WO2017210381A1

WO2017210381A1 - Bioconversion of 1-carbon feedstocks to chemicals and fuels

Info

Publication number: WO2017210381A1
Application number: PCT/US2017/035364
Authority: WO
Inventors: Ramon Gonzalez; Alexander CHOU
Original assignee: William Marsh Rice University
Current assignee: William Marsh Rice University
Priority date: 2016-06-02
Filing date: 2017-06-01
Publication date: 2017-12-07
Anticipated expiration: 2018-12-02

Abstract

A method for the biosynthesis of carbon-based products of interest by assimilation of single carbon feedstocks is described. A reaction pathway that consolidates carbon fixation, central metabolism, and product synthesis is designed and implemented as either a genetically engineered microorganism or as an enzyme reaction mixture. The invention makes use of a carbon-carbon bond formation mechanism that has not been previously exploited for this purpose and which is based on the use of formate acyltransferases. The result is a method that allows for production of chemical products by the utilization of either solely single carbon feedstocks or a mixture of single and multi-carbon feedstocks.

Description

BYCONVERSION OF 1-CARBON FEEDSTOCKS TO CHEMICALS AND FUELS

PRIOR RELATED APPLICATIONS

[0001] This Application claims priority to U.S. Serial No. 62/344,825, filed June 2,

2016, incorporated by reference herein in its entirety for all purposes. PCT/US 15/58121 (WO2016069929) is incorporated by reference herein in its entirety for all purposes.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

FIELD OF THE DISCLOSURE

[0003] The invention relates to biotechnological methods for the production of industrially relevant chemicals from 1-carbon (CI) feedstocks. In particular, methods for the biological production of carbon-based products of interest directly by the assimilation of single carbon units are described.

BACKGROUND OF THE DISCLOSURE

[0004] In the past, much our energy and chemicals have derived from fossilized carbon resources, such as petroleum and coal. However, concerns about climate change, political instability, and depletion and cost of petroleum resources have recently ignited interest in the establishment of a bio-based industry and a number of strategies are being pursued to achieve the sustainable production of fuels and chemicals. One of the most promising approaches is the use of microorganisms to convert biomass into the desired product. The use of these feedstocks, however, is hindered by the difficulty of accessing the resource, their high price, and limited availability. One-carbon compounds, including C0₂, CO, formate, formaldehyde, methanol, and methane have recently emerged as alternative renewable feedstocks. Conversion of these substrates to fuels and chemicals not only offers an attractive alternative in terms of process economics, but also will further contribute to reducing the levels of greenhouse gases, such as C0₂ and methane. [0005] Microbes have been designed and engineered to synthesize products of interest using feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane. For the case of 1 -carbon (C I) feedstocks, such conversions are made possible by a general network of metabolic pathways that are organized as shown in FIG. 1 and include specialized pathways for carbon fixation, central metabolism, and product synthesis. This type of metabolic architecture has been exploited in all metabolic engineering efforts conducted to date to develop microbes for industrial applications. This 'top-down' engineering strategy is highly complex and suffers from inefficiencies arising from need to first produce common metabolic intermediates before eventually forming products. This architecture is also commonly limited to elongation of a carbon backbone by a minimum of two carbons per step, which is a result of the aforementioned use of common metabolic intermediates.

[0006] This disclosure describes an alternative platform for the bioconversion of 1 - carbon substrates to carbon-based products of interest, which consists of a single engineered metabolic pathway that allows for the direct assimilation of one-carbon compounds. The pathway uses single carbon extension units, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single carbon increments.

SUMMARY OF THE DISCLOSURE [0007] The development of a synthetic, biological pathway that consolidates fixation of single carbon compounds, such as C0₂, CO, formate, formaldehyde, methanol, and methane, into the synthesis of carbon-based products is described. The pathway allows for direct synthesis of products from single carbon compounds, avoiding the need to produce the cells' typical intermediates and use of central metabolic pathways, as required by the prior approaches (FIG. 1).

[0008] The new synthetic pathway centers on the ability for formate acyltransferase enzymes, such as pyruvate formate lyase, to catalyze the condensation of a molecule of formate with an acyl-CoA. The resulting 2-ketoacid can then be converted back to an acyl- CoA, now one carbon longer than the originating acyl-CoA, resulting in overall carbon chain elongation and the production of useful products. [0009] The reactions of the pathway are enabled by providing enzymes ito catalyze the needed reactions. In one embodiment of the invention, the necessary gene sequences are provided in an engineered microbial host, such that the microorganism synthesizes the enzymes that comprise the pathway, allowing for this engineered microorganism to synthesize carbon-based products of interest from single carbon molecules. In another embodiment, the enzymes that comprise the pathway are purified and combined in a reaction mixture, providing the ability to synthesize carbon-based products of interest from single carbon molecules. In yet another embodiment, the bacteria themselves can be harvested and used as non-growing bioreactors for the reactions. However, the use of living, growing systems is preferred.

[0010] In one embodiment of the invention, carbon-based products of interest are produced solely from single carbon molecules. In another embodiment, however, products can be produced from a combination of single carbon molecules and multi-carbon molecules. One could provide any intermediate in the designed synthetic pathway as a starting compound, and need not make the entire molecule from scratch. Such embodiments may be useful where an intermediate is plentiful or a waste product from another process, and thus very inexpensive. These additions can also aid the throughput of the designed pathway and allow for the production of more varied products.

[0011] Reactions and the enzymes that catalyze said reactions described herein are understood to operate both in the direction described or illustrated, as well as in the reverse direction unless otherwise stated.

[0012] The use of the word "a" or "an" when used in conjunction with the term

"comprising" in the claims or the specification means one or more than one, unless the context dictates otherwise. [0013] The term "about" means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.

[0014] The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0015] The terms "comprise", "have", "include" and "contain" (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim. [0016] As used herein, the expressions "microorganism," "microbe," "bacteria",

"strain" and the like may be used interchangeably and all such designations include their progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0017] As used herein, reference to a "cell," "microbe," etc. is generally understood to include a culture of such cells, as the work described herein is done in cultures having 10^9" ¹⁵ cells. [0018] As used herein, "growing" cells used it its art accepted manner, referring to exponential growth of a culture of cells, not the few cells that may not have completed their cell cycle at stationary phase or have not yet died in the death phase or after harvesting.

[0019] As used in the claims, "homolog" means an enzyme with at least 40% identity to one of the listed sequences and also having the same general catalytic activity, although of course Km, Kcat, and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it is typical for bacterial sequences to diverge significantly (40-60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).

[0020] Reference to proteins herein can be understood to include reference to the gene encoding such protein. Thus, a claimed "permease" protein can include the related gene encoding that permease. However, it is preferred herein to refer to the protein by standard name per ecoliwiki or HUGO since both enzymatic and gene names have varied widely, especially in the prokaryotic arts.

[0021] Once an exemplary protein is obtained, many additional examples proteins of similar activity can be identified by BLAST search. Further, every protein record is linked to a gene record, making it easy to design expression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them. But, if necessary, new clones can be prepared based on available sequence information using RT-PCR techniques or gene synthesis. Thus, it should be easily possible to obtain all of the needed enzymes for expression or overexpression. [0022] Another way of finding suitable enzymes/proteins for use in the invention is to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme. An enzyme that thus be obtained, e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein.

[0023] Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides that encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be "optimized" for expression in E. coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.

[0024] In calculating "% identity" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI website. The default parameters were used, except the filters were turned OFF. [0025] "Operably associated" or "operably linked", as used herein, refer to functionally coupled nucleic acid or amino acid sequences.

[0026] "Recombinant" is relating to, derived from, or containing genetically engineered material. In other words, the genome or genetic material was intentionally manipulated in some way. [0027] "Reduced activity" or "inactivation" is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species (e.g., the wild type gene in the same host species). Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codons, by frame shift mutation, and the like. [0028] By "null" or "knockout" what is meant is that the mutation produces undetectable active protein. A gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can also completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All null mutants herein are signified by Δ.

[0029] "Overexpression" or "overexpressed" is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any expression in a species that lacks the activity altogether. Preferably, the activity is increased 100-500%). Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. All overexpressed genes or proteins are signified herein by "+". [0030] In certain species it is possible to genetically engineer the endogenous protein to be overexpressed by changing the regulatory sequences or removing repressors. However, overexpressing the gene by inclusion on selectable plasmids that exist in hundreds of copies in the cell may be preferred due to its simplicity and ease of exerting externals controls, although permanent modifications to the genome may be preferred in the long term for stability reasons.

