and vascular repair (see CN105063790A, herein incorporated by reference in its entirety).
The recombinant bacterial cells and methods described herein produce PHBV with a HV content of about 0-50 mol %, about 1-50 mol %, about 0-40 mol %, about 1-40 mol %, about 0-30 mol %, about 1-30 mol %, about 0-20 mol %, about 1-20 mol %, about 20-50 mol %, about 10-20 mol %, about 5-10 mol %, or about 0-5 mol %. In embodiments, the recombinant bacterial cells and methods described herein produce PHBV with a HV content of about 0-50 mol %, about 5-25 mol %, about 1-50 mol %, about 0-40 mol %, about 1-40 mol %, about 0-30 mol %, about 1-30 mol %, about 0-20 mol %, about 1-20 mol %, about 20-50 mol %, about 10-20 mol %, about 5-10 mol %, or about 0-5 mol %. In embodiments, the recombinant bacterial cells and methods described herein produce PHBV with a HV content of at least about 5 mol %, at least about 6 mol %, at least about 7 mol %, at least about 8 mol %, at least about 8.25 mol %, at least about 8.5 mol %, at least about 8.75 mol %, at least about 9 mol %, at least about 10 mol %, at least about 11 mol %, at least about 11 mol %, at least about 11.1 mol %, at least about 11.2 mol %, at least about 11.3 mol %, at least about 11.4 mol %, at least about 11.5 mol %, at least about 11.6 mol %, at least about 11.7 mol %, at least about 11.8 mol %, at least about 11.9 mol %, at least about 12 mol %, at least about 13 mol %, at least about 14 mol %, at least about 15 mol %, at least about 16 mol %, at least about 17 mol %, at least about 18 mol %, at least about 19 mol %, at least about 20 mol %, at least about 25 mol %, at least about 30 mol %, or at least about 35 mol %, and optionally at most about 40 mol %, at most about 45 mol %, or at most about 50 mol %. In embodiments, the recombinant bacterial cell comprises nucleic acid molecule having the sequence of SEQ ID NO: 239 and SEQ ID NO: 240, and the recombinant bacterial cell produces PHBV with a HV content of up to about 40 mol %. In embodiments, the recombinant bacterial cell comprising nucleic acid molecule having the sequence of SEQ ID NO: 239 and SEQ ID NO: 240 produces PHBV by culturing the bacterial cell in a culture medium comprising at least one carbon source. In embodiments, the carbon source comprises glycerol. In embodiments the carbon source comprises at least one VFA. In embodiments, the recombinant bacterial cell comprises nucleic acid molecule having the sequence of SEQ ID NO: 239 and SEQ ID NO: 240, and the recombinant bacterial cell produces PHBV with a HV content from about 15 mol % to about 40 mol %. In embodiments, the recombinant bacterial strain is CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB), intF::(Pgracmax2::(T7.RBS)phaC:(RBS1)phaA) and the bacterial strain produces PHBV with a HV content of up to about 40 mol %. In embodiments, the recombinant bacterial strain is CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB), intF::(Pgracmax2::(T7.RBS)phaC:(RBS1)phaA) and the bacterial strain produces PHBV with a HV content from about 15 mol % to about 40 mol %. In embodiments, the recombinant bacterial cell produces PHBV at a mass yield of up to about 80% of dry cell weight. In embodiments, the HV content of PHBV is adjustable by expression, overexpression, underexpression, attenuation, silencing and/or inactivation of genes or enzymes described herein, optionally the gene is a nonessential gene.
Embodiments of the disclosure will be described in a non-limiting manner by reference to the examples below.
