Carboxylic Acid Platform for Carbon- and Energy-Efficient Production of Fuel and Chemicals

Abstract

The present disclosure provides a novel conceptual framework in which orthogonal and novel carbon and energy conversion pathways facilitate the synthesis of fuels and chemicals from carboxylic acid intermediates (CAi) derived by genetically engineered microorganisms.This allows the CAi platform to produce diverse products with ≧100% carbon yield while retaining the established high product and energy efficiency of fermentative metabolism.In another embodiment, a carboxylic acid platform for fuel and chemical production with high carbon and energy efficiency is also provided.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 220,927, filed July 12, 2021, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Award Nos. DE-AR0001508 and DE-EE0008499 awarded by the Department of Energy. The Government has certain rights in this invention.

[0003] References to sequence listing N / A [Background technology]

[0004] background Fermentative metabolism offers high product flux and energy efficiency through both C-conserving and CO2-releasing pathways. With sugars as the sole source of carbon and energy, the C-conserving pathway is used for the synthesis of neutral or oxidized products (e.g., carboxylic acids), while the CO2-releasing pathway is used for reduced products, including most industrial chemicals and fuels (e.g., alcohols). However, with the availability of electricity / external reducing equivalents, a paradigm shift is needed to focus on C-conservation as opposed to E-conservation, which is often required by metabolism. To develop carbon-optimized fermentation systems for sugar conversion, cellular metabolism must be re-engineered to focus on carbon conservation versus its advanced prioritization of energy conservation.

[0005] Current efforts in this area focus on either recovering carbon lost from the CO2-export pathway or rewiring central metabolism to conserve carbon and prevent carbon loss in the form of CO2 (Bogorad, I., et al. Nature 502:693-697 (2013);Lin, P., et al. Proc. Nat. Acad. Sci. USA 115:3538-3546 (2018);Schuchmann, K., Muller, V. Appl. Environ. Microbiol. 82:4056-4069 (2016);Wang, Q., et al. Metab. Eng. 51: 79-87 (2019);Mainguet, S., et al. Metab. Eng. 19:116-127 (2013);Yu, H., et al. Nat. Commun. 9:10 (2018);Francois, J., et al. Front. Bioeng. Biotechnol. 7:16 (2020)). These approaches require extensive engineering of host metabolic networks that circumvent natural regulations and limit the ability to harness high glycolytic fluxes and generate products efficiently.

[0006] Contrary to modifying native metabolism, we propose a novel conceptual framework in which orthogonal, novel carbon and energy conversion pathways facilitate the synthesis of fuels and chemicals from carboxylic acid intermediates (CAi) induced by exogenously supplied electron donors. This allows the CAi platform to generate diverse products with >= 100% carbon yield while maintaining the established high product and energy efficiency of fermentative metabolism. Moreover, the CAi platform utilizes enzymes and pathways that are ubiquitous in nature, thus enabling its implementation in industrial organisms. Summary of the Invention

[0007] overview As disclosed herein, the carboxylic acid intermediate (CAi) platform is a novel conceptual framework in which novel carbon conversion and energy generation pathways to nature support the synthesis of diverse reductive products with ≧100% carbon harvesting rate. Contrary to current approaches to carbon-optimized fermentation that rely on rewiring central metabolism, the CAi platform strengthens the orthogonality of these pathways to the host metabolism. This allows the established high product flux and energy efficiency of fermentative metabolism to be retained while driving downstream product synthesis from carbon-conserving CAi via externally supplied reductive equivalents. CAi, such as lactate, produced with 100% carbon efficiency, connects upstream pathways and product synthesis pathways based on novel biochemistry that facilitates the production of a range of products in a carbon-conserving or carbon-consuming manner.

[0008] Starting with activation of CAi(s), such as converting lactate to lactoyl-CoA, downstream pathways are based on various novel biochemistries, such as direct reduction reactions for product synthesis or the generation of molecules as precursors for one-carbon (C1) addition via acyloin condensation. The energy required to drive these reactions is derived from reduced C1 compounds via synthetic C1 bioreformation pathways that allow the generation of NADH and ATP (via substrate-level phosphorylation) or H2 for NADH generation via soluble hydrogenase. The ubiquity of fermentative metabolism and the orthogonality of CAi carbon and energy conversion pathways facilitate implementation in various industrial hosts to develop carbon-optimized fermentation systems that avoid carbon losses during reduced product synthesis.

[0009] Therefore, the present specification discloses microorganisms that are not naturally capable of producing reduced products from carboxylic acid intermediates without carbon loss, but are genetically engineered to do so.The genetic engineering of these organisms includes providing a cell system with a first set of metabolic enzymes for activating CA intermediate(s) to corresponding acyl-CoA, a second set of metabolic enzymes for converting acyl-CoA intermediates to products, and a third set of metabolic enzymes for producing reducing equivalents (e.g., NADH) and ATP from an externally supplied energy source (e.g., reduced one-carbon compounds and / or H2), and supplying the system with a carbon source that can be converted to a CA intermediate (e.g., glucose) and an externally supplied energy source under suitable conditions for metabolic enzymes to produce products that are more reduced than the initial CA intermediate.