[0031] The term "endogenous" means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated, or placed under the control of a promoter that results in overexpression or controlled expression of said gene. Thus, genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be overexpressed. "Native" means having a wild type sequence from the species in question.

[0032] "Expression vectors" are used in accordance with the art accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist.

[0033] As used herein, "inducible" means that gene expression can be controlled by the hand-of-man, by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac operon inducible by IPTG, the yeast AOX1 promoter inducible with methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesis may still occur even in expression vectors with tightly controlled promoters.

[0034] As used herein, an "integrated sequence" means the sequence has been integrated into the host genome, as opposed to being maintained on an extra-chromosomal expression vector. It will still be expressible, and preferably is inducible as well.

[0035] As used herein, "carbon based products of interest" refers to products that can be made in microbes, including e.g., alcohols, such as ethanol, butanol, saturated and unsaturated fatty alcohols; diols, such as ethylene glycol, 1,3-propanediol, 1,4-butanediol; polyols, such as glycerol, erythritol; carboxylic acids, such as acetate, propionate, butyrate, crotonate, saturated and unsaturated fatty acids; hydroxycarboxylic acids, such as glycolate, lactate, 3-hydroxybutyrate, polyhydroxybutyrate; dicarboxylic acids, such as adipic acid, succinic acid; alkanes; alkenes; amines; polyketides; fatty acid esters.

BRIEF DESCRIPTION OF FIGURES [0036] FIG. 1. Current 'top-down' metabolic engineering approach based on editing existing architecture of natural metabolism.

[0037] FIG. 2. Single carbon manipulation reactions for the generation of formate and formyl-CoA. Representative enzymes for each reaction are given in the legend.

[0038] FIG. 3. A pathway for the assimilation of single carbon molecules into carbon based products of interest. Representative enzymes for each reaction are given in the legend. Pathway intermediates that are potential products of interest without additional modification are highlighted.

[0039] FIG. 4. An embodiment of the invention that results in the production of saturated or unsaturated carboxylic acids by the action of a thioesterase enzyme. [0040] FIG. 5. An embodiment of the invention that results in the production of alcohols by the action of an alcohol dehydrogenase enzyme.

[0041] FIG. 6. An embodiment of the invention that results in the production of alkanes by the action of an aldehyde decarbonylase enzyme. [0042] FIG. 7. An embodiment of the invention that results in the production of amines by the action of a transaminase enzyme.

[0043] FIG. 8. Vector construct containing the gene encoding formate acyl- transferase pflB and its activating enzyme pflA from E. coli for overexpression in E. coli.

[0044] FIG. 9. Vector construct containing the gene encoding N-terminal HIS-tagged acylating aldehyde reductase Lmol 179 from Lysteria monocytogenes for expression in E. coli.

[0045] FIG. 10. SDS-PAGE showing expression and purification of L. monocytogenes acylating aldehyde reductase Lmol 179 from E. coli.

[0046] FIG. 11. Time course of absorbance at 340 nm corresponding to the production of NADH in the assay of L. monocytogenes acylating aldehyde reductase Lmol 179.

[0047] FIG. 12. ESI-TOF MS data of the -CoA content of L. monocytogenes acylating aldehyde reductase Lmol 179 reaction assay mixtures after solid phase extraction.

[0048] FIG. 13. Vector construct containing the gene encoding LcdABC from Clostridium propionicum for expression in E. coli. LcdABC is an example of a 2- hydroxyacyl-CoA dehydratase.

[0049] FIG. 14. Eadie-Hofstee plot for the determination of Euglena gracilis TER

(egTER) enzyme kinetics. egTER is an example of transenoyl-CoA reductase.

[0050] FIG. 15. Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of MhpF.

[0051] FIG. 16. Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of FucO. [0052] FIG. 17. Time course of absorbance at 340 nm corresponding to the production of NADH in the assay of KoPddABC coupled to acyl-CoA reductase.

[0053] FIG. 18 HPLC chromatogram of the in vitro assembly of the trans-2-enoyl-

CoA reductase and acyl-CoA reductase steps of Scheme B. A peak corresponding to butyraldehyde (butyraldehyde standard in bold) was present in samples containing Treponema denticola TER (tdTER) and E. coli MhpF (solid, thin line), but not the control containing tdTER only (dashed line).

[0054] Table 1. Reaction and enzyme list for the designed pathway. These are only a few of the enzymes that can be used, but provides a representative listing. [0055] Table 2. Summary of enzymes that have been characterized in vitro.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0056] Herein, our focus will be the design of a pathway that allows for the direct assimilation of single carbon molecules into carbon-based products of interest. Such a pathway would allow for a carbon backbone to be elongated by one-carbon units to give the desired product. Details describing the implementation of the proposed invention are given below.

[0057] The first function of the pathway is illustrated in FIG. 2. Single carbon molecules of various reduction levels are interconverted by the illustrated reactions to produce formate, the single carbon unit used to extend a carbon backbone. Details regarding the reactions and exemplary enzymes that accomplish the first function can be found in TABLE 1. Methane can be oxidized to methanol (FIG. 2, reaction 1) by a suitable methane monooxygenase. Methanol can be oxidized to formaldehyde (FIG. 2, reaction 2) by a suitable methanol dehydrogenase. Formaldehyde can be oxidized to formate (FIG. 2, reaction 3) by an aldehyde dehydrogenase. Carbon dioxide can be reduced to formate (FIG. 2, reaction 4) by a formate dehydrogenase or by electrochemical methods.

[0058] In some embodiments of the invention, single carbon molecules are the solely supplied carbon source. In these situations, a one-carbon acyl-CoA, formyl-CoA, is produced. Formate can be converted to formyl-CoA either directly (FIG. 2, reaction 7) by a suitable acetyl-CoA synthetase or through the intermediate formyl-phosphate (FIG. 2, reaction 5-6) by a suitable formate kinase and phosphate acetyl-transferase. Formaldehyde can also be converted to formyl-CoA by a suitable acyl-CoA reductase.

[0059] Combinations of the above reactions can be used to generate formyl-CoA from other single carbon molecules. For example, an implementation that makes use of methane would include the expression of a methane monooxygenase, a methanol dehydrogenase, and an acyl-CoA reductase. Even more combinations of the described reactions and accompanying enzymes can be used to allow for implementations that use a mixture of single carbon units, for example a combination of methane and carbon dioxide through all of the described reactions. At a minimum, this function can be accomplished from either formaldehyde, by the expression of an acylating aldehyde dehydrogenase, or from formate, by a suitable acetyl-CoA synthetase or combined formate kinase and phosphate acetyl-transferase.

[0060] The second function of the pathway is the iterative elongation of a carbon backbone by the single carbon unit formate, known as an "extender unit" herein. This is illustrated in FIG. 3. Details regarding the reactions and exemplary enzymes that accomplish the second function can be found in TABLE 1. First, formate is condensed with an acyl-CoA to give a 2-ketoacid that is one carbon longer than the initial acyl-CoA (FIG. 3, reaction 1) by a suitable formate acyl-transferase. The 2-ketoacid is then reduced to a 2-hydroxyacid (FIG. 3, reaction 2) by a suitable 2-hydroxyacid dehydrogenase. The 2-hydroxyacid is then converted to a 2-hydroxyacyl-CoA (FIG. 3, reaction 3) by a suitable acyl-CoA synthetase.

[0061] In one embodiment of the invention, the 2-hydroxyacyl-CoA is reduced to a 2- hydroxyaldehyde (FIG. 3, reaction 4) by a suitable acyl-CoA reductase. The 2- hydroxyaldehyde is further reduced to a 1,2-diol (FIG. 3, reaction 5) by a suitable 1,2-diol oxidoreductase. The 1,2-diol is then converted to an aldehyde (FIG. 3, reaction 6) by a suitable diol dehydratase. Finally, the resulting aldehyde is converted to an acyl-CoA (FIG. 3, reaction 7) that is one carbon longer than the initial acyl-CoA, by an acyl-CoA reductase. This acyl-CoA can be used for further rounds of elongation.

[0062] In another embodiment of the invention, the 2-hydroxyacyl-CoA produced earlier is converted to a trans-2-enoyl-CoA (FIG. 3, reaction 8) by a suitable 2-hydroxyacyl- CoA dehydratase. The resulting trans-2-enoyl-CoA is converted to an acyl-CoA (FIG. 3, reaction 9) that is one carbon longer than the initial acyl-CoA, by a trans-2-enoyl-CoA reductase. This acyl-CoA can be used for further rounds of elongation.