A two-plasmid system was employed to assess the potential of E. coli to co-produce the monomers of PHBV, i.e. HV and HB, respectively derived from (R)-HV-CoA and (R)-HB-CoA, from propionate and acetate as HV and HB can be readily measured via high performance liquid chromatography (HPLC). The first plasmid contained bktB, hbd (encoding hydroxybutyryl-CoA dehydrogenase Hbd polypeptide that converts 3-ketovaleryl-CoA to (S)-HV-CoA and acetoacetyl-CoA to (S)-HB-CoA), and tesB (encoding acyl-CoA thioesterase II TesB polypeptide that converts (S)-HV-CoA and (R)-HV-CoA to HV, and (S)-HB-CoA and (R)-HB-CoA to HB), i.e. plasmid pK-bktB-hbd-tesB. The second plasmid contained phaA, phaB (PhaB polypeptide converts 3-ketovaleryl-CoA to (R)-HV-CoA and acetoacetyl-CoA to (R)-HB-CoA), and pct(Cp) (from C. propionicum), i.e. plasmid pTrc-phaAB:pct(Cp), which was constructed by amplifying the Ptrc::phaAB fragment (including the plasmid backbone) from plasmid pTrc-phaAB-crt-ter with primers P01 and P02 (SEQ ID NO: 119 and 120), and pct(Cp) from C. propionicum DSM 1682 genomic DNA (gDNA) with primer P03 and P04 (SEQ ID NO: 121 and 122), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs; USA) as per the manufacturers' instructions and readily undertaken by the skilled person. The host cell is E. coli strain CPC-Sbm, which is derived from strain K-12. It is understood that any K-12 derived strain may be useful and the skilled person can readily identify the relevant derivatives of K-12 strain. Plasmids pK-bktB-hbd-tesB and pTrc-phaAB:pct(Cp) (SEQ ID NO: 162) were co-transformed into the host E. coli strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:pct(Cp)), and its ability to produce HV and HB was evaluated in shake flask cultures (see
A two-plasmid system was employed to assess the potential of E. coli to co-produce the monomers of PHBV, i.e. HV and HB, respectively derived from (R)-HV-CoA and (R)-HB-CoA, from propionate and acetate as HV and HB can be readily measured via HPLC. Plasmid pK-bktB-hbd-tesB was the same as in Example 1, and the second plasmid contained phaA, phaB, and pct(Me) (from M. elsdenii), i.e. plasmid pTrc-phaAB:pct(Me) (SEQ ID NO: 163), which was constructed by amplifying the Ptrc::phaAB fragment (including the plasmid backbone) from plasmid pTrc-phaAB-crt-ter with primers P05 and P02 (SEQ ID NO: 123 and 120), and pct(Me) from M. elsdenii DSM 20460 gDNA with primer P06 and P07 (SEQ ID NO: 124 and 125), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-bktB-hbd-tesB and pTrc-phaAB:pct(Me) (SEQ ID NO: 163) were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:pct(Me)), and its ability to produce HV and HB was evaluated in shake flask cultures (see
A two-plasmid system is employed to assess the potential of E. coli to co-produce the monomers of PHBV, i.e. HV and HB, respectively derived from (R)-HV-CoA and (R)-HB-CoA, from propionate and acetate as HV and HB can be readily measured via HPLC. Plasmid pK-bktB-hbd-tesB was the same as in Example 1, and the second plasmid contains phaA, phaB, and prpE(Ec) (from E. coli), i.e. plasmid pTrc-phaAB:prpE(Ec), which is constructed by amplifying the Ptrc::phaAB fragment (including the plasmid backbone) from plasmid pTrc-phaAB-crt-ter, and prpE(Ec) from E coli MG1655 gDNA, followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-bktB-hbd-tesB and pTrc-phaAB:prpE(Ec) were co-transformed into strain CPC-Sbm, resulting in strain CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:prpE(Ec)). This strain produces HV and HB in comparable quantities as strains described in Examples 1 and 2 (
A two-plasmid system is employed to assess the potential of E. coli to co-produce the monomers of PHBV, i.e. HV and HB, respectively derived from (R)-HV-CoA and (R)-HB-CoA, from propionate and acetate as HV and HB can be readily measured via HPLC. Plasmid pK-bktB-hbd-tesB was previously disclosed [13], and the second plasmid contains phaA, phaB, and prpE(Se) (from S. enterica), i.e. plasmid pTrc-phaAB:prpE(Se), which is constructed by amplifying the Ptrc::phaAB fragment (including the plasmid backbone) from plasmid pTrc-phaAB-crt-ter [13], and prpE(Se) from S. enterica DSM 18522 gDNA, followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-bktB-hbd-iesB and pTrc-phaAB:prpE(Se) were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:prpE(Se)). This strain produces HV and HB in comparable quantities as strains described in Examples 1 and 2 (