[0010] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0011] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1. Energy (redox and ATP) requirements for the carboxylate platform and the enzymatic pathway for generating formyl-CoA. [Diagram 2] Figure 2A-2D. Formyl-CoA production from formate. A) Pathway screening of FAE platforms for formyl-CoA production from formate with formaldehyde as a co-substrate; B) Screening results of FAE candidates with formaldehyde and formate using resting cells; C) Pathway screening of FAE platforms for formyl-CoA production from formate with acetone as a co-substrate; D) Screening results of FAE candidates with acetone and formate using growing cultures. [Diagram 3]Figures 3A-3B. Generation of formyl-CoA from formaldehyde using acyl-CoA reductase. Synthesis of glycolyl-CoA-derived glycolate via condensation of formaldehyde and formyl-CoA was used as a proxy for pathway flux. (a) Pathway showing flux from formaldehyde to glycolate in which ACR can act as both ACR1 and ACR2. (b) Screening results of various ACR1 / ACR2 candidates. [Figure 4] Figures 4A-4B. Production of formyl-CoA from methanol-derived formaldehyde using different methanol dehydrogenases (MDH) with LmACR. Synthesis of glycolyl-CoA-derived glycolate via condensation of formaldehyde and formyl-CoA was used as a proxy for pathway flux. (a) Complete pathway showing energy (redox) production (in the form of NADH) and formyl-CoA subsequently fed to glycolate synthesis. Enzymes in bold are overexpressed for the experiments in panels (b) and (c). (b) Resting cell bioconversion of methanol to glycolate using different candidates for MDH. (c) Growing cell bioconversion of methanol to energy (indicated by cell biomass) and formyl-CoA (indicated by glycolate production). [Diagram 5] Figure 5. Carboxylic acid platform using formate as the starting carboxylic acid. [Figure 6] Figure 6. Carboxylic acid platform using formaldehyde as an intermediate aldehyde for C1 elongation. [Figure 7] 7A-7B. Conversion of formate to ethylene glycol. A) Pathway for conversion of formate to ethylene glycol; B) In vitro conversion of formate to ethylene glycol using purified enzymes. [Figure 8]8A-8D. (a) High-throughput resting cell biotransformation platform for screening C1 elongase (HACS) and formate-activating enzyme (FAE; AbfT in this example). (b) Glycolate production from a joint feed of formaldehyde (5 mM) and formate (20 mM). (c) Expression of HACS and FAE (AbfT) is independently controlled by inducible promoters (IPTG and cumate). (d) HACS screening results for C1-C1 condensation reaction as shown by glycolate productivity. [Figure 9] Figures 9A-9B. Cell-free pathway prototyping of the complete α-reduction pathway starting with formaldehyde as a surrogate. (a) The complete iteration to the C2 aldehyde, acetaldehyde via α-reduction and the relevant enzymes used for cell-free prototyping. (b) Changes in substrate and product concentrations over time with different combinations of enzymes added to the reaction mixture. [Figure 10] Figures 10A-10B. (a) Synthesis of glycine from glycolate as an alternative to C1-C1 condensation followed by CoA hydrolysis. An E. coli strain was constructed that is unable to synthesize glycine. This strain can only grow if either supplemented with glycine or provided with an alternative substrate (glycolate) along with the appropriate enzymes to synthesize glycine. (b) The strain can only grow with glycolate supplementation showing higher growth rates with increasing glycolate concentrations. [Figure 11] Figure 11. Carboxylic acid platform using formamide as an intermediate (substituted) aldehyde for C1 elongation. [Figure 12] Figure 12. Formamidase (MmFmdA) under the control of the T7lac promoter in the expression vector (pCDFDuet-1). [Figure 13] Figure 13. Carboxylic acid platform using C2+ carboxylic acids as substrates. [Figure 14-1] Figures 14A-4B. (a) Carboxylic acid platform using acetic acid as a substrate. [Figure 14-2](b) HACS screening results for acetaldehyde-C1 condensation reaction represented by lactic acid productivity. [Figure 15] Figures 15A-15B. (a) High-throughput resting cell biotransformation platform for screening C1 elongase (HACS). (b) Expression of HACS and FAE (AbfT) is independently controlled by inducible promoters (IPTG and cumate). [Figure 16-1] Figures 16A-16B. (a) Carboxylic acid platform using propionic acid as a substrate. [Figure 16-2] (b) HACS screening results for propionaldehyde-C1 condensation reaction represented by 2-hydroxybutyric acid productivity. [Figure 17] Figure 17. Carboxylic acid platform using butyric acid as substrate. [Figure 18-1] Figures 18A-18B. (a) Carboxylic acid platform using glycolic acid as the substrate. [Figure 18-2] (b) HACS screening results for glycolaldehyde-C1 condensation reaction represented by glyceric acid productivity. [Figure 19] Figure 19. Carboxylic acid platform using lactate as substrate. [Figure 20] Figure 20. Carboxylic acid platform using glyceric acid as a substrate. [Figure 21] Figure 21. Carboxylic acid platform using 3-hydroxypropionic acid as substrate. [Figure 22] 22A-22B. (a) Carboxylic acid platform using oxalic acid as substrate. (b) HACS screening results for glyoxylic acid-C1 condensation reaction represented by tartronic acid productivity. [Figure 23] Figure 23. Carboxylic acid platform using malonic acid as substrate. [Figure 24] Figure 24. Carboxylic acid platform using succinic acid as substrate. [Diagram 25] Figure 25. Carboxylic acid platform using isobutyric acid as substrate. [Figure 26] Figure 26. Carboxylic acid platform using isovaleric acid as a substrate. [Figure 27] Figure 27. Preparation of 2-hydroxy acids, 3-hydroxy acids, alcohols, 1,2-diols, and α,β-unsaturated acids from the condensation of carboxylic acid-derived ketones and formyl-CoA. [Figure 28] Figure 28. Preparation of 2-hydroxyisobutyric acid, 3-hydroxyisobutyric acid, isobutanol, isobutene glycol, and methacrylic acid from the condensation of lactic acid-derived acetone and formyl-CoA. [Figure 29] Figure 29. Preparation of 2-hydroxy-2-methylbutanoic acid, 3-hydroxy-2-methylbutanoic acid, 2-methylbutan-1-ol, 2-methylbutane-1,2-diol, and 2-methylbut-2-enoic acid from the condensation of 2-hydroxybutanoic acid-derived butanone and formyl-CoA. [Diagram 30] Figure 30. Preparation of 2-hydroxy-2-methylpentanoic acid, 3-hydroxy-2-methylpentanoic acid, 2-methylpentan-1-ol, 2-methylpentane-1,2-diol, and 2-methylpent-2-enoic acid from the condensation of 2-hydroxypentanoic acid-derived pentanone and formyl-CoA. [Diagram 31] Figure 31. Preparation of 2-hydroxy-2-methylheptanoic acid, 3-hydroxy-2-methylheptanoic acid, 2-methylheptan-1-ol, 2-methylheptane-1,2-diol, and 2-methylhept-2-enoic acid from the condensation of 2-hydroxyheptanoic acid-derived heptanone and formyl-CoA. [Diagram 32] Figure 32. Preparation of 2,3-hydroxy-2-methylpropanoic acid, 2-methylpropane-1,3-diol, 2-methylpropane-1,2,3-triol, and 3-hydroxy-2-methylacrylic acid from the condensation of hydroxyacetone derived from 2,3-hydroxypropanoic acid and formyl-CoA. [Diagram 33] Figure 33. Preparation of 2-hydroxy-2,3-dimethylbutanoic acid, 3-hydroxy-2,3-dimethylbutanoic acid, 2,3-dimethylbutan-1-ol, 2,3-dimethylbutane-1,2-diol, and 2,3-dimethylbut-2-enoic acid from the condensation of 2-hydroxy-3-methylbutanoic acid-derived 3-methyl-2-butanone and formyl-CoA. [Diagram 34] Figure 34. Preparation of 2-hydroxy-2-methyl-3-oxopropanoic acid, 2-methylpropane-1,3-diol, and 2-methylheptane-1,2,3-triol from the condensation of 2-hydroxy-3-oxopropanoic acid-derived methylglyoxal and formyl-CoA. [Diagram 35] Figure 35. Preparation of 2-hydroxy-2-methyl-4-oxopentanoic acid, 3-hydroxy-2-methyl-4-oxopentanoic acid, 5-hydroxy-4-methylpentan-2-one, 4,5-dihydroxy-4-methylpentan-2-one, and 2-methyl-4-oxopent-2-enoic acid from the condensation of 2-hydroxy-4-oxopentanoic acid-derived pentane-2,4-dione and formyl-CoA. [Diagram 36] Figures 36A-36B. Methyl ketones as substrates for condensation with formyl-CoA using purified enzymes. A) Pathway for condensation of methyl ketones and formyl-CoA from formate to 2-hydroxy-2-methyl acid; B) GC-MS results of in vitro assay with different methyl ketones and formates. The desired product is indicated with a blue arrow. [Figure 37] Figures 37A-37B. Acetone as a substrate for condensation with formyl-CoA using growing cultures. A) Pathway for condensation of acetone and formyl-CoA from formate to 2HIB; B) Preparation of 2HIB from acetone and formation in E. coli. [Figure 38]FIG. 38 illustrates a generalized carboxylic acid (CA) platform for reduced product synthesis without carbon loss. Carboxylic acids are activated to the corresponding acyl-CoA, which can be reduced to a variety of products, many of which can also serve as intermediates for one-carbon (C1) elongation. Reducing equivalents and ATP required for product synthesis are generated via reduced C1 compounds or H2. Exemplary products are shown and represent only a subset of the potential products. [Figure 39] Figure 39 illustrates a generalized carboxylic acid (CA) platform for reduced product synthesis without carbon loss when using biomass-derived sugars as the carbon source. Carboxylic acids are generated from the supplied sugar(s) and then activated to the corresponding acyl-CoA, which can be reduced to a variety of products, many of which can also serve as intermediates for one-carbon elongation. Reducing equivalents and ATP required for product synthesis are generated via reduced C1 compounds or H2. Exemplary products are shown and represent only a subset of the potential products. [Diagram 40] FIG. 40 shows the activation, product synthesis and energy generation pathways for making reduction products from supplied carboxylic acids. [Diagram 41] FIG. 41 shows the activation, product synthesis and energy production pathways for making reduction products from a supplied 2-hydroxycarboxylic acid. [Diagram 42]FIG. 42 illustrates the formyl-CoA elongation pathway for one-carbon elongation. A reduced one-carbon substrate, such as methanol, is activated to formyl-CoA, a C1 elongation unit, via various redox reactions. Formyl-CoA serves to elongate carbonyl-containing compounds in a reaction catalyzed by HACL, resulting in the creation of 2-hydroxyacyl-CoA. 2-hydroxyacyl-CoA can be further reduced to a 2-hydroxyaldehyde. 2-hydroxyaldehydes can be reduced to 1,2-diols and dehydrated to aldehydes. Various intermediates in these elongation pathways can be converted to desired chemical products, such as 2-hydroxyacids, diols, polyols, and alcohols. Abbreviations: MDH: methanol dehydrogenase; ACR: acyl-CoA reductase; HACL: 2-hydroxyacyl-CoA lyase; ADH: alcohol dehydrogenase; DDR: diol dehydratase; TES: thioesterase. Examples of compounds made from carboxylic acid activation and reduction that serve as intermediates in C1 elongation are shown (acetone, propionaldehyde, lactaldehyde). Products that can be derived from these exemplary compounds are also shown, along with the concentrations made. [Diagram 43] Figure 43 illustrates the energy generation pathways for reducing equivalents (NADH) and ATP synthesis required to drive product synthesis pathways. Pathways from reduced one-carbon compounds (e.g., methanol) and H2 are shown. [Diagram 44] Figure 44 shows an example of the production of lactate from sugars as carboxylic acid intermediates via activation, product synthesis and energy production pathways. Illustrative products are shown and represent only a subset of the potential products. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description Before describing the present disclosure in more detail, it is to be understood that the present disclosure is not limited to particular embodiments described, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0013] Where a range of values ​​is provided, it is understood that, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range and any other stated or intervening values ​​within the stated range is encompassed in the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed in the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0015] All publications and patents cited herein are incorporated by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference as if to disclose and describe the methods and / or materials for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0016] Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0017] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other several aspects without departing from the scope or spirit of the disclosure. Any described method may be carried out in the order of events described or in any other order which is logically possible.

[0018] Aspects of the present disclosure employ, unless otherwise indicated, chemical, biological and other techniques within the skill of the art.

[0019] The following examples are provided to provide those skilled in the art with a complete disclosure and description of how to carry out the methods and how to use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g. amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperatures are in °C, and pressures are at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0020] Before the aspects of the present disclosure are described in detail, it is understood that unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, etc., as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. It is also possible that in the present disclosure, steps may be performed in different sequences where this is logically possible.

[0021] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] As defined herein, the phrases "recombinant host microorganism", "genetically engineered host microorganism", "genetically engineered host microorganism" and "genetically modified host microorganism" may be used interchangeably and refer to a host microorganism that has been genetically modified to (a) express one or more exogenous polynucleotides, (b) overexpress one or more endogenous and / or one or more exogenous polynucleotides, such as those contained in a vector or with modified expression of an endogenous gene, or (c) knock out or downregulate an endogenous gene. Also, certain genes may be physically removed from the genome (e.g., knocked out), or they may be genetically engineered to have reduced, modified or enhanced activity.