[0063] In other embodiments of the invention, a combination of the above routes can be implemented at the same time such that for some carbon chain lengths, elongation takes place through FIG. 3 reactions 4-7, whereas for other carbon chain lengths, elongation takes place through FIG. 3 reactions 8 and 9. However, both routes can be simultaneously present at the same time in the same system.

[0064] In some embodiments of the invention, the carbon-based products of interest are the intermediates of the above reactions. Examples of these products are highlighted in FIG. 3 and include ketoacids, hydroxyacids, aldehydes, diols and polyols. In other embodiments of the invention, products are obtained by producing products of interest from the intermediates of the above reactions. These products of interest include, but are not limited to alcohols, such as ethanol, butanol, saturated and unsaturated fatty alcohols; diols, such as ethylene glycol, 1,3 -propanediol, 1,4-butanediol; polyols, such as glycerol, erythritol; carboxylic acids, such as acetate, propionate, butyrate, crotonate, saturated and unsaturated fatty acids; hydroxy carboxylic acids, such as glycolate, lactate, 3-hydroxybutyrate, polyhydroxybutyrate; dicarboxylic acids, such as adipic acid, succinic acid; alkanes; alkenes; amines; polyketides; and fatty acid esters.

[0065] To give some examples, products containing carboxylic acids can be produced by providing suitable thioesterase enzymes, which convert acyl-CoAs into carboxylic acids (FIG. 4). Alcohols can be produced by providing a suitable alcohol dehydrogenase enzyme, which converts aldehydes into alcohols (FIG. 5). Alkanes can be produced by providing suitable aldehyde decarbonylase enzymes, which convert aldehydes into alkanes (FIG. 6). Amines can be produced by providing suitable transaminase enzymes, which convert aldehydes into amines (FIG. 7).

[0066] In one embodiment of the invention, the described pathway is provided within the context of a microbial host. The pathway in a living system is generally made by transforming the microbe with one or more expression vector(s) containing a gene encoding one or more of the enzymes, but the genes can also be added to the chromosome by recombineering, homologous recombination, gene editing, and similar techniques. Where the needed protein is endogenous, as is the case in some instances, it may suffice as is, but is usually overexpressed for better functionality and control over the level of active enzyme. Preferable, one or more, or all, such genes are under the control of an inducible promoter.

[0067] Initial cloning experiments have proceeded in E. coli for convenience since most of the required genes are already available in plasmids suitable for bacterial expression, but the addition of genes to bacteria is of nearly universal applicability. Indeed, since recombinant methods were invented in the 70' s and are now so commonplace, even school children perform genetic engineering experiments using bacteria. Such species include e.g., Bacillus, Streptomyces, Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus, or any of the completely sequenced bacterial species. Indeed, hundreds of bacterial genomes have been completely sequenced, and this information greatly simplifies both the generation of vectors encoding the needed genes, as well as the planning of a recombinant engineering protocol. Such species are listed along with links at en.wikipedia.org/wiki/List_of_sequenced_bacterial_genomes. [0068] Additionally, yeasts are common species used for microbial manufacturing, and many species can be successfully transformed. In fact, the alpha oxidation pathway is present in yeast and thioesterases, and the reverse beta oxidation pathways have been successfully expressed in yeast Saccharomyces . Other species include but are not limited to Candida, Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, and Yarrowia lipolytica, to name a few.

[0069] It is also possible to genetically modify many species of algae, including e.g.,

Spirulina, Apergillus, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica, and the like. Indeed, the microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas. [0070] Furthermore, a number of databases include vector information and/or a repository of vectors and can be used to choose expression vectors suitable for the chosen host species. See e.g., AddGene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See also Plasmid Information Database (PlasmID) and DNASU having over 191,000 plasmids. A collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.

[0071] The enzymes can be added to the genome or via expression vectors, as desired. Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via plasmid or other vector. Initial experiments may employ expression plasmids hosting 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for stability reasons.

[0072] Still further improvements in yield can be had by reducing competing pathways, such as those pathways for making e.g., acetate, formate, ethanol, and lactate, and it is already well known in the art how to reduce or knockout these pathways. See e.g., the Rice patent portfolio by Drs. San and Bennett (US7569380, US7262046, US8962272, US8795991) and patents by these inventors (US8129157 and US8691552) (each incorporated by reference herein in its entirety for all purposes). Many others have worked in this area as well.

[0073] Following the construction of a suitable strain containing the engineered pathway, culturing of the developed strains can be performed to evaluate the effectiveness of the pathway at its intended goal— the production of products from single carbon compounds. The organism can be cultured in a suitable growth medium, and can be evaluated for product formation on single carbon substrates, from methane to C0₂, either alone or in combination with multi-carbon molecules. The amount of products produced by the organism can be measured by UPLC or GC, and indicators of performance such as growth rate, productivity, titer, yield, or carbon efficiency can be determined. [0074] Further evaluation of the interaction of the pathway enzymes with each other and with the host system can allow for the optimization of pathway performance and minimization of deleterious effects. Because the pathway is under synthetic control, rather than under the organism's natively evolved regulatory mechanisms, the expression of the pathway is usually manually tuned to avoid potential issues that slow cell growth or production and to optimize production of desired compounds.

[0075] Additionally, an imbalance in relative enzyme activities might restrict overall carbon flux throughout the pathway, leading to suboptimal production rates and the buildup of pathway intermediates, which can inhibit pathway enzymes or be cytotoxic. Analysis of the cell cultures by HPLC or GC can reveal the metabolic intermediates produced by the constructed strains. This information can point to potential pathway issues.

[0076] As an alternative to the in vivo expression of the pathway, a cell free in vitro version of the pathway can be constructed. By purifying the relevant enzyme for each reaction step, the overall pathway can be assembled by combining the necessary enzymes in a reaction mixture. With the addition of the relevant cofactors and substrates, the pathway can be assessed for its performance independently of a host.

[0077] In one embodiment of the invention, single carbon molecules, such as carbon dioxide, formate, formaldehyde, methanol, methane, and carbon monoxide are solely used in the production of products containing at least one carboxyl group. In this embodiment, both formate and the priming acyl-CoA, in the form of formyl-CoA, are produced from single carbon molecules as described earlier.

[0078] In another embodiment of the invention, the priming acyl-CoA can be derived from multi-carbon substrates. In this embodiment, the invention is implemented in an engineered microbial host that is designed to be able to convert the multi-carbon molecules into an acyl-CoA. Some exemplary substrates include glucose and other sugars or glycerol and other sugar alcohols, which may be converted to acyl-CoAs via a pathway such as glycolysis, resulting in the production of acetyl-CoA or malonyl-CoA. Still additional examples include substrates that contain carboxyl groups, which can be directly converted to acyl-CoAs by providing a suitable acyl-CoA synthetase. This further increases the diversity of products that can be produced by the invention. For example, by providing a carboxylic acid with an co-phenyl group, different phenylated products of different length and functionality can be produced. In addition to increasing product diversity, the multi-carbon substrates may provide additional carbon and energy for microbial survival.

[0079] The following description of experiments provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other details, as it would be of inordinate length if one were to list every possible combination of genes/vectors/enzymes/hosts that can be made to implement the engineered pathway.

GENERAL METHODS [0080] Enzymes of interest can be expressed from vectors such as pCDFDuet-1

(Merck KGaA, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience. [0081] The genes can be amplified by PCR using primers designed with 15-25 base pairs of homology for the appropriate vector cut site. For enzymes that will not require a 6X- histadine tag fusion for purification, pCDFDuet-1 can be linearized with Ncol and EcoRI. Enzymes that will be purified by Ni-NTA column will make use of the 6X-HIS tag in pCDFDuet-1. The vector can be linearized using only EcoRI in this case. [0082] The PCR product can be inserted into the vector using e.g., the In-Fusion HD

EcoDry Cloning System and the vector transformed by heat shock into competent E. coli cells. Transformants can be selected on solid media containing the appropriate antibiotic. Plasmid DNA can be isolated using any suitable method, including QIAprep Spin Miniprep Kit (QIAGEN, Limburg), and the construct confirmed by PCR and sequencing. Confirmed constructs can be transformed by e.g., electroporation into a host strain such as E. coli for expression, but other host species can be used with suitable expression vectors and possible codon optimization for that host species.

[0083] Expression of the desired enzymes from the constructed strain can be conducted in liquid culture, e.g., shaking flasks, bioreactors, chemostats, fermentation tanks and the like. Gene expression is typically induced by the addition of a suitable inducer, when the culture reaches an OD550 of approximately 0.5-0.8. Induced cells can be grown for about 4-8 hours, at which point the cells can be pelleted and saved to -20°C. Expression of the desired protein can be confirmed by running cell pellet samples on SDS-PAGE or by enzymatic assay.