A two-plasmid system was employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contained lvaE and tesB, i.e. plasmid pK-lvaE:tesB, and was constructed by amplifying lvaE from P. putida KT2440 gDNA with primers P08 and P09 (SEQ ID NO: 116 and 117), and the Plac-tesB fragment (including plasmid backbone) from pK-bktB-hbd-tesB with primers P10 and P11 (SEQ ID NO: 128 and 129), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. The second plasmid contained PP_2216 (gene encoding a short-chain acyl-CoA dehydrogenase polypeptide) and H16_RS27940, i.e. plasmid pTrc-PP_2216:H16_RS27940, and was constructed by amplifying PP_2216 from P. putida KT2440 gDNA with primers P12 and P13 (SEQ ID NO: 130 and 131), H16_RS27940 from C. necator H16 gDNA with primers P14 and P15 (SEQ ID NO: 122 and 123), and Ptrc (including plasmid backbone) from Ptrc99a (as detailed in Amann E et al., Gene 1988, 69:301-315, the contents of which are incorporated herein by reference in its entirety for all purposes) with primers P16 and P17 (SEQ ID NO: 124 and 125), followed by subsequent assembly of the three fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. lvaE and PP_2216 that have been codon optimized for expression in E. coli can also be used. Plasmids pK-lvaE:tesB and pTrc-PP_2216:H16_RS27940 (SEQ ID NO: 165) were co-transformed into strain CPC-Sbm, resulting in strain CPC-Sbm(pK-lvaE:tesB, pTrc-PP_2216:H16_RS27940), and its ability to produce HB was evaluated in shake flask cultures (
A two-plasmid system was employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contained lvaE and tesB, i.e. plasmid pK-lvaE:tesB, and its construction was described in Example 5. The second plasmid contained BC_5341 (gene encoding a short-chain acyl-CoA dehydrogenase polypeptide) and H16_RS27940, i.e. plasmid pTrc-BC_5341:H16_RS27940, and was constructed by amplifying BC_5341 from B. cereus DSM 31 gDNA with primers P18 and P19 (SEQ ID NO: 136 and 137), and the Ptrc-H16_RS27940 fragment (including plasmid backbone) from plasmid pTrc-PP_2216:H16_RS27940 with primers P20 and P21 (SEQ ID NO: 138 and 139), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-lvaE:tesB and pTrc-BC_5341:H16_RS27940 were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-lvaE:tesB, pTrc-BC_5341:H16_RS27940), and its ability to produce HB was evaluated in shake flask cultures (
A two-plasmid system was employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contained atoDAE (atoE encodes putative short-chain fatty acid transporter AtoE) and tesB, i.e. plasmid pK-atoDAE:tesB, and was constructed by amplifying atoDAE from E. coli MG1655 gDNA with primers P22 and P23 (SEQ ID NO: 140 and 141), and the Plac-tesB fragment (including plasmid backbone) from pK-bktB-hbd-tesB with primers P10 and P24 (SEQ ID NO: 128 and 142), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. The second plasmid contained PP_2216 and H16_RS27940, i.e. plasmid pTrc-PP_2216:H16_RS27940, and its construction was described in Example 5. Plasmids pK-atoDAE:tesB and pTrc-PP_2216:H16_RS27940 were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-atoDAE:tesB, pTrc-PP_2216:H16_RS27940), and its ability to produce HB was evaluated in shake flask cultures (
A two-plasmid system was employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contained atoDAE (atoE encodes putative short-chain fatty acid transporter AtoE) and tesB, i.e. plasmid pK-atoDAE:tesB, and was described in Example 7. The second plasmid contained BC_5341 and H16_RS27940, i.e. plasmid pTrc-BC_5341:H16_RS27940, and its construction was described in Example 6. Plasmids pK-atoDAE:tesB and pTrc-BC_5341:H16_RS27940 were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-atoDAE:tesB, pTrc-BC_5341:H16_RS27940), and its ability to produce HB was evaluated in shake flask cultures (
A two-plasmid system was employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contained lvaE and tesB, i.e. plasmid pK-lvaE:tesB, and its construction was described in Example 5. The second plasmid contained PP_2216 and phaJ(Ac), i.e. plasmid pTrc-PP_2216:phaJ(Ac), and was constructed by amplifying the Ptrc::PP_2216 fragment (including plasmid backbone) from plasmid pTrc-PP_2216:H16_RS27940 with primers P25 and P26 (SEQ ID NO: 143 and 144), and phaJ(Ac) from A. caviae DSM 7323 gDNA with primers P27 and P28 (SEQ ID NO: 145 and 146), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-lvaE:tesB and pTrc-PP_2216:phaJ(Ac) were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-lvaE:tesB, pTrc-PP_2216:phaJ(Ac)), and its ability to produce HB was evaluated in shake flask cultures (
A two-plasmid system is employed to assess the potential of E. coli to produce the monomer of PHBV, i.e. HB, derived from (R)-HB-CoA, from butyrate as HB can be readily measured via HPLC. The first plasmid contains lvaE and tesB, i.e. plasmid pK-lvaE:tesB, and its construction was described in Example 5. The second plasmid contains fadE and phaJ(Ac), i.e. plasmid pTrc-fadE:phaJ(Ac), and is constructed by amplifying fadE from E. coli MG1655 gDNA and the Ptrc-phaJ(Ac) fragment (including plasmid backbone) from plasmid pTrc-PP_2216:phaJ(Ac), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-lvaE:tesB and pTrc-fadE:phaJ(Ac) are co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-lvaE:tesB, pTrc-fadE:phaJ(Ac)). This strain produces HB in comparable quantities as strains listed in Examples 5-8 (