[0023] The terms "engineer", "genetically engineer" or "genetically modified" refer to any manipulation of a microorganism that results in a detectable change in the microorganism, including but not limited to introducing a non-native metabolic function via a heterologous (exogenous) polynucleotide or removing a native function via polynucleotide deletion, mutation or knockout. The term "metabolically engineered" generally includes rational pathway design for the production of a desired metabolite and the assembly of biosynthetic genes (or ORFs), genes associated with operons and regulators of such polynucleotides. "Metabolically engineered" can further include genetic engineering, such as reducing, disrupting or knocking out competing metabolic pathways that compete with intermediates leading to the desired pathway, and optimizing metabolic flux by controlling and optimizing transcription, translation, protein stability and protein function using appropriate culture conditions.

[0024] The phrases "metabolically engineered microorganism" and "modified microorganism" are used interchangeably herein and refer not only to a particular host cell of interest, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still fall within the scope of the terms used herein.

[0025] The term "mutation" as used herein refers to any modification of a nucleic acid and / or polypeptide that results in an altered nucleic acid or polypeptide (i.e., relative to a wild-type nucleic acid or polypeptide sequence). Mutations include point mutations, substitutions, deletions, or insertions of single or multiple residues in a polynucleotide (or encoded polypeptide), including, for example, modifications occurring within the protein-coding region of a gene and modifications in regions outside the protein-coding sequence, such as, but not limited to, in regulatory or promoter sequences. A genetic modification can be any type of mutation. For example, a mutation can constitute a point mutation, a frameshift mutation, an insertion, or a deletion of part or all of a gene. In certain embodiments, a portion of the genome of a genetically modified microorganism can be replaced with one or more heterologous (exogenous) polynucleotides. In some embodiments, the mutation occurs naturally. In other embodiments, the mutation is the result of artificial selection pressure. In still other embodiments, the mutation in the microorganism genome is the result of genetic re-engineering.

[0026] The term "expression" or "expressed" refers to the transcription of a gene, ORF or polynucleotide sequence and, where appropriate, the translation of the resulting mRNA transcript into a protein. Thus, as the context will indicate, protein expression occurs by transcription and translation of an open reading frame sequence. The level of expression of a desired product in a host microorganism can be determined based on either the amount of corresponding mRNA present in the host or the amount of the desired product encoded by the selected sequence. For example, the mRNA transcribed from the selected sequence can be quantified by PCR or Northern hybridization (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). The protein encoded by the selected sequence can be quantified by various methods, such as by ELISA, by assays for the biological activity of the protein, or by using assays independent of such activity, such as Western blotting or radioimmunoassays using antibodies that recognize and bind to the reactive protein.

[0027] The term "endogenous," as used herein with respect to polynucleotides (and polypeptides encoded therein), refers to polynucleotides and polypeptides that are expressed in the organism in which they occur (i.e., they are native to the organism). In contrast, the terms "heterologous" and "exogenous," as used interchangeably and defined herein with respect to polynucleotides (and polypeptides encoded therein), refer to polynucleotides and polypeptides that are expressed in an organism other than the organism in which they (i.e., the polynucleotide or polypeptide sequence) occur or from which they are derived.

[0028] The term "feedstock" is defined as a raw material or mixture of raw materials that are fed to a microorganism or fermentation process from which other products can be produced. For example, as described in the present invention, a glucose carbon source or a xylose carbon source, either alone or in combination, is a biomass-derived feedstock for a microorganism that produces a biofuel or biochemical in a fermentation process. However, in addition to the feedstock of the present invention (e.g., glucose substrate), the fermentation medium contains appropriate minerals, salts, cofactors, buffers and other components known to those skilled in the art that are suitable for the growth of the culture and for promoting the enzymatic pathways required for multi-carbon compound production.

[0029] The term "substrate" refers to any substance or compound that is converted or is intended to be converted into another compound by the action of enzyme. The term includes not only single compounds, but also combinations of compounds, such as solutions, mixtures, and other materials that contain at least one substrate or its derivatives. Furthermore, the term "substrate" encompasses not only compounds that provide a suitable carbon source (e.g., methane) for use as starting material, but also intermediates and end-product metabolites used in the pathways related to the metabolically engineered microorganisms described herein.

[0030] The term "fermentation" or "fermentation process" is defined as a process in which a host microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, where the microorganism converts raw materials, such as the feedstock, into a product.

[0031] The term "polynucleotide" is used interchangeably herein with the term "nucleic acid" and refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides, or their analogs, such as, but not limited to, single- or double-stranded, sense or antisense deoxyribonucleic acid (DNA) of any length, and single- or double-stranded, sense or antisense ribonucleic acid (RNA), including siRNA, where appropriate. The term "nucleotide" refers to any of several compounds that consist of a ribose or deoxyribose sugar linked to a purine or pyrimidine base and to a phosphate group, and are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound that consists of a purine or pyrimidine base linked to a deoxyribose or ribose, and is found especially in nucleic acids, such as guanosine or adenosine. The terms "nucleotide analog" or "nucleoside analog" refer to a nucleotide or nucleoside, respectively, in which one or more individual atoms have been replaced with a different atom or a different functional group. Thus, the term polynucleotide includes nucleic acids of any length, including DNA, RNA, ORFs, analogs and fragments thereof.

[0032] As defined herein, the term "open reading frame" (hereinafter "ORF") refers to any open reading frame (whether naturally occurring, non-naturally occurring or synthetic). ORF means a nucleic acid or nucleic acid sequence that contains an uninterrupted reading frame consisting of (i) a start codon, (ii) a series of two (2) or more codons representing amino acids, and (iii) a stop codon, and is read (translated) in the 5' to 3' direction.

[0033] It is understood that the polynucleotides described herein include "genes," and that the nucleic acid molecules described herein include "vectors" or "plasmids."

[0034] Thus, the term "gene" refers to a polynucleotide that encodes a specific sequence of amino acids that comprises all or part of one or more proteins or enzymes, and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, that determine the conditions under which the gene is expressed. The transcribed regions of a gene may include untranslated regions, such as introns, 5'-untranslated regions (UTRs) and 3'-UTRs and coding sequences.

[0035] The term "promoter" refers to a nucleic acid sequence that can control the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. Promoters can be driven in their entirety from a natural gene or can be composed of different elements from different promoters found in nature, or can even include synthetic nucleic acid segments. It is understood by those skilled in the art that different promoters can induce the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions. A promoter that causes a gene to be expressed in most cell types most of the time is usually referred to as a "constitutive promoter". It is further understood that DNA fragments of different lengths can have the same promoter activity, since in most cases the exact boundaries of the control sequence have not been completely defined.

[0036] The term "operably linked" refers to the relationship of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0037] The term "codon optimization," as it refers to the coding region (or ORF) of a gene or nucleic acid molecule for transformation of various hosts, refers to the modification of codons in the coding region of a gene or nucleic acid molecule to reflect the typical codon usage of the host organism, without altering the polypeptide encoded by the DNA.

[0038] The term "operon" refers to two or more genes that are transcribed as a single transcription unit from a common promoter. In certain embodiments, the genes, polynucleotides, or ORFs that comprise an operon are contiguous genes. It is understood that transcription of the entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Alternatively, any gene, polynucleotide, or ORF, or any combination thereof, in an operon can be modified to modify the function or activity of the encoded polypeptide. The modification can result in an increase or decrease in the activity or function of the encoded polypeptide. Additionally, the modification can confer a novel activity to the encoded polypeptide.

[0039] A "vector" is any means by which a nucleic acid can be propagated and / or transported between organisms, cells or cellular components. Vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) and PLACs (plant artificial chromosomes), and are "episomes" that can replicate autonomously or integrate with the chromosome of a host microorganism. A vector can also be a naked RNA polynucleotide that is not naturally episomal, a naked DNA polynucleotide, a polynucleotide that is composed of both DNA and RNA in the same strand, polylysine-conjugated DNA or RNA, peptide-conjugated DNA or RNA, liposome-conjugated DNA, and the like, or an organism that contains one or more of the above-mentioned polynucleotide constructs, such as Agrobacterium or bacteria.

[0040] The term "homolog" when used in reference to an original enzyme, polypeptide, gene or polynucleotide (or ORF encoding the same) of a first family or species, refers to a separate enzyme, gene or polynucleotide of a second family or species that is determined by functional, structural or genomic analysis to be an enzyme, gene or polynucleotide of the second family or species that corresponds to the original enzyme or gene of the first family or species. Most often, a "homolog" has functional, structural or genomic similarity. Techniques are known by which homologs of enzymes, genes or polynucleotides can be readily cloned using genetic probes and PCR. The identity of the cloned sequence as a "homolog" can be confirmed using functional assays and / or by genomic mapping of the gene.

[0041] A polypeptide (or protein or enzyme) has "homology" or is "homologous" to a second polypeptide if the nucleic acid sequence encoding the polypeptide has a similar sequence as the nucleic acid sequence encoding the second polypeptide.

[0042] Alternatively, a polypeptide has homology to a second polypeptide if the two proteins have "similar" amino acid sequences. Thus, the term "homologous protein" or "homologous polypeptide" is defined to mean that two polypeptides have similar amino acid sequences. In certain embodiments of the present invention, polynucleotides and polypeptides that are homologous to one or more polynucleotides and / or polypeptides listed in Table 1 can be easily identified using methods known in the art for sequence analysis and comparison.

[0043] Homologous polynucleotide or polypeptide sequences of the present invention can also be determined or identified by BLAST analysis (Basic Local Alignment Search Tool) or similar bioinformatics tools that compare a query nucleotide or polypeptide sequence to a database of known sequences. For example, a search analysis can be done using BLAST to determine sequence identity or similarity to previously published sequences, which can provide a relevant investigation into the function of a DNA or protein sequence if the sequence has not yet been published.