[0084] The expressed enzyme can be directly assayed in crude cell lysates, simply by breaking the cells by chemical, enzymatic, heat or mechanical means. Depending on the expression level and activity of the enzyme, however, purification may be required to be able to measure enzyme activity over background levels. Purified enzymes can also allow for the in vitro assembly of the pathway, allowing for its controlled characterization. N-terminal or C-terminal HIS-tagged proteins can be purified using e.g., a Ni-NTA Spin Kit (Qiagen, Venlo, Limburg) following the manufacturer's protocol, or other methods could be used. The HIS-tag system was chosen for convenience only, and other tags are available for purification uses. Further, the proteins in the final assembled pathway need not be tagged if they are for in vivo use. Tagging was convenient, however, for the enzyme characterization work performed herein.

[0085] The reaction conditions for enzyme assays can vary greatly with the type of enzyme to be tested. In general, however, enzyme assays follow a similar general protocol. Purified enzyme or crude lysate is added to suitable reaction buffer. Reaction buffers typically contain salts, necessary enzyme cofactors, and are at the proper pH. Buffer compositions often change depending on the enzyme or reaction type. The reaction is initiated by the addition of substrate, and some aspect of the reaction related either to the consumption of a substrate or the production of a product is monitored.

[0086] Choice of the appropriate monitoring method depends on the compound to be measured. Spectrophotometric assays are convenient because they allow for the real time determination of enzyme activity by measuring the concentration dependent absorbance of a compound at a certain wavelength. There are not always compounds with a measureable absorbance at convenient wavelengths in the reaction, unfortunately. In these situations, other methods of chemical analysis may be necessary to determine the concentration of the involved compounds. [0087] Gas chromatography (GC) is convenient for the quantification of volatile substances, of which fatty acids and aldehydes are of particular relevance. Internal standards, typically one or more molecules of similar type not involved in the reaction, is added to the reaction mixture, and the reaction mixture is extracted with an organic solvent, such as hexane. Fatty acid samples, for example, can be dried under a stream of nitrogen and converted to their trimethylsilyl derivatives using N,0-Bis(trimethylsilyl)trifluoroacetamide ("BSTFA") and pyridine in a 1 : 1 ratio. After 30 minutes incubation, the samples are once again dried and resuspended in hexane to be applied to the GC. Aldehyde samples do not need to be derivatized. Samples can be run e.g., on a Varian CP-3800 gas chromatograph (VARIAN ASSOC., CA) equipped with a flame ionization detector and HP-5 capillary column (AGILENT TECH., CA).

[0088] Once the pathway has been fully studied in vitro, the pathway can be constructed in vivo with greater confidence. The strain construction for the in vivo pathway operation should allow for the well-defined, controlled expression of the enzymes of the pathway. As before, E. coli, B. subtilus or yeast will be a host of choice for the in vivo pathway, but other hosts could be used. The Duet system (MERCK KGaA, Germany), allows for the simultaneous expression of up to eight proteins by induction with IPTG in E. coli, and initial experiments will use this host.

[0089] Pathway enzymes can also be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of plasmids. There are also, theoretically, an infinite number of genes that can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with plasmid expression.

[0090] DNA constructs for chromosomal integration usually include an antibiotic resistance marker with flanking FRT sites for removal, as described by Datsenko and Wanner, a well characterized promoter, a ribosome binding site, the gene of interest, and a transcriptional terminator. The overall product is a linear DNA fragment with 50 base pairs of homology for the target site on the chromosome flanking each side of the construct.

[0091] However, the Flp-FRT recombination method is only one system for adding genes to a chromosome, and other systems are available, such as the RecBCD pathway, the RecF pathway, RecA recombinase, non-homologous end joining (NHEJ), Cre-Lox recombination, TYR recombinases and integrases, SER resolvases/invertases, SER integrases, PhiC31 Integrase, and the like. Chromosomal modifications in E. coli can also achieved by the method of recombineering, as originally described by Datsenko and Wanner.

[0092] In a recombineering method, for example, the cells are prepared for electroporation following standard techniques, and the cells transformed with linear DNA that contains flanking 50 base pair targeting homology for the desired modification site. For seamless integration of a DNA construct, a two-step approach can be taken using a cassette that contains both positive and negative selection markers, such as the combination of cat and sacB. In the first round of recombineering, the cat-sacB cassette with targeting homology for the desired modification site is introduced to the cells. The cat gene provides resistance to chloramphenicol, which allows for positive recombinants to be selected for on solid media containing chloramphenicol. A positive isolate can be subjected to a second round of recombineering introducing the desired DNA construct with targeting homology for sites that correspond to the removal of the cat-sacB cassette. The sacB gene encodes for an enzyme that provides sensitivity to sucrose. Thus, growth on media containing sucrose allows for the selection of recombinants in which the cat-sacB construct was removed. PI phage ly sates can be made from isolates confirmed by PCR and sequencing. The lysates can be used to transduce the modification into desired strains, as described previously.

[0093] Engineered strains expressing the designed pathway can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for autotrophic growth can be tested by the use of formate or formaldehyde as a substrate in MOPS minimal media, as described by Neidhardt, supplemented with appropriate antibiotics, and inducers. Mixotrophic growth can be characterized by the addition of both single carbon compounds and glucose or glycerol.

[0094] Analysis of culture media after fermentation provides insight into the performance of the engineered pathway. Quantification of longer chain products can be analyzed by GC. Other metabolites, such as short chain organic acids and substrates such as glucose or glycerol can be analyzed by UPLC. Once the pathway is fully functional, the cultures can be grown in chemostat, providing continuous uninterrupted production of product in a living, growing system if desired.

[0095] Various -omics techniques, such as microarray or 2D-PAGE can give information about gene expression or protein expression, respectively. Genome scale modeling allows for the identification of additional modifications to the host strain that might lead to improved performance. Deletion of competing pathways, for example, might increase carbon flux through the engineered pathway for product production.

[0096] Standard molecular biology techniques were used for gene cloning, plasmid isolation, and E. coli transformation. Native E. coli genes were amplified from E. coli MG1655 genomic DNA using primers to append 15 bp of homology on each end of the gene insert for recombination into the vector backbone. Genes from other organisms were codon optimized and synthesized by either GeneArt (LIFE TECH., CA or GENSCRJPT, NJ). Plasmids were linearized by the appropriate restriction enzymes and recombined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system (CLONTECH LAB. CA,). The mixture was subsequently transformed into Stellar competent cells (CLONTECH LAB.).

[0097] Transformants that grew on solid media (LB+Agar) supplemented with the appropriate antibiotic were isolated and screened for the gene insert by PCR. Plasmid was isolated from the verified transformants and the sequence of the gene insert was further confirmed by DNA sequencing (LONE STAR LABS, TX). Plasmids (also referred to as vectors) in each case contain at least one promoter, a ribosome binding site for each gene, the gene(s) of interest, at least one terminator, an origin of replication, and an antibiotic resistance marker. Exemplary plasmids are shown in FIG. 8, 9, and 13.

PROOF OF CONCEPT

[0098] A plasmid containing the codon optimized gene encoding 6X HIS-tagged Lmol l79 from E monocytogenes was constructed as described above. The resulting construct, FIG. 9, was transformed into E. coli BL21(DE3) for expression. The resulting strain was cultured in 50 mL of TB media containing 50 μg/mL spectinomycin in a 250 mL flask. When the culture reached an OD550 of approximately 0.6, expression was induced by the addition of 0.1 mM IPTG, and the cells were harvested by centrifugation after overnight incubation at room temperature. [0099] The resulting cell pellet was resuspended in Bacterial Protein Extraction

Reagent (B-PER) (THERMO SCI, MA) to an OD550 of approximately 40, to which approximately 5000 U of lysozyme and approximately 250 U of Benzonase nuclease (SIGMA-ALDRICH CO., MO) were added. The cell mixture was left at room temperature until completely clarified to give the cell extract. 1 M stock solution of imidazole was added to provide a final concentration of 10 mM imidazole in the cell extract.