A two-plasmid system was employed to assess the potential of E. coli to produce succinate, i.e. an intermediate in the biosynthesis of (R)-HV-CoA from butyrate. The first plasmid contained lvaE and gadAe, i.e. plasmid pK-lvaE:gadAe, and was constructed by amplifying lvaE from P. putida KT2440 gDNA with primers P08 and P09 (SEQ ID NO: 116 and 117), gadAe from a gBlock® gene fragment synthesized by Integrated DNA Technologies (USA) with primers P29 and P30 (SEQ ID NO: 147 and 148), and the Plac fragment (including plasmid backbone) from pK184 (further details in Jobling M G et al., Nucleic Acids Research 1990, 18:5315, the contents of which are incorporated herein by reference in its entirety for all purposes) with primers P31 and P11 (SEQ ID NO: 149 and 129), followed by subsequent assembly of the three fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. The second plasmid contained FG99_15380, pduP(Se), and gabD, i.e. plasmid pTrc-FG99_15380:pduP(Se):gabD, and was constructed by amplifying FG99_15380 from a gBlock® gene fragment synthesized by Integrated DNA Technologies (FG99_15380 was codon optimized for expression in E. coli) with primers P32 and P33 (SEQ ID NO: 150 and 151), pduP(Se) from S. enterica DSM 18522 gDNA with primers P34 and P35 (SEQ ID NO: 152 and 153), gabD from E. coli MG1655 gDNA with primers P36 and P37 (SEQ ID NO: 154 and 155), and Ptrc (including plasmid backbone) from Ptrc99a [15] with primers P38 and P39 (SEQ ID NO: 156 and 157), followed by subsequent assembly of the four fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-lvaE:gadAe and pTrc-FG99_15380:pduP(Se):gabD were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-lvaE:gadAe, pTrc-FG99_15380:pduP(Se):gabD), and its ability to produce succinate was evaluated in shake flask cultures (
A two-plasmid system was employed to assess the potential of E. coli to produce succinate, i.e. an intermediate in the biosynthesis of (R)-HV-CoA from butyrate. The first plasmid contained lvaE and gadAe, i.e. plasmid pK-lvaE:gadAe (SEQ ID NO: 169), and its construction was described in Example 11. The second plasmid contained FG99_15380, pduP(Kp), and gabD, i.e. plasmid pTrc-FG99_15380:pduP(Kp):gabD, and was constructed by amplifying the Ptrc::FG99_15380-gabD fragment (including plasmid backbone) from pTrc-FG99_15380:pduP(Se):gabD with primers P40 and P41 (SEQ ID NO: 158 and 159), and pduP(Kp) from K. pneumoniae DSM 2026 gDNA with primers P42 and P43 (SEQ ID NO: 160 and 161), followed by subsequent assembly of the two fragments via the NEBuilder HiFi DNA Assembly Master Mix as per the manufacturers' instructions. Plasmids pK-lvaE:gadAe (SEQ ID NO: 169) and pTrc-FG99_15380:pduP(Kp):gabD (SEQ ID NO:171) were co-transformed into strain CPC-Sbm [14], resulting in strain CPC-Sbm(pK-lvaE:gadAe, pTrc-FG99_15380:pduP(Kp):gabD), and its ability to produce succinate was evaluated in shake flask cultures (
Genes that encode enzymes that convert propionate to propionyl-CoA, or comprise a pathway for the conversion of butyrate to (R)-HB-CoA are stably integrated into the genome of E. coli to avoid the use of antibiotics for plasmid maintenance and chemical inducers of protein expression, and plasmid instability (i.e. plasmid loss from the engineered cell). The expression of pct(Cp), is controlled by any one of a plethora of synthetic promoters that have been previously disclosed, for example but not limited to those described in Puigbo et al (2007), Nakamura et al (2000), and Jobling et al (1990), herein incorporated by reference. For instance, synthetic promoters can be derived by altering the upstream, −35 or −10, or spacer (i.e. the sequence between the −35 and −10) (further details in Hwang H J et al., Biotechnology for Biofuels 2018, 11:103, the contents of which are incorporated herein by reference in its entirety for all purposes) sequences of promoters recognized by σ70 (a protein that initiates the transcription of most genes in E. coli). Constitutive promoters with activities spanning at least one order of magnitude are also tested to determine the required promoter activity for each genomically integrated expression cassette to achieve the desired HV content and/or PHBV yield. The Design of Experiment (DoE) approach can be used to reduce the number promoters that must be tested for each genomically integrated expression cassette, and the number of experiments to be conducted, while identifying important interactions that may be observed upon altering the promoter activities of multiple expression cassettes simultaneously. Inducible promoters, for example, but not limited to, IPTG-inducible promoter Ptrc, arabinose-inducible promoter PBAD, and tetracycline-inducible promoter PtetA can also be employed to tune the expression of genomically integrated operons, but without wishing to be bound by theory, are considered a less favorable option due to the cost associated with inducer chemicals.