[0044] In various embodiments, the present invention provides carboxylic acid (CA) compounds, either directly or produced from compounds such as biomass-derived sugar(s) that serve as carbon sources for organisms. The CA intermediates are activated to the corresponding acyl-CoA intermediates by multiple C-storing or C-fixing downstream product synthesis pathways engineered to produce a variety of reduced products. To generate the energy required to drive these reactions and avoid CO2 release, compounds such as reduced one-carbon (C1) molecules (e.g., methanol) or hydrogen (H2) serve as energy sources for organisms by synthetic pathways that utilize these compounds to generate NADH and ATP. Together, these allow the disclosed system to generate reduced compounds without carbon loss.

[0045] In some embodiments, a carboxylic acid (CA) molecule is the only carbon source provided. In some embodiments, a sugar such as glucose may be the only carbon source provided and is converted to a carboxylic acid (CA) molecule prior to activation. In these situations, the A3 molecule is first activated to the corresponding acyl-CoA by the appropriate acyl-CoA synthetase, or acyl-CoA transferase, or carboxylate kinase and phosphotransacylase, or carboxylate reductase and acyl-CoA reductase, or aldehyde dehydrogenase and acyl-CoA reductase.

[0046] Acyl-CoA initiates product synthesis pathways to allow further biochemical reactions to create a range of products and target intermediates amenable to elongation. For example, a C1 unit (formyl-CoA) can be added to aldehydes and ketones via 2-hydroxyacyl-CoA lyase. These reactions and combinations thereof result in intermediates amenable to further biochemical reactions that significantly expand the range of achievable products.

[0047] For the energy production required to drive these reactions and avoid CO2 release, reducing equivalents in the form of NAD(P)H and ATP are generated from an externally supplied energy source. In some embodiments, reduced one-carbon (C1) molecules, such as methanol, are provided as energy sources. In these situations, C1 molecules are oxidized to CO2 by appropriate enzymes with the simultaneous generation of NADH. For example, methanol can be oxidized to CO2 via methanol dehydrogenase, which converts methanol to formaldehyde, formaldehyde dehydrogenase, which converts formaldehyde to formate, and formate dehydrogenase, which converts formate to CO2. In some embodiments, reduced one-carbon (C1) is oxidized using appropriate enzymes that generate both NAD(P)H and ATP. For example, methanol can be oxidized to CO2 via methanol dehydrogenase, which converts methanol to formaldehyde, acylated formaldehyde dehydrogenase, which converts formaldehyde to formyl-CoA, phosphate formyltransferase, which converts formyl-CoA to formyl-phosphate, formate kinase, which converts formyl-phosphate to formate, and formate dehydrogenase, which converts formate to CO2. In some embodiments, hydrogen (H2) is provided as an energy source. In these situations, H2 is converted to NAD(P)H via the appropriate NAD+-reducing hydrogenase.

[0048] Thus, disclosed herein are methods for enabling the production of reduced products from CA intermediates without carbon loss, the methods comprising a cellular system comprising a first set of metabolic enzymes that activate the CA intermediate to the corresponding acyl-CoA intermediate, a second set of metabolic enzymes that convert the acyl-CoA intermediate to a product, and a third set of metabolic enzymes that generate reducing equivalents and ATP from an externally supplied energy source, and supplying to the system a CA intermediate or a carbon source from which the CA intermediate can be made and an external energy source under conditions suitable for the metabolic enzymes to make the desired reduced compound.

[0049] The first step in the disclosed system and method is the conversion of CA intermediates (e.g., lactate) to the corresponding acyl-CoA (e.g., lactoyl-CoA). This step is referred to herein as CA activation. Generally, CA activation involves the conversion of a carboxylic acid group to a CoA thioester and requires at least one of the following steps: (1) direct conversion of the carboxylic acid to the corresponding acyl-CoA by acyl-CoA synthetase or acyl-CoA transferase; (2) conversion of the carboxylic acid to a phosphate intermediate by carboxylate kinase and then conversion of the corresponding acyl-CoA by phosphotransacylase; (3) reduction of the carboxylic acid to the corresponding aldehyde by carboxylate reductase and then conversion of the corresponding acyl-CoA by acyl-CoA reductase; or (4) reduction of the carboxylic acid to the corresponding aldehyde by aldehyde dehydrogenase and then conversion of the corresponding acyl-CoA by acyl-CoA reductase.

[0050] The second step in the disclosed system and method is the conversion of acyl-CoA to the desired reduction product(s). This step is referred to herein as product synthesis. In general, a variety of reduction products with different functional groups and chain lengths can be synthesized from acyl-CoA intermediates. For example, reduction of acyl-CoA by acyl-CoA reductase can produce an aldehyde. This aldehyde can be further reduced to an n-alcohol by alcohol dehydrogenase or can serve as a precursor for formyl-CoA elongation, using formyl-CoA as the C1 building block in a reaction catalyzed by 2-hydroxyacyl-CoA lyase (HACL) or oxalyl-CoA decarboxylase (OXC). These enzymes can link formyl-CoA to a variety of carbonyl-containing acceptors with a wide range of chain lengths and functionalities, such as aldehydes and ketones. In some embodiments, the initial carboxylic acid and the corresponding acyl-CoA contain a hydroxy (-OH) group at the second carbon (2-hydroxyacyl-CoA). In these situations, reduction of the CoA group by acyl-CoA reductase and alcohol dehydrogenase results in the formation of a 1,2-diol, which can be further dehydrated to the corresponding ketone or aldehyde by diol dehydratase. The ketone or aldehyde can also serve as a precursor for formyl-CoA elongation using formyl-CoA as the C1 building block.

[0051] In some embodiments, 2-hydroxyacyl-CoA formed by the addition of formyl-CoA to an aldehyde or ketone is reduced to a 2-hydroxyaldehyde by acyl-CoA reductase (ACR; EC 1.2.1.-, e.g., 1.2.1.10, 1.2.1.76, 1.2.1.84). Further reduction of the 2-hydroxyaldehyde to give a 1,2-diol is possible by an appropriate 1,2-diol oxidoreductase (DOR; EC 1.1.1.77) or alcohol dehydrogenase (ADH; EC 1.1.1.71). Dehydration of the 1,2-diol can be catalyzed by the activity of a diol dehydratase (DDR; EC 4.2.1.28) to give an aldehyde, which can be further reduced to an alcohol by an alcohol dehydrogenase (ADH; EC 1.1.1.71).

[0052] In some embodiments, combinations of the above pathways can be implemented simultaneously such that for some molecules, products of the same chain length as the acyl-CoA are formed, while for other molecules elongation occurs. Both pathways can co-exist in the same system at the same time.

[0053] In some embodiments, the product of interest is the product of the above-mentioned reaction.Examples of these products include, but are not limited to, alcohols, such as ethanol, propanol and butanol; diols, such as 1,2-propanediol, 1,2-butanediol and 1,3-butanediol; triols, such as 1,2,3-butanetriol; hydroxycarboxylic acids, such as lactate, 2-hydroxybutyrate and 2,3-dihydroxybutyrate; and ketones, such as acetone.

[0054] The third step in the disclosed system and method is the generation of reducing equivalents and ATP from an external energy source. This step is referred to herein as energy generation. Generally, energy generation involves the oxidation of an external energy source provided, resulting in the generation of reducing equivalents and ATP. In some embodiments, a reduced one-carbon (C1) molecule, such as methanol, is provided as an energy source. In these situations, the C1 molecule is oxidized to CO2 by a suitable enzyme with the simultaneous generation of NADH. For example, methanol can be oxidized to CO2 via methanol dehydrogenase, which converts methanol to formaldehyde, formaldehyde dehydrogenase, which converts formaldehyde to formate, and formate dehydrogenase, which converts formate to CO2. In some embodiments, the reduced one-carbon (C1) is oxidized using a suitable enzyme to generate both NAD(P)H and ATP. For example, methanol can be oxidized to CO2 via methanol dehydrogenase, which converts methanol to formaldehyde, acylated formaldehyde dehydrogenase, which converts formaldehyde to formyl-CoA, phosphate formyltransferase, which converts formyl-CoA to formyl-phosphate, formate kinase, which converts formyl-phosphate to formate, and formate dehydrogenase, which converts formate to CO2. In some embodiments, hydrogen (H2) is provided as an energy source. In these situations, H2 is converted to NAD(P)H by an appropriate NAD+-reducing hydrogenase.

[0055] In some embodiments, the described pathways are provided in terms of a microbial host. In some embodiments, the microbial host is cultured in a fermentation system to produce the desired product. In other embodiments, the microbial system is used to produce enzymes that are then extracted from the microorganisms for use in the cell-free system. In other embodiments, the enzymes are made separately and added individually to the system.

[0056] Pathways in biological systems are generally created by transforming microorganisms with one or more expression vectors (one or more) that contain genes encoding one or more enzymes, although genes can also be added to chromosomes by recombinant genetic engineering, homologous recombination, gene editing and similar techniques. If the required protein is endogenous, as in some cases, the protein may be sufficient, but is usually overexpressed for better functionality and control over the level of active enzyme. In some embodiments, one or more or all of such genes are under the control of an inducible promoter.

[0057] The enzymes can be added to the genome or via an expression vector if desired. Preferably, multiple enzymes are expressed in one vector, or multiple enzymes can be combined into an operon by adding the necessary signals between the coding regions. Further improvements can be made by overexpressing one or more or even all of the enzymes, for example by adding extra copies to the cell via a plasmid or other vector. Initial experiments can use an expression plasmid with three or more ORFs for convenience, but it may be preferable to insert an operon or individual genes into the genome for stability reasons.