[00100] The HIS-tagged Lmol l79 protein was purified from the cell extract using

Talon Metal Affinity Resin (CLONTECH LAB.). In short, a 250 μΐ. resin bed was equilibrated twice using 2.5 mL of a buffer containing 50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole at pH 7.5 (NPI-10). The cell extract was added to the resin and the mixture shaken gently for 20 minutes on ice. The resin was then washed twice with 2.5 mL buffer NPI-20 (same as NPI-10 but with 20 mM imidazole), shaking gently on ice for 15 minutes each wash. The resin was then transferred to a gravity column and washed once with 1.25 mL NPI-20. Finally, the desired protein was eluted using 1.25 mL of buffer NPI-250 (same as buffer NPI-10 but with 250 mM imidazole), and the eluate collected in 500 μΕ fractions. A sample purification of Lmol 179 is shown in FIG. 10.

[00101] The genes that encode the enzymes of the engineered pathway were cloned and expressed as described above. The purified enzymes were assessed for their ability to catalyze the proposed reactions as summarized in Table 2. Here we describe testing in vitro the steps that make up an exemplary pathway.

[00102] Lmol 179 was cloned (FIG. 9), expressed, and purified (FIG. 10) in E. coli as described above. The purified enzyme was evaluated for its ability to convert formaldehyde into the extender unit formyl-CoA. Enzyme assays were performed in 23 mM potassium phosphate buffer pH 7.0, 1 mM CoASH, 0.5 mM NAD⁺, 20 mM 2-mercaptoethanol, and 50 mM formaldehyde. The reaction was monitored by measuring absorbance at 340 nm, corresponding to the production of NADH. CoA compounds were extracted from the reaction mixture by solid phase extraction (SPE) using a CI 8 column, and the mass of the extracted CoAs were determined by ESI-TOF MS. The inclusion of Lmol 179 in the reaction mixture resulted in the conversion of formaldehyde to formyl-CoA as indicated by the coproduction of NADH (FIG. 11). Mass spectrometry analysis confirmed the production of formyl-CoA by Lmol 179. A peak at the expected mass of formyl-CoA (796) was identified in the sample incubated with enzyme, as shown in FIG. 12. The peak was not present in the no-enzyme control (FIG. 12), indicating that Lmol 179 produced formyl-CoA.

[00103] In some embodiments, the 2-hydroxyacyl-CoA undergoes dehydration to its corresponding trans-2-enoyl-CoA. A 2-hydroxyacyl-CoA dehydratase was identified as LcdABC from C. propionicum. LcdABC has been characterized by Hofmeister and Buckel for the dehydration of 2-hydroxybutyryl-CoA to crotonyl-CoA, with specific activity corresponding to 1.21 ± 0.08

protein (Hofmeister & Buckel, 1992). The genes encoding LcdABC were cloned as described above (FIG. 13).

[00104] The trans-2-enoyl-CoA is then reduced to the saturated acyl-CoA by a trans-2- enoyl-CoA reductase. A variant from E. gracilis, EgTER, was identified and cloned as described above. After expression and purification of EgTER, in vitro assays were performed by monitoring the loss of NADH absorbance in the presence of 100 mM Tris HCL pH 7.5 and 0.2 mM NADH in a final volume of 200 [iL at 25°C. This revealed that the enzyme is capable of catalyzing the conversion of crotonyl-CoA to butyryl-CoA with specific activity corresponding to 1.21 ± 0.08 μιηοΐ/min/mg protein (FIG. 14).

[00105] The saturated acyl-CoA is finally converted to its corresponding aldehyde by an acyl-CoA reductase. Native E. coli acyl-CoA reductase MhpF was tested for its ability to convert butyryl-CoA to butyraldehyde (FIG. 15). E. coli MhpF was expressed from an ASKA collection strain. The ASKA collection is a set of ORFs cloned from E. coli K12 W3110 into the high copy number plasmid pCA24N. This plasmid has a modified pMBl replication origin (same as pQE30 from Qiagen) and CamR marker. The cloned ORF is under control of the IPTG-inducible T5-lac promoter.

[00106] Strains were grown anaerobically in 10 mL LB with 100 mM Tris pH 8.0, 10 g/L glucose, 50 μΜ FeS0₄, 5 μΜ NaH₂Se0₃, and 5 μΜ (NH₄)₆Mo₇0₂₄. The cells were grown to 0.4 OD₅₅₀ at which point protein expression was induced by the addition of 0.1 mM IPTG. After 3 hours growth, cells were pelleted and the pellet was resuspended to 40 OD in 100 mM MOPS pH 7.5. 1 mL of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor (SCI. INDUS., NY). The cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays. The assay mixture consisted of 100 mM MOPS pH 7.5, 6 mM DTT, 5 mM MgS0₄, 0.3 mM Fe(NH₄)₂(S0₄)₂, 0.3 mM NADH, and 0.2 mM butyryl- CoA. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of NADH, as shown in FIG. 15. MhpF was capable of catalyzing the conversion with a specific activity of 0.009 ± 0.003

protein.

[00107] E. coli FucO was assayed for its ability to perform an analogous reaction— the reduction of butyraldehyde to butanol. FucO was expressed from an ASKA collection strain and purified as described above. Purified FucO was assayed in a buffer containing 100 mM Tris-HCl pH 7.5, 0.3 mM NADH, and 10 mM butyraldehyde. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of NADH, as shown in FIG. 16. FucO was capable of catalyzing the reduction of butyraldehyde to butanol with a specific activity of 5.08 ± 0.08 μιηοΐ/min/mg protein. Furthermore, FucO was tested for the ability to convert glycoaldehyde, a 2-hydroxyaldehyde, to ethane- 1,2-diol, as necessary for Scheme A. FucO was capable of catalyzing the reduction with a specific activity of 4.060 ± 0.004 μιηοΐ/min/mg protein.

[00108] KoPddABC, a diol dehydratase from Klebsiella oxytoca, was cloned, expressed, and purified as described. Cell extracts of E. coli BL21(DE3) expressing KoPddABC were prepared by resuspending a pellet of said E. coli to an OD 550 of 40 in 60 mM potassium phosphate buffer pH 7.4 with 200 mM 1,2-ethanediol. 1 mL of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor (SCI. INDUS. NY). The cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays.

[00109] The cell extract was incubated at 30°C for 3 hours in the presence of 10 μΜ coenzyme B 12. The reaction was terminated by the addition of 1% sulfuric acid, and the precipitant was pelleted by centrifugation. The supernatant was analyzed by HPLC. Acetaldehyde was detected in extracts of cells expressing KoPddABC, indicating that the dehydratase can convert 1,2-diols to their corresponding aldehydes. Diol dehydratase was further assayed by coupling the dehydration of ethylene glycol to acetaldehyde to an acyl- CoA reductase (LmACR) to give acetyl-CoA with the reduction of NAD+ to NADH, which was monitored at 340 nm.

[00110] The final assay mixture was 250 μΐ. and contained 50 mM potassium phosphate pH 7.5, 5 mM CoASH, 0.5 mM NAD+, 0.2 M ethylene glycol, 7 iL purified LmACR (from frozen stock purified previously), 50 iL cell lysate, and 15 μΜ coenzyme B 12. The relevant controls included were no cell lysate (replaced with 50 μΐ. of buffer) and no coenzyme B12. The assays were performed at 28°C. The results are shown in FIG. 17.

[00111] A portion of the proposed invention was assembled in vitro to demonstrate the functionality of the combined pathway steps. The trans-2-enoyl-CoA reductase and acyl-CoA reductase steps were combined to assess the overall conversion of crotonyl-CoA to butyraldehyde. Experiments were carried out carried out in 50 mM Tris buffer, pH 7.5 containing 1 mM DTT at 37°C. The reaction with MhpF contained 0.15 g/L TdTer, 0.1 g/L of MhpF, 7.5 mM NADH and 1.7 mM crotonyl-CoA was added to the media for use as a primer in the new reaction pathway. Reaction with Lmol l79 contained 0.15 g/L TdTer, 0.9 g/L Lmol l79, 7.5 mM NADH and 1.7 mM crotonyl-CoA. The assay was monitored by measuring production of butyraldehyde with FIPLC. When assayed under the conditions described above, the combination of trans-2-enoyl-CoA reductase from Treponema denticola and E. coli enzyme MhpF was shown to produce butyraldehyde from crotonyl-CoA to a final concentration of 0.55 mM (FIG. 18). (PROPHETIC) IN VIVO PATHWAY

[00112] The purpose of this example is to demonstrate the biosynthesis of chemicals by implementation of the described invention. In this implementation, an engineered E. coli strain MG1655(DE3) AfrmA AfdhFON serves as the host strain. This strain contains deletions of genes that compete for one carbon substrates formate and formaldehyde. Genes for overexpression are cloned into vectors or inserted into the chromosome using standard methods described above. Vectors and chromosomal constructs harbored by the host strain to give the engineered strains of interest.