To facilitate the conversion of propionate to propionyl-CoA, the constitutive expression cassette consisting of pct(Cp) and synthetic promoter is integrated into the genome of strain CPC-Sbm, or any strain derived from it, at a locus corresponding to a nonessential gene, i.e. genes that can be silenced or inactivated, or its activity attenuated, without significantly affecting cell viability. Examples of nonessential genes include but are not limited to, cadA (encoding lysine decarboxylase 1 polypeptide), yjcS (encoding linear primary-alkylsulfatase polypeptide), endA (encoding DNA-specific endonuclease I polypeptide), intF (encoding putative phage integrase), bcsA (encoding cellulose synthase catalytic subunit), bcsC (encoding cellulose synthase outer membrane channel), and lacI (encoding the transcriptional repressor of the lac operon). In addition, nonessential genes that encode enzymes that inhibit or reduce the dissimilation of VFAs and/or PHBV production can be used as genomic integration sites, or can be silenced or inactivated for the purpose of improving VFA dissimilation and/or PHBV production. Examples of such nonessential genes can include but are not limited to ghrB (encoding glyoxylate reductase polypeptide that consumes both glyoxylate needed for growth on acetate and NADPH, a cofactor required by PhaB); gcl (encoding glyoxylate carboligase polypeptide that consumes glyoxylate); gabT and puuE (encoding 4-aminobutyrate aminotransferase polypeptides that consume 4-aminobutyrate needed to produce succinate semialdehyde by KES23458); gadC (encoding L-glutamate:4-aminobutyrate antiporter that exports 4-aminobutyrate out of the cell); sad (encoding NAD(+)-dependent succinate semialdehyde dehydrogenase polypeptide); atoB and yqeF (encoding acetyl-CoA acetyltransferase polypeptides that consume acetyl-CoA); fadA (encoding 3-ketoacyl-CoA thiolase polypeptide that may consume butyryl-CoA and acetyl-CoA); fadB, fadJ, and paaZ (encoding enzymes with significant 3-hydroxyacyl-CoA dehydrogenase activity that can consume crotonyl-CoA and/or (R)-HB-CoA); fadE (encoding acyl-CoA dehydrogenase polypeptide that can consume butyryl-CoA and/or crotonyl-CoA); fadR (encoding DNA-binding transcriptional dual regulator that represses transcription of fadA, fadB, fadE, etc.), ybgC, yigI, tesA, tesB, and yciA (encoding thioesterase polypeptides that can consume HB-CoA and HV-CoA); arcA and fnr (encoding global regulatory protein polypeptides that can regulate carbon flux through the TCA cycle); prpBCD (encoding enzymes that comprise the 2-methylcitrate cycle that converts propionyl-CoA to succinate); and yqhD (encoding NADPH-dependent aldehyde reductase that can convert butyraldehyde to butanol). Subsequently, one or more constitutive expression cassettes consisting of lvaE and phaJ(Ac) and one or more synthetic promoters are integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp) expression cassette at one or more loci corresponding to one or more nonessential genes to facilitate the conversion of butyrate to (R)-HB-CoA as previously outlined. In this case, however, fadR is inactivated by inventor through fadR gene knockout to derepress expression of fadE to facilitate the conversion of butyryl-CoA to crotonyl-CoA. In addition, atoC (encoding DNA-binding transcriptional activator/ornithine decarboxylase inhibitor that activates transcription of the atoDAEB operon for enhanced VFA uptake and conversion to acyl-CoAs) is mutated to confer constitutive expression of the atoDAEB operon by introducing the amino acid substitution I129S, yielding atoC(Con). The resulting strain containing genomically-integrated pct(Cp), lvaE, and phaJ(Ac) expression cassettes, and constitutively expressed fadE and atoDAEB are subsequently co-transformed with plasmids pPhaCAB (encoding phaA, phaB, and phaC) and pKBktB (encoding bktB) [18], and the resulting strain is evaluated for PHBV production in shake flask and/or bioreactor cultures. The strain produces PHBV with a HV content of 1-30 mol % at a mass yield of 5-80% of dry cell weight.
Genes that encode enzymes that 1) convert propionate to propionyl-CoA, 2) comprise a pathway for the conversion of butyrate to (R)-HB-CoA, or 3) comprise a pathway for the conversion of butyrate to succinate are stably integrated into the genome of E. coli. The expression of pct(Cp) is controlled by a synthetic promoter and the corresponding constitutive expression cassette is integrated into the genome of strain CPC-Sbm, or any strain derived from it, at a locus corresponding to a nonessential gene to facilitate the conversion of propionate to propionyl-CoA as outlined in Example 13. Subsequently, one or more constitutive expression cassettes consisting of lvaE, PP_2216, and phaJ(Ac) and one or more synthetic promoters are integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp) expression cassette at one or more loci corresponding to one or more nonessential genes to facilitate the conversion of butyrate to (R)-HB-CoA. Subsequently, one or more constitutive expression cassettes consisting of gadAe, FG99_15380, pduP(Se), and gabD and one or more synthetic promoters are integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp), lvaE, PP_2216, and phaJ(Ac) expression cassettes at one or more loci corresponding to one or more nonessential genes to facilitate the conversion of butyryl-CoA to succinate. Finally, the resulting strain containing genomically-integrated pct(Cp), lvaE, PP_2216, phaJ(Ac), gadAe, FG99_15380, pduP(Se), and gabD expression cassettes are subsequently co-transformed with plasmids pPhaCAB (encoding phaA, phaB, and phaC) and pKBktB (encoding bktB) [18], and the resulting strain is evaluated for PHBV production in shake flask and/or bioreactor cultures in which cyanocobalamin has been added to activate the Sbm pathway for the conversion of succinyl-CoA to propionyl-CoA. The strain produces PHBV with a HV content of 1-30 mol % at a mass yield of 5-80% of dry cell weight.