[0058] Further improvement in yield can be achieved by reducing competing pathways, such as those for making acetate, formate, ethanol and lactate, and how to reduce or knock out these pathways is already well known in the art.See, for example, U.S. Patent Nos. 7,569,380, 7,262,046, 8,962,272, 8,795,991, 8,129,157 and 8,691,552, each of which is incorporated by reference in its entirety for all purposes.Many others have also been made in this field.

[0059] After construction of a suitable strain containing an engineered pathway, cultures of the developed strains can be carried out to evaluate the effectiveness of the pathway in producing a product that is less than its intended target-carboxylic acid intermediate. The organisms can be cultured in a suitable culture medium and evaluated for product formation from a carboxylic acid substrate, where the carboxylic acid is supplied as a carbon source or is produced from a different supplied carbon source (e.g., glucose). The amount of product produced by the organisms can be measured by UPLC or GC, and performance indicators such as growth rate, productivity, titer, yield, or carbon efficiency can be determined.

[0060] Further evaluation of the interactions of pathway enzymes with each other and with the host system can allow for optimization of pathway performance and minimization of adverse effects. Because pathways are under synthetic control rather than the organism's naturally occurring control mechanisms, expression of the pathway is usually manually tuned to optimize production of the desired compound, avoiding potential problems that slow cell growth or production.

[0061] Furthermore, an imbalance in relative enzyme activity can limit the overall carbon flux through the pathway, resulting in suboptimal production rates and the build-up of pathway intermediates that can inhibit pathway enzymes or render them toxic. Analysis of cell cultures by HPLC or GC can reveal metabolic intermediates made by the constructed strains. This information can point to potential pathway problems.

[0062] As an alternative to in vivo expression of a pathway, a cell-free in vitro version of the pathway can be constructed. By purifying the relevant enzymes for each reaction step, the entire pathway can be assembled by combining the required enzymes in a reaction mixture. By adding the relevant cofactors and substrates, the pathway can be evaluated for its performance independent of the host.

[0063] General methods for gene synthesis and DNA cloning, as well as vector and plasmid construction, are well known in the art and are described in several publications. More specifically, techniques such as digestion and ligation-based cloning and in vitro and in vivo recombination methods can be used to assemble DNA fragments that code for polypeptides that catalyze substrate to product conversion into suitable vectors. These methods include restriction digestion cloning, sequence and ligation independent cloning (SLIC), Golden Gate cloning, Gibson assembly, etc. Some of these methods can be automated and miniaturized for high-throughput applications.

[0064] Genetic cassettes for expressing engineered metabolic pathways in host microorganisms are known in the art. The cassette may contain one or more open reading frames (ORFs) that code for the enzymes of the introduced pathway, a promoter for directing the transcription of downstream ORF(s) in the operon, a ribosome binding site for directing the translation of the mRNA encoded by the individual ORF(s) and a transcription termination sequence. Due to the modular nature of the various components of the expression cassette, permutations of these aligned combinations can be created by substituting different components at one or more positions. The orientation of one or more ORFs can also be reversed to determine whether any of these alternative orientations improve product yield.

[0065] In some embodiments, host microorganisms for expressing metabolic pathway genes contain a plasmid vector(s) carrying a metabolic pathway expression cassette that is transferred to these organisms via conjugation.

[0066] In an alternative method for expressing metabolic pathway genes in a microbial host, the biosynthetic pathway genes can be directly inserted into the chromosome. Methods for chromosomal modification include both untargeted and targeted deletions and insertions.

[0067] In some embodiments, the disclosed system and method also includes recovering and purifying the desired product from the fermentation broth. The method used depends on the physicochemical properties of the product and the nature and composition of the fermentation medium and cells. For example, U.S. Patent No. 8,101,808 describes a method for recovering C3-C6 alcohols from the fermentation broth using continuous flash evaporation and phase separation processes. In some embodiments, solids can be removed from the fermentation medium by centrifugation, filtration and decantation. In some embodiments, multi-carbon compounds are isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation or pervaporation.

[0068] Some exemplary, non-limiting embodiments of the systems, methods and compositions disclosed herein are disclosed herein. In these instances where a convention similar to "at least one of A, B and C, etc." is used, such constructs are generally intended in the sense that one skilled in the art would understand the convention (e.g., "a system having at least one of A, B and C" includes, but is not limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B and C together). It will be further understood by one skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibility of "A" or "B" or "A and B".

[0069] For example, in some embodiments, the microorganism is modified to express one or more enzymes responsible for catalytic reactions, as shown in Figures 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37. Suitably, the microorganism is modified to express two or more enzymes, three or more enzymes, four or more enzymes, five or more enzymes, and may be determined from the pathways shown in the figures and not limited by the examples described herein. Furthermore, one skilled in the art may select a combination of enzymes to be introduced into the microorganism to create a genetically modified organism capable of modifying a carbon source to a desired chemical outcome.

[0070] In some embodiments, the microorganism is modified with enzymes responsible for energy, redox and ATP and formyl-CoA production. In any and all combinations, the microorganism is modified to include one or more of the following enzymes, such as at least one of mmoXyBZCD, pmoA1A2B1B2, BmMDH2, CnMCH2, BsMDH, LmACR, StEutE, CbA1d, EcMhpF, PsDmpF, EcFrmA, PpFdhA, CcPta-Ack, EcACS, StACSstab, MhACS, ArACS, CaAbft, OfFrc, PsFdh and / or CbFdh.

[0071] In some embodiments, the microorganism is modified with an enzyme responsible for the carboxylic acid platform that uses formaldehyde as an intermediate aldehyde for C1 elongation. In any and all combinations, the microorganism is modified to include one or more of the following enzymes, such as at least one of CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, ArACS, CaAbfT, OfFrc, LmACE, RuHACL, BsmHACL, AcHACK, MeOXC4, LmACR, StPduP, EcAldA, EcfucO, KoPddABC, EcAdhE, CcPta-Ack, EcPta-Ack, EcYciA, EcGlcD, MtAld, BsAld, ALAT1, aldH1, dhaS, EcSerC, GOT1, and / or EcYdfG.

[0072] In some embodiments, the microorganism is modified with an enzyme responsible for the carboxylic acid platform that uses formamide as the intermediate aldehyde for C1 elongation. In any and all combinations, the microorganism is modified to include one or more of the following enzymes, such as at least one of MmFmdA, RuHACL, BsmHACL, AcHACL, MeOXC4, LmACR, EcAldA, EcfucO, KoPddA BC, EcAdhE, CcPta-Ack, EcPta-Ack, EcYciA, EcGlcD, MtAld, BsAld, ALA T1, aldH1, dhaS, EcSerC, GOT1, EcYdfG, and / or EcGldA.

[0073] In some embodiments, the microorganism is modified to express enzymes responsible for the energy (redox and ATP) and formyl-CoA production pathways. In any and all combinations, the microorganism is modified to include one or more of the following enzymes, such as at least one of mmoB, mdh2, adh, frmA, LmACR, mhpF, dmpF, eutE, CcPta-AcK, EcPta-AcK, EcACS, StACSstabm MhACS, ArACS, abfT, frc, and / or fdh.

[0074] In some embodiments, the microorganism is modified with enzymes responsible for the activation-reduction and C1 elongation pathways. In any and all combinations, the microorganism is modified to include one or more of the following enzymes, such as at least one of ackA, tdcD, buk1, pta, ptb, yfaC, prpE, IvaE, AAE3, sucC, sucD, cat1, scoC, cat3, yfdE, pct, eutE, pduP, adhE2, sucD, RuHACL, MeOXC4, JGI15, ydiF, tesA, ldh, ald, pdh, pduP, yahK, pduC, pduD, pduE, pddA, pddB, pddC, PiDD, yahk, lcdAB, acuN, gbuF, acuK, crt, lvaC, acuK, tesB, pcs, bcd, fade, pduP, and / or ald.

[0075] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses methyl ketones for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0076] In some embodiments, the microorganism is modified with enzymes that are responsible for the carboxylic acid platform that uses acetone for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, such as mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0077] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses butanone for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0078] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses pentanone for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0079] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses heptanone for the C-1 elongation pathway. In any and all combinations, these enzymes include the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcFrmA, P and / or at least one of pFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0080] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses hydroxyacetone for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0081] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses 3-methyl-2-butanone for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, such as mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0082] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses methylglyoxal for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, for example, mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0083] In some embodiments, the microorganism is modified with enzymes responsible for the carboxylic acid platform that uses acetylacetone (pentane-2,4-ddione) for the C-1 elongation pathway. In any and all combinations, the microorganism may be modified with the following enzymes, e.g., mmoXYBDCD, pmoA1A2B1B2, BmMDH2, CnMDH2, BsMDH, LmACR, StEutE, CbAld, EcMhpF, PsDmpF, pduP, AtAdhE, EcMhpF, CcPta-Ack, EcPta-Ack, EcACS, StACSstab, MhACS, arACS, RuHACS, BsmHACS, DbhACS, AcHACS, EcF and / or at least one of EcYciA, PpFdhA, fucO, gldA, rhaZ, yahK, adhA, yjgB, yqhD, pduq, YLL056C, RiDD, pduCDE, pddABC, BpCaiD_2, CdHadBC, EcPaaZ, AtACX4, EgTER, CaCRT, PfECH, CaHbd, EcAldA, CaAbfT, OfFrc, PsFdh, CbFdh, and / or EcYciA.