[00113] When formate is the intended one carbon substrate, an acyl-CoA synthetase is selected for overexpression to catalyze the conversion of formate into formyl-CoA. A vector, for example pETDuet-l-Pl-acs, expressing an acyl-CoA synthetase from E. coli, is constructed and transformed into the host strain. When carbon dioxide is the intended one carbon substrate, a carbon dioxide reductase, catalyzing the conversion of carbon dioxide to formate is added. For example, pETDuet-l-Pl-acs-P2-AwFdhF2-hycB2-hycB3-hydA2 expressing carbon dioxide reductase from Acetobacterium woodii is constructed and transformed into the host strain. When formaldehyde is the intended one carbon substrate, an acyl-CoA reductase is selected for overexpression to catalyze the conversion of formaldehyde to formyl-CoA. Hydrolysis of formyl-CoA to formate is expected to occur spontaneously, but can be aided by expression of a thioesterase such as E. coli tesA. Here, a vector such as pETDuet-l-Pl-Lmol l79, expressing an acyl-CoA reductase from L. monocytogenes is constructed and transformed into the host strain. When methanol is the intended one carbon substrate, a methanol dehydrogenase is added. A vector such as pETDuet-l-Pl-Lmol 179-P2-BsMdh2, expressing a methanol dehydrogenase from Bacillus stearothermophilus is constructed and transformed into the host strain. When methane is the intended one carbon substrate, a methane monooxygenase is added, giving for example pETDuet-l-Pl-Lmol l79-P2-BsMdh2-MtMmoXYZBC-orfY, which further expresses a methane monooxygenase from Methylosinus trichosporium OB3b.

[00114] To initiate product generation, the condensation of formyl-CoA and formate is catalyzed by overexpression of a formate acyltransferase. A vector such as pRSFDuet-l-Pl- pflBA, expressing a formate acyltransferase from E. coli, is constructed and transformed into the host strain. This vector provides the means to produce glyoxylic acid from one-carbon substrates. 2-hydroxyacids, such as glycolic acid, can be produced by the additional overexpression of a 2-hydroxyacid dehydrogenase. A vector such as pRSFDuet-l-Pl-pflAB- P2-glcD, expressing a 2-hydroxyacid dehydrogenase from E. coli is constructed. When 1,2- diols are the desired product, overexpression of an acyl-CoA synthetase, acyl-CoA reductase, and 1,2-diol oxidoreductase are added. A vector such as pRSFDuet-l-Pl-pflAB-P2-glcD-acs- Lmol l79-fucO is constructed containing an acyl-CoA synthetase from E. coli, an acyl-CoA reductase from J. monocytogenes, and a 1,2-diol oxidoreductase ΐχονα Ε. coli.

[00115] To obtain longer carbon chain products, an acyl-CoA must be regenerated for further condensation with formate by formate acyltransferase. The acyl-CoA can be produced by overexpression of diol dehydratase and an acyl-CoA reductase. A vector such as pRSFDuet-l-Pl-pflAB-P2-glcD-acs-Lmol l79-fucO-KoPddABC is constructed containing a diol dehydratase from K. oxytoca. Here, the acyl-CoA reductase Lmol l79 catalyzes two acyl-CoA reductase reactions. Longer carbon chain products can also be obtained through a separate route, by overexpression of a 2-hydroxyacyl-CoA dehydratase and a trans-2-enoyl- CoA reductase to give the acyl-CoA. A suitable example vector would be pRSFDuet-l-Pl- pflAB-P2-glcD-acs-CpLcdABC-EgTER, containing genes for the formate acyltransferase, 2- hydroxyacid dehydrogenase, acyl-CoA synthetase, along with a 2-hydroxyacyl-CoA dehydratase from C. propionicum, and a trans-2-enoyl-CoA reductase from E. gracilis.

[00116] To catalyze a substrate to product conversion, any combination of a 'substrate conversion vector' and a 'product conversion vector' described above can be co-transformed into the host strain. pETDuet-1 and pRSFDuet-1 are compatible for simultaneous maintenance an E. coli. Thus, for example, for the conversion of methanol to glycolic acid, the host strain would be transformed with pETDuet-l-Pl-Lmol 179-P2-BsMdh2 and pRSFDuet-l-Pl-pflAB- P2-glcD to give the strain with genotype MG1655(DE3) AfrmA AfdhFON pETDuet-l-Pl- Lmol 179-P2-BsMdh2 pRSFDuet-l-Pl-pflAB-P2-glcD. [00117] An exemplary substrate to product conversion is performed as follows. The engineered E. coli is grown overnight in LB medium at 37°C. 250 μΐ. of the overnight culture is used to inoculate 25 mL of MOPS-LB-Glycerol medium (125 mM MOPS, 4 mM Tricine, 39.52 mM H₄C1, 5 mM ( H₄)₂S0₄, 0.276 mM K₂S0₄, 0.523 mM MgCl₂, 50 mM NaCl, 0.01 mM FeS0₄, 2.8 mM Na₂HP0₄, 0.5 μΜ CaCl₂, 0.004 μΜ ( H₄)₆Mo₇0₂₄, 0.4 μΜ H₃B0₃, 0.03 uM CoCl₂, 0.01 μΜ CuS0₄, 0.08 μΜ MnCl₂, 0.01 μΜ ZnS0₄, 15 uM thiamine, 10 g/L tryptone, 5 g/L yeast extract, 20 g/L glycerol) in a 125 mL baffled flask. When the culture OD550 reaches 0.4 to 0.6, IPTG is added to 0.1 mM to induce gene expression. 24 hours after inoculation, cells are harvested by centrifugation and the pellet is washed twice with MOPS minimal medium (the composition of MOPS minimal medium is the same as MOPS-LB-Glycerol without the tryptone, yeast extract, and glycerol components). Finally, the pellet containing the engineered E. coli is resuspended in MOPS minimal medium and the one carbon substrate of choice is added.

[00118] For gaseous substrates, the substrate is provided by bubbling into the cell suspension or by adding to the headspace of a sealed container. After 24 hours, the culture medium containing the desired product is recovered and saved for analysis or product isolation.

(PROPHETIC) IN VITRO PATHWAY

[00119] The purpose of this example is to demonstrate the biosynthesis of chemicals by implementation of the described invention. Vectors expressing the necessary enzymes to catalyze the desired substrate to product conversions are prepared as described above. E. coli strain BL21(DE3) is transformed with the necessary vectors to catalyze the desired substrate to product conversion using standard methods. The resulting engineered strain is grown overnight in LB medium at 37°C. The overnight culture is used to inoculate a larger volume of LB medium to 1%. The culture is grown at 30°C to an OD550 of 0.4 to 0.6 at which point gene expression is induced by adding IPTG to a concentration of 0.1 mM. 24 hours after inoculation, cells are harvested by centrifugation and the pellet is saved by freezing. When ready, the pellet is thawed and the cells are resuspended in 50 mM potassium phosphate buffer pH 7.4. The cells are broken by glass beads and the cell extract containing the expressed proteins is collected after centrifugation. The one carbon substrate, as well as any necessary cofactors, are added to the cell extract and the reaction mixture is incubated at room temperature for 16 hours. The reaction mixture is saved for analysis or product isolation.

[00120] A portion of the proposed invention was assembled in vitro to demonstrate the functionality of the combined pathway steps. The trans-2-enoyl-CoA reductase and acyl-CoA reductase steps were combined to assess the overall conversion of crotonyl-CoA to butyraldehyde. Experiments were carried out carried out in 50 mM Tris buffer, pH 7.5 containing 1 mM DTT at 37°C. The reaction with MhpF contained 0.15 g/L TdTer, 0.1 g/L of MhpF, 7.5 mM NADH and 1.7 mM crotonyl-CoA was added to the media for use as a primer in the new reaction pathway. Reaction with Lmol l79 contained 0.15 g/L TdTer, 0.9 g/L Lmol l79, 7.5 mM NADH and 1.7 mM crotonyl-CoA. The assay was monitored by measuring production of butyraldehyde with a HPLC. When assayed under the conditions described above, the combination of trans-2-enoyl-CoA reductase from T. denticola and E. coli enzyme MhpF was shown to produce butyraldehyde from crotonyl-CoA to a final concentration of 0.55 mM (FIG. 18). (PROPHETIC) OTHER SPECIES

[00121] The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease-deficient strain like WB800N is preferably used for greater stability of heterologous protein. The E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easily to a more suitable vector for Bacillus. Alternatively, two vectors pHTOl and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids ρΑΜβΙ and pBS72 can be used. Several other suitable expression systems are available. Since the FAS and BOX-R enzymes are ubiquitous, the invention is predicted to function in Bacillus.