Genes that encode enzymes that 1) convert propionate to propionyl-CoA, 2) comprise a pathway for the conversion of butyrate to succinate, 3) comprise a pathway for the conversion of butyrate to acetyl-CoA, and 4) facilitate the conversion of succinate to succinyl-CoA are stably integrated into the genome of E. coli. The expression of lvaE and pct(Cp) is controlled by a synthetic promoter and the corresponding constitutive expression cassette is integrated into the genome of strain CPC-Sbm, or any strain derived from it, at a locus corresponding to a nonessential gene to facilitate the conversion of butyrate to butyryl-CoA and propionate to propionyl-CoA, respectively. Subsequently, a constitutive expression cassette consisting of fadE, fadB, and atoB and a synthetic promoter is integrated into a locus corresponding to a nonessential gene in the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated lvaE:pct(Cp) expression cassette to facilitate the conversion of butyryl-CoA to acetyl-CoA. One or more constitutive expression cassettes consisting of gadAe, FG99_15380, pduP(Se), and gabD and one or more synthetic promoters are then integrated into the genome of a derivative of strain CPC-Sbm containing genomically-integrated lvaE:pct(Cp) and fadE:fadB:atoB expression cassettes at one or more loci corresponding to one or more nonessential genes to facilitate the conversion of butyryl-CoA to succinate. Subsequently, a constitutive expression cassette consisting of CKL_RS14680 and a synthetic promoter is integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated lvaE:pct(Cp), fadE:fadB:atoB, gadAe, FG99_15380, pduP(Se), and gabD expression cassettes at a locus corresponding to a nonessential gene to facilitate the conversion of succinate to succinyl-CoA. Finally, the resulting strain containing genomically-integrated lvaE:pct(Cp), fadE:fadB:atoB, gadAe, FG99_15380, pduP(Se), gabD, and CKL_RS14680 expression cassettes are subsequently co-transformed with plasmids pPhaCAB (encoding phaA, phaB, and phaC) and pKBktB (encoding bktB) [18], and the resulting strain is evaluated for PHBV production in shake flask and/or bioreactor cultures in which cyanocobalamin has been added to activate the Sbm pathway for the conversion of succinyl-CoA to propionyl-CoA. The strain produces PHBV with a HV content of 1-40 mol % at a mass yield of 5-80% of dry cell weight.
Genes that encode enzymes that 1) convert propionate to propionyl-CoA, 2) comprise a pathway for the conversion of butyrate to (R)-HB-CoA, 3) comprise a pathway for the conversion of butyrate to succinate, or 4) facilitate the conversion of succinate to succinyl-CoA are stably integrated into the genome of E. coli. Inventor has determined that inactivation of iclR, encoding a transcriptional repressor that regulates the glyoxylate shunt in E. coli, can stimulate propionyl-CoA production from acetate when the Sbm pathway is activated (
Genes that encode enzymes that 1) convert propionate to propionyl-CoA, 2) comprise a pathway for the conversion of butyrate to succinate, or 3) facilitate the conversion of succinate to succinyl-CoA are stably integrated into the genome of E. coli. The expression of pct(Cp) is controlled by a synthetic promoter and the corresponding constitutive expression cassette is integrated into the genome of strain CPC-Sbm, or any strain derived from it, at a locus corresponding to a nonessential gene to facilitate the conversion of propionate to propionyl-CoA as outlined in Example 13. Subsequently, a constitutive expression cassette consisting of lvaE and a synthetic promoter is integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp) expression cassette at a locus corresponding to a nonessential gene to facilitate the conversion of butyrate to butyryl-CoA. Subsequently, the native fadR promoter is replaced with the rhamnose-inducible promoter Prha from the rhaBAD operon of E. coli in the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp) and lvaE expression cassettes to facilitate inducible derepression of fadE, which will restrict the conversion of butyryl-CoA to crotonyl-CoA to reduce butyrate dissimilation for biomass accumulation in a tunable manner. In addition, an atoS:atoC(I129S) expression cassette containing the native promoter is integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp), lvaE, and Prha::fadR expression cassettes to confer constitutive expression of the atoDAEB operon. Subsequently, one or more constitutive expression cassettes consisting of gad(Ls), FG99_15380, pduP(Se), and gabD and one or more synthetic promoters are integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp), lvaE, Prha::fadR, and atoS:atoC(I129S) expression cassettes at a locus corresponding to one or more nonessential genes to facilitate the conversion of butyryl-CoA to succinate. Subsequently, a constitutive expression cassette consisting of CKL_RS14680 and a synthetic promoter is integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated pct(Cp), lvaE, Prha::fadR, atoS:atoC(I129S), gad(Ls), FG99_15380, pduP(Se), and gabD expression cassettes at a locus corresponding to a nonessential gene to facilitate the conversion of succinate to succinyl-CoA. Finally, the resulting strain containing genomically-integrated pct(Cp), lvaE, Prha::fadR, atoS:atoC(I129S), gad(Ls), FG99_15380, pduP(Se), gabD, and CKL_RS14680 expression cassettes are subsequently co-transformed with plasmids pPhaCAB (encoding phaA, phaB, and phaC) and pKBktB (encoding bktB) [18], and the resulting strain is evaluated for PHBV production in shake flask and/or bioreactor cultures in which cyanocobalamin has been added to activate the Sbm pathway for the conversion of succinyl-CoA to propionyl-CoA. The strain produces PHBV with a HV content of 1-50 mol % at a mass yield of 5-80% of dry cell weight.