[0084] In certain embodiments, the microorganism is modified with enzymes responsible for the production of formyl-CoA from formate, such as at least one of AbfT, CcPta-Ack, CaAbfT, OfFrc, EcACS, MhACS, MhACS3, ArACS, StACS6, and / or StACS, in any and all combinations.

[0085] In certain embodiments, the microorganism is modified with enzymes responsible for the production of formyl-CoA from formaldehyde using acyl-CoA reductase, such as at least one of LmACR, StEutE, EcmhpF, and / or PsDmpF, in any and all combinations.

[0086] In certain embodiments, the microorganism is modified with enzymes responsible for the production of formyl-CoA from methanol-derived formaldehyde using a different methanol dehydrogenase relative to LmACR, such as, for example, at least one of BmMDH, CnMdh, RuHACL, and / or EcAldA, in any and all combinations.

[0087] In certain embodiments, the microorganism is modified with enzymes responsible for the conversion of formate to ethylene glycol, such as, for example, at least one of LmACR, OfFrc, and / or HACS, in any and all combinations.

[0088] In certain embodiments, the microorganism is modified with enzymes responsible for the conversion of glycolate production, such as, for example, at least one of AbfT, HACS, RuHACL, BsmHACL, MeOXC4, and / or AcHACL, in any and all combinations.

[0089] A specific embodiment shows a cell-free method for prototyping a C1 elongation pathway for formaldehyde as a starting substrate, which can be generated by activation and reduction of a carboxylic acid, formate. In any and all combinations, the enzymes can include LcACR, RuHACL, EcFucO and / or KoPddABC.

[0090] In certain embodiments, the microorganism is modified with enzymes responsible for the synthesis of glycine from glycolate, such as, for example, at least one of AbfT, HACS, RuHACL, BsmHACL, MeOXC4 and / or AcHACL, in any and all combinations.

[0091] In certain embodiments, the microorganism is modified with enzymes responsible for the implementation of a carboxylic acid (CA) platform that uses acetate (R=H) as a CA intermediate via the C1 elongation pathway. In any and all combinations, the enzymes may include, for example, JGI15, RuHACL, and / or MeOXC4.

[0092] In certain embodiments, the microorganism is modified with an enzyme responsible for the implementation of a carboxylic acid (CA) platform that uses propionate (R=CH3) as a CA intermediate via the C1 elongation pathway. In any and all combinations, the enzyme may include, for example, JGI15, RuHACL, and / or AcHACL.

[0093] In certain embodiments, the microorganism is modified with an enzyme responsible for the implementation of a carboxylic acid (CA) platform that uses glycolic acid (R=OH) as a CA intermediate via the C1 elongation pathway. In any and all combinations, the enzyme may include, for example, JGI15, RuHACL, and / or AcHACL.

[0094] In certain embodiments, the microorganism is modified with an enzyme responsible for the implementation of a carboxylic acid (CA) platform that uses oxalic acid (R=OOH) as a CA intermediate via the C1 elongation pathway. In any and all combinations, the enzyme may include, for example, JGI15 and / or RuHACL.

[0095] In certain embodiments, the microorganism is modified with an enzyme responsible for the condensation of methyl ketones with formyl-CoA using purified enzymes. In any and all combinations, the enzymes may include, for example, CaAbfT and / or BsmHACS.

[0096] In certain embodiments, the microorganism is modified with enzymes responsible for carrying out the condensation of methyl ketones (from formate) with formyl-CoA using cells growing in vivo, with acetone being used as a representative methyl ketone. In any and all combinations, the enzymes may include, for example, BsmHACS and / or AcHCS.

[0097] Several embodiments of the invention have been described. Nevertheless, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. EXAMPLES

[0098] Working Example Example 1: This example shows the implementation of a carboxylic acid (CA) platform using lactic acid (lactate) as a CA intermediate. Lactate can be supplied as a carbon source or can be made from carbon sources such as glucose and / or other biomass-derived sugars. The conversion of sugars to lactate can enhance the efficiency of fermentation metabolism, i.e., (L- or D-) lactic acid fermentation, which works at high titers (>200g / L), rates (>3g / L / h) and yields (near the theoretical maximum of 1g / g) and has been shown in many microorganisms at industrial scale (Rawoof, S., et al. Environ. Chem. Lett. 19: 539-556 (2021);Mora-Villalobos, JA, et al. Fermentation 6:21 (2020);de Oliveira, RA, et al. Biochem. Eng. J. 133:219-239 (2018)). In this example, we use established knowledge about high titer, rate and yield production of D- and L-lactate in E. coli (Mazumdar, S., et al. Microb. Cell Fact. 12:7 (2013);Mazumdar, S., et al. Appl. Environ. Microbiol. 76:4327-4336 (2010);Zhu, Y., et al. Appl. Environ. Microbiol. 73:456-464 (2007);Zhao, JF, et al. Microb. Cell Fact. 12: (2013)) as well as previous work on engineering Zymomonas mobilis strains for lactate production (Liu, Y., et al. Metab. Eng. 61:261-274 (2013)). (2020)) is utilized to generate host strains for D- or L-homolactate fermentation from sugars. It will be understood by those skilled in the art that other microbial hosts are suitable for direct supply and conversion of carboxylic acids or production of carboxylic acids from other carbon sources such as sugars.

[0099] Activation of lactate to lactoyl-CoA utilizes acyl-CoA synthetase from Thermococcus kodakarensis (Awano, T., et al. J. Bacteriol. 196:140-147 (2014)), and the subsequent product synthesis pathway can be engineered to generate products with different functionality and chain length in the host strain.

[0100] The reduction of lactoyl-CoA to lactaldehyde proceeds via the CoA-dependent aldehyde dehydrogenase PduP from Salmonella enterica (Niu, W. and Guo, J., ACS Syn. Biol. 4:378-382 (2015)). The specific lactaldehyde enantiomer, determined by the enantiomer of lactate, is then reduced to the corresponding 1,2-propanediol enantiomer by the action of the lactaldehyde reductase YahK from Escherichia coli (Niu, W. and Guo, J., ACS Syn. Biol. 4:378-382 (2015)). 1,2-PDO is then dehydrated to either propanal or acetone, depending on the 1,2-PDO enantiomer and diol dehydratase used. Diol dehydratases such as those from Roseburia inulinivorans (LaMattina, JW, et al. J. Biol. Chem. 291:15515-15526 (2016)) and Klebsiella oxytoca (Tobimatsu, T., et al. Arch. Biochem. Biophys. 347:132-40 (1997)) are used for this conversion. Propanal is then further reduced to 1-propanol by alcohol dehydrogenases such as PduQ from Salmonella enterica (Cheng, S., et al. PLoS ONE 7:e47144 (2012)). Alternatively, when acetone is formed from 1,2-PDO dehydration, further reduction by an alcohol dehydrogenase, such as the secondary alcohol dehydrogenase from Trichomonas vaginalis, yields 2-propanol (Sutak, R., et al. FEBS J. 279:2768-2780 (2012)).

[0101] Methanol is provided as an external energy source to generate reducing equivalents and ATP required for reduction product synthesis. Methanol oxidation proceeds via NADH-dependent methanol dehydrogenase 2 from Bacillus methanolicus (Roth, TB, et al. ACS Syn. Biol. 8:796-806 (2019)) to form formaldehyde and generate NADH. To enable direct ATP generation from the provided C1, formaldehyde is converted to formate via formyl-CoA and formyl-phosphate intermediates using CoA-acylated formaldehyde dehydrogenase, phosphate formyltransferase, and formate kinase. Here, CoA-acylated formaldehyde dehydrogenase from Listeria monocytogenes (Chou, A., et al. Nat. Chem. Biol. 15:900-906 (2019)) and phosphate formyltransferase and formate kinase enzymes from Clostridium cylindrosporum (Sly, WS and Stadtman, ERJ Biol. Chem. 238:2639-2647 (1963)) are used to generate additional NADH and ATP during this conversion. Formate oxidation to CO2 by NADH-dependent formate dehydrogenase from Pseudomonas sp. (strain 101) is used to generate additional NADH (Tishkov, VI, et al. Biochem. Biophys. Res. Commun. 192:976-81 (1993)).

[0102] Genes for overexpression are either cloned into a suitable vector or inserted into the chromosome with a strong synthetic constitutive promoter such as M1-93. When cloned into a vector, these genes are amplified by PCR using appropriate primers to add homology to each end for recombination into the vector backbone with, for example, Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as gene inserts. Plasmids are linearized with appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA) and recombined with the gene insert using the In-Fusion HD Eco-Dry cloning system. The mixture is then transformed into Stellar competent cells. Transformants growing on solid medium (LB+agar) supplemented with the appropriate antibiotic are isolated and screened for gene inserts by PCR. Plasmids from verified transformants are isolated and the sequences of the gene inserts are further confirmed by DNA sequencing. The sequence-confirmed plasmids are then introduced into the host strain via electroporation.