[00122] The above experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Several yeast E. co\\ shuttle vectors are available for ease of the experiments.

[00123] The following references are incorporated by reference in their entirety herein for all purposes:

[00124] 61/440, 192, WO2012109176 US20130316413 : Reverse beta oxidation pathway

[00125] 61/531/911, 61/440, 192, WO2013036812 US20140273110: Functionalized carboxylic acids and alcohols by reverse fatty acid oxidation [00126] 62/069,850, WO2016069929: Biosynthesis of products from 1-carbon compounds

[00127] WO2015191422 Omega-hydroxylated carboxylic acids

[00128] 62/011,474, 62/012,113, 62/011,465, WO2015191972: Omega-carboxylated carboxylic acids and derivatives [00129] WO2015191422: Omega-hydroxylated carboxylic acids

[00130] Binstock, J.F. & Schulz, H. (1981) Fatty acid oxidation complex from

Escherichia coli. Methods in Enzymology, 71 Pt C(1967), 403-411.

[00131] Blecher, M. (1981) Synthesis of long-chain fatty acyl-CoA thioesters using N- hydroxysuccinimide esters. Methods in Enzymology, 72(1967), 404-408. [00132] Cintolesi, A., et al. (2014) In silico assessment of the metabolic capabilities of an engineered functional reversal of the β-oxidation cycle for the synthesis of longer-chain (C>4) products. Metabolic Engineering, 23, 100-15. [00133] Datsenko, K.A. & Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America, 97(12), 6640-5.

[00134] Hofmeister, A. & Buckel, W. (1992) (R)-Lactyl-CoA dehydratase from Clostridium propionicum. European J. Biochem., 552, 547-552.

[00135] Kitagawa, M., et al. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Research : An International Journal for Rapid Publication of Reports on Genes and Genomes, 12(5), 291-9. [00136] Neidhardt, F. C, et al. (1974) Culture Medium for Enterobacteria. Journal Of

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[00138] Parasaran, T., & Tarbell, D. S. (1964) Formic Ethylcarbonic Anhydride. The Journal of Organic Chemistry, 29(11), 3422-3423.

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C02 to formate by a bacterial carbon dioxide reductase. Science (New York, N.Y.), 342(6164), 1382-5.

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Claims

1. A genetically engineered microorganism, comprising overexpressed polypeptides catalyzing a pathway converting formate and a C(n)-acyl-CoA to a C(n+l)-acyl- CoA, said pathway selected from:

a. a formate acyltransferase catalyzing the conversion of formate and a C(n)- acyl-CoA to a 2-keto-C(n+l)-acid;

b. a 2-hydroxy-C(n+l)-acid dehydrogenase catalyzing the conversion of a 2- keto-C(n+l)-acid to a 2-hydroxy-C(n+l)-acid;

c. an acyl-CoA synthetase catalyzing the conversion of a 2-hydroxy-C(n+l)-acid a 2-hydroxy-C(n+l)-acyl-CoA;

d. an acyl-CoA reductase catalyzing the conversion of a 2-hydroxy-C(n+l)-acyl- CoA to a 2-hydroxy-C(n+l)-aldehyde;

e. a 1,2-diol oxidoreductase or alcohol dehydrogenase catalyzing the conversion of a 2-hydroxy-C(n+l)-aldehyde to a l,2-C(n+l)-diol;

f. a diol dehydratase catalyzing the conversion of a l,2-C(n+l)-diol to a C(n+1)- aldehyde;

g. an acyl-CoA reductase catalyzing the conversion of a C(n+l)-aldehyde to a C(n+l)-acyl-CoA;

wherein n refers to the number of carbons, and

wherein said microorganism assimilates single carbon molecules into carbon-based products of interest.

2. A genetically engineered microorganism, comprising overexpressed polypeptides catalyzing a pathway converting formate and a C(n)-acyl-CoA to a C(n+l)-acyl- CoA, said pathway selected from:

a. a formate acyltransferase catalyzing the conversion of formate and a C(n)- acyl-CoA to a 2-keto-C(n+l)-acid,;

d. a 2-hydroxyacyl-CoA dehydratase catalyzing the conversion of 2-hydroxy- C(n+l)-acyl-CoA to a trans-2-C(n+l)-enoyl-CoA; e. a trans-2-enoyl-CoA reductase catalyzing the conversion of a trans-2-C(n+l)- enoyl-CoA to a C(n+l)-acyl-CoA;

wherein n refers to the number of carbons, and

3. A genetically engineered microorganism, comprising overexpressed polypeptides catalyzing a pathway converting formate and a C(n)-acyl-CoA to a C(n+l)-acyl- CoA, said pathway selected from:

h. a 2-hydroxyacyl-CoA dehydratase catalyzing the conversion of 2-hydroxy- C(n+l)-acyl-CoA to a trans-2-C(n+l)-enoyl-CoA;

i. a trans-2-enoyl-CoA reductase catalyzing the conversion of a trans-2-C(n+l)- enoyl-CoA to a C(n+l)-acyl-CoA;

wherein n refers to the number of carbons, and

4. The microorganism of claims 1-3 wherein said formate is derived from

formaldehyde by the conversion of formaldehyde to formate by an aldehyde dehydrogenase.

5. The microorganism of claim 4, wherein said formaldehyde is derived from methanol by the conversion of methanol to formaldehyde by a methanol dehydrogenase.

6. The microorganism of claim 5, wherein said methanol is derived from methane by conversion of methane to methanol by a methane monooxygenase.

7. The microorganism of claims 1-3, wherein said formate is derived from carbon

dioxide by the conversion of carbon dioxide to formate by a formate dehydrogenase.

8. The microorganism of claims 1-3, wherein said C(n)-acyl-CoA is derived from a carboxylic acid by the conversion of the carboxylic acid to an acyl-CoA by an acyl- CoA synthetase.

9. The microorganism of claims 1-3, wherein said C(n)-acyl-CoA is derived from a carboxylic acid by the conversion of the carboxylic acid to an acyl-CoA by a kinase and phosphate acyl-transferase.

10. The microorganism of claims 1-3, wherein said formate acyltransferase is selected from £. colipflB ( P_415423), or E. coli tdcE (WP_000861734.1).

11. The microorganism of claims 1-3, wherein said 2-hydroxyacid dehydrogenase is selected from E. coli IdhA ( P_415898.1), E. coli did NP_416637.1), or E. coli lldD ( P_418062.1).

12. The microorganism of claims 1-3 and 8, wherein said acyl-CoA synthetase is

selected from E. coli acs ( P_418493.1), or E. coli fadK (NP_416216).

13. The microorganism of claims 1 and 3, wherein said acyl-CoA reductase is selected from E coli mhpF ( P 414885.1), Pseudomonas sp. CF600 dmpF (Q52060), Lysteria monocytogenes Lmoll79 ( P 464704), Salmonella typhimurium eutE ( P_461398), Acinetobacter calcoaceticus acrl (AAC45217.1), Acinetobacter sp Strain M-l acrM (BAB85476.1), Clostridium beijerinckii aid (AAT66436.1), or is. coli eutE ( P_416950.1).

14. The microorganism of claims 1 and 3, wherein said 1,2-diol oxidoreductase or

alcohol dehydrogenase is selected from E. coli betA ( P_414845.1), E. coli dkgA ( P_417485.4), E. coli eutG ( P_416948.4), E. colifucO ( P_417279.2), E. coli ucpA ( P_416921.4), E. coliyahK ( P_414859.1), E. coliybbO ( P_415026.1), E. coliybdH ( P_415132.1), £. coliyiaY (YP_026233.1), or E. coliyjgB

( P_418690.4).

15. The microorganism of claims 1 and 3, wherein said diol dehydratase is selected from Klebsiella ocytoca pddABC (Q59470, Q59471, Q59472), E. coli pduCDE

(CAS09680, CAS09681, CAS09682), or S. enterica pduCDE ( P_456590,

P_456591, P_456592).

16. The microorganism of claims 2 and 3, wherein said 2-hydroxyacyl-CoA dehydratase is selected from Clostridium propionicum IcdABC (G3KIM3, G3KTM4, G3KIM5), or Clostridium difficile hadBCI (AAV40818.1, AAV40819.1, AAV40820.1).