Genes that encode enzymes that 1) convert propionate to propionyl-CoA, 2) comprise a pathway for the conversion of butyrate to (R)-HB-CoA, 3) comprise a pathway for the conversion of butyrate to succinate, 4) facilitate the conversion of succinate to succinyl-CoA, 5) comprise the pathways for the conversion of acetyl-CoA to (R)-HB-CoA, and acetyl-CoA and propionyl-CoA to (R)-HV-CoA, or 6) facilitate the polymerization of (R)-HB-CoA and (R)-HV-CoA to PHBV are stably integrated into the genome of E. coli. The construction of a strain containing genomically-integrated lvaE:pct(Cp), fadE:fadB:atoB, gadAe, FG99_15380, pduP(Se), gabD, and CKL_RS14680 expression cassettes was described in Example 15. A constitutive expression cassette consisting of phaC, phaB, bktB, phaA and one or more synthetic promoters is integrated into the genome of a derivative of strain CPC-Sbm that contains the genomically-integrated lvaE:pct(Cp), fadE:fadB:atoB, gadAe, FG99_15380, pduP(Se), gabD, and CKL_RS14680 expression cassettes at loci corresponding to nonessential genes to facilitate the conversion of acetyl-CoA to (R)-HB-CoA, acetyl-CoA and propionyl-CoA to (R)-HV-CoA, and the polymerization of (R)-HB-CoA and (R)-HV-CoA to PHBV. Finally, the resulting strain containing genomically-integrated lvaE:pct(Cp), fadE:fadB:atoB, gadAe, FG99_15380, pduP(Se), gabD, CKL_RS14680, phaC, phaB, bktB, and phaA expression cassettes is evaluated for PHBV production in shake flask and/or bioreactor cultures in which cyanocobalamin has been added to activate the Sbm pathway for the conversion of succinyl-CoA to propionyl-CoA. The strain produces PHBV with a HV content of 1-40 mol % at a mass yield of 5-80% of dry cell weight.
Carbon flux through the Sbm pathway primarily occurs through the reductive TCA cycle under low oxygenic conditions. However, high carbon flux through the Sbm pathway was achieved under aerobic conditions by simultaneously blocking the oxidative TCA cycle and deregulating the glyoxylate shunt through respective inactivation of sdhA and iclR. Accordingly, strains CPC-Sbm, CPC-Sbm(ΔiclR), and CPC-Sbm(ΔiclR ΔsdhA) were tested for their ability to consume acetate under aerobic and microaerobic conditions. These strains were cultivated in the base medium supplemented with 20 g/L sodium acetate, 0.3 mM IPTG, and 0.6 μM vitamin B12 in capped (microaerobic) and vented (aerobic) 125 mL polycarbonate flasks (
Strains CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:pct(Cp)) and CPC-Sbm(pK-bktB-hbd-tesB, pTrc-phaAB:pct(Me)) were evaluated for their ability to co-produce HB and HV from acetate and propionate, with or without glycerol. These strains were cultivated in the base medium supplemented with 5 g/L sodium acetate, 4 g/L sodium propionate, 0.3 mM IPTG, 30 mg/L kanamycin, and 60 mg/L ampicillin, with or without 5 g/L glycerol in 125 mL Erlenmeyer flasks with foam stoppers (i.e. under aerobic conditions;
Strains CPC-Sbm(pK-lvaE:tesB, Ptrc-PP_2216:H16_RS27940), CPC-Sbm(pK-lvaE:tesB, Ptrc-BC_5341:H16_RS27940), CPC-Sbm(pK-atoDAE:tesB, Ptrc-PP_2216:H16_RS27940), CPC-Sbm(pK-atoDAE:tesB, Ptrc-BC_5341:H16_RS27940), and CPC-Sbm(pK-lvaE:tesB, Ptrc-PP_2216:phaJ(Ac)) were evaluated for their ability to produce HB from butyrate. These strains were cultivated in the base medium supplemented with 3 g/L sodium butyrate, 10 g/L glucose (as carbon source for growth), 0.3 mM IPTG, 30 mg/L kanamycin, and 60 mg/L ampicillin in 125 mL Erlenmeyer flasks with foam stoppers (
Strains CPC-Sbm(pK-lvaE:gadAe, PTrc-FG99_15380:pduP(Se):gabD) and CPC-Sbm(pK-lvaE:gadAe, PTrc-FG99_15380:pduP(Kp):gabD) were evaluated for their ability to produce succinate from butyrate. These strains were cultivated in the base medium supplemented with 3 g/L sodium butyrate, 10 g/L glucose, 0.3 mM IPTG, 30 mg/L kanamycin, and 60 mg/L ampicillin in 125 mL Erlenmeyer flasks with foam stoppers (
An expression cassette containing 1) promoter Pgracmax2, a stronger derivative of promoter Pgrac, 2) the strong RBS from gene 10 of Phage T7 (T7.RBS) that can significantly enhance translation efficiency relative to the consensus RBS of E. coli, 3) bktB, 4) a strong Gram-positive RBS coupled with a nine bp sequence derived from T7.RBS (i.e. TTAACTTTA) that facilitates base-pairing with the 16S rRNA of E. coli to enhance translation efficiency (RBS1), 5)phaB, and 6) a strong transcriptional terminator was genomically integrated into the bcsA locus of CPC-Sbm, resulting in strain CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB). An expression cassette containing the same elements as previously described, except that bktB and phaB were respectively replaced with phaC and phaA, was subsequently genomically integrated into the intF