[0103] When inserted into the chromosome, CRISPR is used, and the gene sites of tesB and adhE are in the appropriate locus, but others can be used. The CRISPR method is based on the method developed by Jiang et al. (Jiang, Y., et al. Appl. Environ. Microbiol. 81:2506-2514 (2015)). First, the host strain is transformed with the plasmid pCas and the vector for the expression of Cas9 and λ-red recombinase. The resulting strain is grown at 30°C with L-arabinose for induction of λ-red recombinase expression, and when the OD reaches about 0.6, competent cells are prepared and transformed with pTargetF (AddGene 62226) that expresses sgRNA and the N20 spacer that targets the locus and the template for the insertion of the target gene. The template is an M1-93 promoter with approximately 500 bp sequence homology to the inserted gene plus the upstream and downstream of the insertion locus, constructed by overlap PCR using Phusion polymerase or synthesized by GenScript (Piscataway, NJ) or GeneArt® (Life Technologies, Carlsbad, CA). The method for replacing the N20 spacer of the pTargetF plasmid is inverse PCR with a modified N20 sequence hanging at the 5' end of the primer using Phusion polymerase followed by self-ligation using T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants growing below 30°C on solid medium (LB+agar) supplemented with spectinomycin and kanamycin (or other appropriate antibiotics) are isolated and screened for chromosomal gene inserts by PCR. The sequences of gene inserts amplified from genomic DNA by PCR using Phusion polymerase are further confirmed by DNA sequencing. pTargetF can then be cured by IPTG induction and pCas can be cured by growth at a higher temperature, such as 37-42°C.

[0104] All molecular biology techniques are performed using standard methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989).) or according to manufacturer's protocols. Strains are stored in glycerol stocks at -80°C. Plates are prepared using LB medium with 1.5% agar and containing the appropriate antibiotics at the following concentrations: ampicillin (100 μg / mL), kanamycin (50 μg / mL), spectinomycin (50 μg / mL), and chloramphenicol (12.5 μg / mL).

[0105] Use MOPS minimal medium (Neidhardt et al. J. Bacteriol. 119736-47 (1974)) with 125 mM MOPS, and Na2HPO4 instead of K2HPO4 (2.8 mM), and supplemented with 20 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 100 μM FeSO4, 5 mM (NH4)2SO4 and 30 mM NH4Cl for fermentation. If necessary, also supplement with 55 g / L CaCO3 as a pH buffer. Supplement with 20 mM lactate if not synthesized intracellularly and required for the experiment. Also supplement with 500 mM methanol. Include antibiotics (50 μg / mL carbenicillin, 50 μg / mL spectinomycin and 50 μg / mL kanamycin) if appropriate. All chemicals were obtained from Fisher Scientific Co. (Pittsburg, PA) and Sigma-Aldrich Co. (St. Louis, MO).

[0106] Fermentations are performed in 25 mL Pyrex Erlenmeyer flasks (narrow mouth / sturdy rim, Corning Inc., Corning, NY) filled with an appropriate volume of fermentation medium and sealed with a foam plug filling the neck. For anaerobic conditions, 17.5 mL Hungate tubes are filled completely with fermentation medium and sealed with a rubber (tubber) septum. Single colonies of the desired strains are grown overnight (14-16 h) in LB medium with the appropriate antibiotic to an initial OD of approximately 0.05. 600 The plasmid is used as inoculum with . After inoculation, the flasks are incubated at 200 rpm in an NBS I24 benchtop incubator shaker (New Brunswick Scientific Co., Inc., Edison, NJ) at 37 °C or 30 °C. When the optical density (550 nm, OD550) reaches approximately 0.3-0.5, an appropriate concentration of isopropyl β-D-1-thiogalactopyranoside (IPTG) (or other appropriate inducer) is added for plasmid gene induction. Further fermentations are performed in a SixFors multiple fermentation system (Infors HT, Bottmingen, Switzerland) with a flow rate of 2 N L / hr of air or argon, independent control of temperature (37 °C), pH (controlled at 7.0 by NaOH and H2SO4) and appropriate agitator speed. Precultures are grown in 25 mL Pyrex Erlenmeyer flasks as described above and incubated for 4 h after induction. An appropriate amount of this preculture is centrifuged, washed twice with fresh medium and used for inoculation (400 mL initial volume). Fermentation in bioreactors uses the described fermentation medium with 40 g / L glucose and appropriate IPTG and antibiotics. If required, lactic acid (20 mM) is added at 0, 24 and 48 hours.

[0107] After fermentation, the supernatant obtained by centrifugation of 2 mL culture at 5000 g for 5 min in an Optima L-80XP Ultracentrifuge (Beckman-Coulter, Schaumburg, IL) is prepared for GC-FID / GC-MS analysis. A 2 mL aliquot of the supernatant is transferred to a 5 mL glass vial (Fisher Scientific Co., Pittsburgh, PA). An organic solvent (typically ethyl acetate) is then added in a 1:1 ratio to the fermentation broth sample for extraction (e.g., 2 mL for 2 mL aqueous solution). After appropriate extraction (vortex the sample for 15 s, spin on a rotator at 60 rpm for 2 h, vortex again for 15 s), 1 mL of the organic phase is removed. 50 μL of pyridine and 50 uL BSTFA are then added to the 1 mL of organic phase for derivatization, and the reaction is allowed to proceed at 70° C. for 30 min. After cooling to room temperature, the mixture is used for GC analysis.

[0108] GC analysis is performed on an Agilent Intuvo 9000 Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector Turbo EI Bundle (for identification) or a Flame Ionization Detector (for quantification) and an Agilent HP-5 capillary column (0.25 mm internal diameter, 0.25 μm film thickness, 30 m length). The following temperature profile is used with helium as carrier gas at a flow rate of 1.5 mL / min: initial 50°C (3 min hold); ramp to 270°C at 20°C / min (6 min hold). Injector and detector temperatures are 250°C and 350°C, respectively. 1 uL of sample is injected with a separation ratio of 20:1.

[0109] Example 2: This example shows the implementation of a carboxylic acid (CA) platform that uses lactic acid (lactate) as a CA intermediate via the C1 elongation pathway. As described above, lactate can be supplied as a carbon source or can be made from carbon sources such as glucose and / or other biomass-derived sugars. Activation and reduction of lactate to various compounds such as lactaldehyde, acetone or propanal proceeds via the reactions and associated enzymes described above.

[0110] For C1 elongation, the pathway is based on 2-hydroxyacyl-CoA (HACL) enzymes that condense formyl-CoA with carbonyl-containing compounds such as propanal, lactaldehyde, and acetone (Chou, A., et al. Nat. Chem. Biol. 15:900-906 (2019)). Here, these intermediates are selectively generated by lactate reduction and serve as substrates for C1 addition, and the resulting 2-hydroxyacyl-CoA is converted to various products. Formyl-CoA is generated from formaldehyde by CoA-acylated formaldehyde dehydrogenase from Listeria monocytogenes (Chou, A., et al. Nat. Chem. Biol. 15:900-906 (2019)).

[0111] When lactaldehyde is made from lactoyl-CoA, HACL from the Rhodospirillales bacterium URHD0017 is used to condense lactaldehyde with formyl-CoA to form 2,3-dihydroxybutyryl-CoA. This C4 intermediate can then be converted to a variety of products. 2,3-Dihydroxybutyrate is made from 2,3-dihydroxybutyryl-CoA by the action of thioesterases such as TesB from Escherichia coli or Pseudomonas putida (McMahon, MD and Prather, KLJ Appl. Environ. Microbiol. 80:1042-1050 (2014)). Alternatively, 2,3-dihydroxybutyryl-CoA can be reduced to 2,3-dihydroxybutyraldehyde by an acyl-CoA reductase such as Clostridium beijerinckii ALD (Kim, S., et al. J. Ind. Microbiol. Biotechnol. 42:465-475 (2015)). 2,3-Dihydroxybutyraldehyde can be further reduced to 1,2,3-butanetriol by an appropriate alcohol dehydrogenase such as E. coli FucO, AdhP, or YqhD (Kim, S., et al. J. Ind. Microbiol. Biotechnol. 42:465-475 (2015)).

[0112] Dehydration of 1,2,3-butanetriol to 3-hydroxybutyraldehyde is catalyzed by K. oxytocandiol dehydratase (Yamanishi, M., et al. FEBS J. 279: 793-804 (2012)). Further reduction of 3-hydroxybutyraldehyde by the action of an appropriate alcohol dehydrogenase such as E. coli FucO, AdhP or YqhD (Kim, S., et al. J. Ind. Microbiol. Biotechnol. 42:465-475 (2015)) results in the production of 1,3-butanediol.

[0113] When acetone is made from lactoyl-CoA, HACL from the Rhodospirillales bacterium URHD0017 is used to condense acetone with formyl-CoA to form 2-hydroxyisobutyryl-CoA. This C4 intermediate can then be converted to a variety of products, such as 2-hydroxyisobutyric acid, 2-hydroxyisobutyraldehyde, 2-methyl-1,2-propanediol, isobutyraldehyde, and isobutanol, by the pathways described above using appropriate enzymes.

[0114] When propanal is made from lactoyl-CoA, HACL from the Rhodospirillales bacterium URHD0017 is used to condense propanal with formyl-CoA to form 2-hydroxybutyryl-CoA. This C4 intermediate can then be converted to a variety of products, such as 2-hydroxybutyric acid, 2-hydroxybutyraldehyde, 1,2-butanediol, butyraldehyde, and n-butanol, by the pathways described above using appropriate enzymes.