17. The microorganism of claims 2 and 3, wherein said trans-2-enoyl-CoA reductase is selected from Rhodobacter sphaeroides acul (Q3 J6K9.1), E. coliyhdH

( P_417719.1), E. coli fabl (NV_415804.1), Enterococcus faecalis fabK

( P 816503.1), Bacillus subtilis fabL (KFK80655.1), Euglena gracilis egTER (Q5EU90.1), Treponema denticola tdTER ( P 97121 1.1), or Vibrio cholera fabV (B 1P0R8).

18. The microorganism of claim 4, wherein said aldehyde dehydrogenase is selected from E. coli adhE (NP_415757), E. coli aldA ( P_415933), or E. coli aldB

( P_418045).

19. The microorganism of claim 5, wherein said methanol dehydrogenase is selected from Bacillus methanolicus mdh (P31005), Mycobacterium sp. DSM 3803 mdo (C5MRT8), or Methylobacterium extorquens moxl, moxF (P14775, P16027).

20. The microorganism of claim 6, wherein said methane monooxygenase is selected from Me thy losinus trichosporium OB3b mmoXYZBC, orfY (P27353, P27354, P27355, Q53563, P27356, Q53562), or Methylococcus capsulatus Bath mmoXYZBC, or/7 (P22869, P18798, PI 1987, P18797, P22868, P22867).

21. The microorganism of claim 7, wherein said formate dehydrogenase is selected from Acetobacterium woodii fdhF2, hycB2, hycB3, hydA2 (YP_005268502.1,

YP_005268503.1, YP_005268505.1, YP_005268506.1), E. colifdhF (NP_418503), E. colifdh-0 ( P_418330, P_418329, P_418328), or E. coli fdh-N (NV_4\ 5993, P_415992, P_415991).

22. The microorganism of claim 9, wherein said kinase is selected from Salmonella

typhimurium stAckA (Ί>6341 1), or E. coli AckA (NP_416799.1).

23. The microorganism of claim 9, wherein said phosphate acyl-transferase is an amino acid sequence selected from E. coli eutD ( P_416953.1j, E. colipta ( P_416800.1), or Salmonella typhimurium PduL (Q9XDN5).

24. A method for the production of carbon-based products of interest from single carbon molecules comprising:

a. providing a microorganism of any one of claims 1-23;

b. growing said microorganism in a culture comprising single-carbon molecules and a growth media under conditions whereby the microorganism assimilates said single-carbon molecules to produce a carbon-based product of interest; and,

c. isolating said product from said microorganism, or said growth media, or both.

25. A method for the production of carbon-based products of interest from a mixture of single-carbon molecules and multi-carbon substrates comprising:

a. providing the microorganism of claim 1-23 wherein said microorganism has been engineered to convert or natively converts said multi-carbon substrates into a priming acyl-CoA;

b. growing said microorganism in a culture comprising single-carbon molecules, multi-carbon substrates, and a growth media under conditions whereby the microorganism condenses the single-carbon molecules with said priming acyl- CoA to produce a carbon-based product of interest; and,

26. A method for the production of a carboxylic acid from single-carbon molecules

comprising:

a. providing a reaction mixture comprising purified polypeptides isolated from a microorganism of claim 1-23 and buffers, minerals, vitamins, and cofactors; b. contacting said reaction mixture with single-carbon molecules under

conditions whereby said single-carbon molecules are converted to a carbon based product of interest; and,

c. isolating said product from said reaction mixture.

27. A method according to claims 24-26, wherein the single-carbon molecule substrate is selected from the group consisting of methane, methanol, formaldehyde, formate, carbon monoxide, and carbon dioxide, and combinations thereof.

28. A method according to claims 24-27, wherein the carbon-based products of interest are selected from a group consisting of alcohols, such as ethanol, butanol, saturated and unsaturated fatty alcohols; diols, such as ethylene glycol, 1,3 -propanediol, 1,4- butanediol; polyols, such as glycerol, erythritol; carboxylic acids, such as acetate, propionate, butyrate, crotonate, saturated and unsaturated fatty acids;

hydroxy carboxylic acids, such as glycolate, lactate, 3-hydroxybutyrate,

polyhydroxybutyrate; dicarboxylic acids, such as adipic acid, succinic acid; alkanes; alkenes; amines; polyketides; fatty acid esters.

29. A method according to claims 24-28, wherein the product is a 1,2-diol, for example ethanediol, 1,2-propanediol, 1,2-butanediol, which is produced by the reduction of a 2-hydroxyaldehyde by a 1,2-diol oxidoreductase.

30. A method according to claims 24-28, wherein the product is a 2-hydroxyacid, for example glycolic acid, lactic acid, 2-hydroxybutyric acid, which is produced by the reduction of a 2-ketoacid by a 2-hydroxyacid dehydrogenase.

31. A method according to claims 24-28, wherein the product is a 2-ketoacid, for

example glyoxalic acid, pyruvic acid, 2-ketobutyric acid, which is produced by the condensation of formate with an acyl-CoA by a formate acyltransferase.

32. A method according to claims 24-28, wherein the product is a saturated or

unsaturated carboxylic acid, for example acetic acid, propionic acid, butyric acid, crotonic acid, which is produced from the acyl-CoA and trans-2-enoyl-CoA intermediates of the pathway by the action of a thioesterase.

33. A method according to claims 24-28, wherein the product is an n-alcohol, for

example ethanol, propanol, butanol, which is produced from the aldehyde intermediates of the pathway by the action of an alcohol dehydrogenase.

34. A method according to claims 24-28, wherein the product is an n-alkane, for

example ethane, propane, butane, which is produced from the aldehyde intermediates of the pathway by the action of an aldehyde decarbonylase.

35. A method according to claims 24-28, wherein the product is an amine, for example ethylamine, propylamine, butylamine, which is produced from the aldehyde intermediates of the pathway by the action of a transaminase.

36. A method according to claim 32, wherein said overexpressed thioesterase is selected from E. coli tesA ( P_415027.1), E. coli tesB ( P_414986.1), E. coliyciA ( P_415769.1), £. coli fadM (N? 14911 A\ E. coli ydil (NP_416201.1), E. coli ybgC ( P_415264.1), Alcanivorax borkumensis tesB2 (YP_692749.1) Fibrobacter succinogenes Fs2108 (YP 005822012.1), Prevotella ruminicola Pr655

(YP_003574018.1), or Prevotella ruminicola Pr 1687 (YP_003574982.1).

37. A method according to claim 33, wherein said overexpressed alcohol dehydrogenase is selected from E. coli betA ( P_414845.1), E. coli dkgA ( P_417485.4), E. coli eutG ( P_416948.4), E. colifucO ( P_417279.2), E. coli ucpA ( P_416921.4), E. coli yahK (NP_4\4859A\ E. coliybbO ( P_415026.1 ), E. coliybdH

( P_415132.1), £. coliyiaY (YP_026233.1), or E. coliyjgB ( P_418690.4).

38. A method according to claim 34, wherein said aldehyde decarbonylase is selected from Synechococcus elongatus PCC7942 orfl593 (Q54764.1), Nostoc punctiforme PCC73102 npun_R1711 (B2J1M1.1), or Prochlorococcus marinus MIT9313 pmtl231 (Q7V6D4.1).

39. A method according to claim 35, wherein said overexpressed transaminase is

selected from Arabidopsis thaliana At3g22200 ( P_001189947.1), Alcaligenes denitrificans AptA (AAP92672.1), Bordetella bronchiseptica BB0869

(WP 015041039.1 ), Bordetella parapertussis BPP0784 (WP 010927683.1 ), Brucella melitensis BAWG 0478 (EEW88370.1), Burkholderia pseudomallei BP1026B I0669 (AFI65333.1), Chromobacterium violaceum CV2025

(AAQ59697.1), Oceanicola granulosus OG2516_07293 (WP_007254984.1), Paracoccus denitrificans PD 1222 Pden_3984 (ABL72050.1), Pseudogulbenkiania ferrooxidans ω-ΤΑ (WP 008952788.1), Pseudomonas putida ω-ΤΑ (P28269.1), Ralstonia solanacearum co-TA (YP 002258353.1), Rhizobium meliloti SMc01534 ( P_386510.1), Vibrio fluvialis ω-ΤΑ (AEA39183.1), Mus musculus abaT

(AAH58521.1), or E. coli gabT (YP_490877.1).

40. A method according to claim 24-26, wherein said microorganism is a member of a genus selected from the group consisting of Escherichia, Saccharomyces,

Zymomonas, Bacillus, Lactobacillus, Corynebacterium, Brevibacterium, Pichia, Candida, Clostridium, Salmonella, Rhodococcus, Pseudomonas, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, and Hansenula.