locus of CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB), resulting in strain CPC-Sbm(bcsA::(Pgracmax2::(T7.RBS)bktB:(RBS1)phaB), intF::(Pgracmax2::(T7.RBS)phaC:(RBS1)phaA). This strain was fermented in a medium containing 30 g/L glycerol, 10 g/L yeast extract, 10 mM NaHCO3, 0.4 μM vitamin B12, and 1000th dilution (i.e. 1 mL/L) trace elements (2.86 g/L H3BO3, 1.81 g/L MnCl2·4H2O, 0.222 g/L ZnSO4·7H2O, 0.39 g/L Na2MoO4·2H2O, 79 μg/L CuSO4·5H2O, 49.4 μg/L Co(NO3)2·6H2O), 0.1 mM IPTG, 0.23 g/L K2HPO4, 0.51 g/L NH4Cl, 49.8 mg/L MgCl2, 48.1 mg/L K2SO4, 2.78 mg/L FeSO4·7H2O, 0.055 mg/L CaCl2, 2.93 g/L NaCl, and 0.72 g/L tricine under different aeration conditions, resulting in the production of PHBV with a HV content of 15-40 mol % at a mass yield of up to 80% of dry cell weight. Further details are provided in Phan T T P et al., Protein expression and purification 2006, 46:189-195, the contents of which are incorporated herein by reference in its entirety for all purposes.
To analyze the factors that possibly contribute to the production of PHBV with a Mw of 1-1.5 MDa, the following experiments were performed to test the effect of different variables, such as, the use of thermostable enzymes, the order of the genes in an operon, ribosomal binding sites and genome integration sites.
Strains listed in Table 7 below were analyzed for their ability to produce PHBV using the methods described herein. While GEN-EC-GLY-01 strain was engineered to comprise nucleic acid molecules encoding the Cupriavidus necator PhaA protein, the Cupriavidus necator PhaB protein, the Cupriavidus necator PhaC protein and the Cupriavidus necator BtkB protein, the GEN-EC-GLY-17 strain was engineered to comprise nucleic acid molecules encoding the Cupriavidus sp. S-6 PhaA protein, the Cupriavidus sp. S-6 PhaB protein, the Cupriavidus sp. S-6 PhaC protein and the Cupriavidus gilardii QJ1 BtkB protein.
Without being bound by a theory, it is thought that, because Cupriavidus necator is a mesophile, the Cupriavidus necator PhaA, PhaB, PhaC and BtkB proteins would be thermostable at a temperature of about 28° C. to about 30° C., and thereby be capable of promoting the production of PHBV in the bacterial host cell at this temperature range. On the other hand, it is thought that since Cupriavidus sp. S-6 and Cupriavidus gilardii QJ1 are moderate thermophiles, the PhaA, PhaB, PhaC and BtkB proteins of these organisms would be thermostable at temperature higher than 30° C. (such as, at a temperature in the range of about 37° C. to about 50° C.), and thereby be capable of promoting the production of PHBV in the bacterial host cell at this higher temperature range.
Analysis of PHBV produced by the strains listed in Table 7 shows that GEN-EC-GLY-17 is indeed capable of producing PHBV at 37° C. However, surprisingly, it was seen that the molecular weight of PHBV produced varied based on the strain (
Next, the strains listed in Table 8 below, which differ in the order and combination of phaA, phaB and phaC genes in the operons, were analyzed for their ability to produce PHBV using the methods described herein.
As shown in
Next, the strains listed in Table 9 below, which differ in the ribosomal binding site (RBS) used in the phaCAB expression cassette, were analyzed for their ability to produce PHBV using the methods described herein.
While GEN-EC-GLY-13 comprises a nucleic acid molecule encoding PhaA, PhaB and PhaC proteins operably linked to a Pgracmax2 promoter and a RBS-5 ribosomal binding site, the GEN-EC-GLY-11 strain comprises a similar nucleic acid molecule encoding PhaA, PhaB and PhaC proteins operably linked to a Pgracmax2 promoter and a RBS-T7 ribosomal binding site. When the production of PHBV from glycerol by either of these strains was evaluated, the molecular weight of the PHBV produced was seen to differ. As shown in
While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The following list of embodiments is included herein for illustration purposes only and is not intended to be comprehensive or limiting. The subject matter to be claimed is expressly not limited to the following embodiments.
The following list of embodiments is included herein for illustration purposes only and is not intended to be comprehensive or limiting. The subject matter to be claimed is expressly not limited to the following embodiments.
The present application claims the benefit of priority to U.S. Provisional Application No. 63/342,707, filed on May 17, 2022, and U.S. Provisional Application No. 63/426,558, filed on Nov. 18, 2022, the contents of each of which are hereby incorporated by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| 63342707 | May 2022 | US | |
| 63426558 | Nov 2022 | US |