[0115] Methanol is provided as an external energy source to generate reducing equivalents and ATP required for reduction product synthesis. Methanol oxidation proceeds via the NADH-dependent methanol dehydrogenase 2 from Bacillus methanolicus (Roth, TB, et al. ACS Syn. Biol. 8:796-806 (2019)) to form formaldehyde and generate NADH. To enable direct ATP generation from the provided C1, formaldehyde is converted to formate via formyl-CoA and formyl-phosphate intermediates using CoA-acylated formaldehyde dehydrogenase, phosphate formyltransferase, and formate kinase. Here, CoA-acylated formaldehyde dehydrogenase from Listeria monocytogenes (Chou, A., et al. Nat. Chem. Biol. 15:900-906 (2019)) and phosphate formyltransferase and formate kinase enzymes from Clostridium cylindrosporum (Sly, WS and Stadtman, ERJ Biol. Chem. 238:2639-2647 (1963)) are used to generate additional NADH and ATP during this conversion. Formate oxidation to CO2 by NADH-dependent formate dehydrogenase from Pseudomonas sp. (strain 101) is used to generate additional NADH (Tishkov, VI, et al. Biochem. Biophys. Res. Commun. 192:976-81 (1993)).

[0116] Example 3: Overview of enzymes catalyzing energy (redox and ATP) and formyl-COA production The purpose of this example is to provide exemplary genes, enzymes, and pathways involved in the interconversion of one-carbon (C1) molecules to provide energy (in terms of NADH and ATP) and formyl-CoA required for the C1 elongation reaction.

[0117] The preferred embodiments of the present invention are provided as examples that allow those skilled in the art to make further modifications in combination with the present disclosure.The examples teach how to confer the ability to microorganisms, such as specific pathway design, the genes / enzymes required to construct the pathway, methods for cloning and transformation, and the use of engineered microorganisms to monitor and produce product formation.Although the examples show modified E. coli strains, these modifications can be easily carried out in other microorganisms of the same family of Enterobacteriaceae and other bacterial species as well as yeast and fungi.

[0118] Methane is a readily available C1 source derived from natural gas, landfill waste and agriculture. The biological oxidation of methane to methanol is catalyzed by methane monooxygenase. Functional expression of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus is shown in Escherichia coli as a host (bioRxiv 2021.08.05.455234) (Figure 1 and Table 1). The subsequent oxidation of methanol to formaldehyde is catalyzed by NAD +MDHs from Bacillus methanolicus MGA3 (BmMDH), Bacillus stearothermophilus (BsMDH) (Metab. Eng. 39:49-59, 2017) and Cupriavidus necator (Appl. Microbiol. Biotechnol. 100:4969-4983, 2016) were expressed in E. coli and characterized (Figure 1 and Table 1). Formaldehyde can be directly oxidized to formic acid, catalyzed by the E. coli formaldehyde detoxification system (frmA). Alternatively, formaldehyde oxidation to formyl-CoA can be catalyzed by various acylating aldehyde dehydrogenase candidates (Nat. Chem. Biol. 15:900-906, 2019) (Figure 1 and Table 1). Formyl-CoA can either be fed to the C1 elongation platform or further converted to produce energy. Formyl-CoA hydrolysis to formate can be catalyzed by two different families of enzymes. Acyl-CoA transferases (ACTs) can catalyze the reversible CoA transfer from various CoA donors such as glycolyl-CoA, acetyl-CoA or succinyl-CoA to formate. Phosphotransacylase-formate kinase (PTA-FOK) pairs catalyze the reversible phosphorylation of formyl-CoA to formyl-phosphate and then dephosphorylation to formate to generate one ATP. Although these two reactions are believed to be fully reversible, AMP-forming acyl-CoA synthetase is preferred for formate activation to formyl-CoA. Finally, formate can be further oxidized to CO2 catalyzed by soluble formate dehydrogenase to generate NADH, or the same enzyme can be used to reduce CO2 to formate for the generation of formyl-CoA (Figure 1 and Table 1). [Table 1]

[0119] Example 4: Preparation of formyl-COA from formic acid The purpose of this example is to illustrate the aspects of the invention pertaining to the production of formyl-CoA from formate in vivo using both resting cells and growing cultures. Formyl-CoA produced by formate-activating enzyme (FAE) is further condensed with formaldehyde to produce glycolic acid. We engineered a vector to independently control the expression of BsmHACS (UniProt accession: A0A3C0TX30), which is sourced from beach sand metagenome along with FAE. BsmHACS is under the control of IPTG-inducible T7 promoter in pCDFduet-1, and FAE is under the control of cumate-inducible T5 promoter in pETDuet-1 (Figure 2A). The FAEs were selected to represent the three major formate activation pathways, including acyl-CoA synthase ACS (Nmar0206 and EcACS from Nitrosopumilus maritimus), acyl-CoA transferase ACT (AbfT from Clostridium aminobutyricum) and phosphotransacylase (PTA)-FOK (CcPta+CcAck from Clostridium cylindrosporum). As hosts for these vectors, we used genetically engineered strains of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhF ΔfdnG ΔfdoG) oxidation as well as for glycolate utilization (ΔglcD), which we predicted might compete or interfere with our analysis of the pathways.

[0120] The cells from the precultures described above were then centrifuged (5000×g, 22° C.), washed twice with the minimal medium described above without a carbon source, and resuspended to an optical density of about 10. Five mL of this cell suspension and the indicated amount of carbon source (e.g., formaldehyde) were added to a 25 mL Pyrex Erlenmeyer flask (Corning Inc., Corning, NY) and sealed with a foam plug filling the neck. 10 mM formaldehyde and 50 mM formate were added at 0 hours. The flasks were incubated at 30° C. and 200 rpm in an NBS I24 benchtop incubator shaker (New Brunswick Scientific Co., Inc., Edison, NJ). After 24 hours of incubation at 30° C., the cells were centrifuged at 20817×g for 15 minutes, and the supernatants were analyzed by HPLC as described below.

[0121] Quantification of product and substrate concentrations (formic acid, formaldehyde, and glycolic acid) was determined by HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) with operating conditions to optimize peak separation (0.3 ml / min flow rate, 30 mM H2SO4 mobile phase, column temperature 42°C). Compound identification and analysis were performed by GC-MS using an Agilent 7890B Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector Turbo EI Bundle (for identification) and an Agilent HP-5-ms capillary column (0.25 mm inner diameter, 0.25 μm film thickness, 30 m length).

[0122] The expected product glycolate was detected in the medium, indicating the performance of the corresponding formate-activating enzymes. Among the formate CoA transferases tested, CaAbfT has the best performance (Figure 2B).

[0123] Since formate activation is a required step for formate utilization, we further evaluated the enzymes that catalyze this reaction. Acyl-CoA synthetase (ACS) enzyme is one of the three examined pathways for formate activation, which can directly generate formyl-CoA from formate at the expense of 2 ATP equivalents (ATP is converted to AMP). ACS evaluated using resting cells showed relatively poor activity as mentioned above, which may be due to the limited availability of ATP in resting cell experiments. Therefore, ACS variants were further evaluated using a novel established growing cell platform with BsmHACS overexpression. In this platform, formyl-CoA generated by formate-activating enzyme is further condensed with the non-toxic substrate acetone to generate 2-hydroxyisobutyrate (2HIB) (Figure 2C).

[0124] Screening of a larger panel of formate-activating enzymes in actively growing cells was performed using a medium containing 6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2, 15 μM thiamine-HCl, 10 g / L tryptone, and 5 g / L yeast extract as described by Neidhardt et al. 68A single colony of the desired strain was grown overnight (14-16 h) in LB medium with the appropriate antibiotic and used as an inoculum (1%) into a 50 mL centrifuge tube containing 5 mL of M9-LB medium. Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. The culture was then incubated at 30 °C, 250 rpm in a Lab Companion SI-600 rotary shaker (Jeio Tech, Seoul, South Korea) until it reached an OD550 of approximately 0.4, at which point appropriate amounts of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) and substrate (100 mM acetone and 20 mM formate) were added. The tube was clamped and incubated for a total of 48 h after inoculation. Cells were pelleted by centrifugation and the supernatant was analyzed by HPLC as described below. The expected product 2-hydroxyisobutyric acid was detected in the medium, indicating the performance of the corresponding formate-activating enzyme.

[0125] Quantification of product and substrate concentrations (formic acid, acetone, and 2HIB) was determined by HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) under operating conditions that optimized peak separation (0.3 ml / min flow rate, 30 mM H2SO4 mobile phase, column temperature 42°C). Compound identification and analysis were performed by GC-MS using an Agilent 7890B Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector Turbo EI Bundle (for identification) and an Agilent HP-5-ms capillary column (0.25 mm inner diameter, 0.25 μm film thickness, 30 m length).

[0126] To test ACS activity, we also included two CoA transferases with good performance for formate activation to serve as positive controls. By screening a larger group of FAEs in actively growing cells, the CoA transferases (CaAbfT and OfFrc) have better performance for formate activation than the other tested formate-activating enzymes. Of the ACSs tested, StACS has the best performance, with its activity comparable to CoA transferases (Figure 2D).

[0127] Example 5: Preparation of Formyl-COA from Formaldehyde The purpose of this example is to illustrate the use of various acylated formaldehyde dehydrogenases / acyl-CoA reductases (ACR1) to catalyze the interconversion between formaldehyde and formyl-CoA. We substituted glycolate production by adding active 2-hydroxyacyl-CoA lyase, HACL (RuHACL) from the Rhodospirillales bacterium URHD0017, along with different ACR1 candidates (Figure 3A).

[0128] Cell-free reactions for pathway prototyping were performed in 50 mM KPi pH 7.4, 4 mM MgCl2, 0.1 mM TPP, 2.5 mM CoASH, 5 mM NAD + and 50 mM formaldehyde. Individual cell extract loadings were approximately 4.4 g / L protein (1 / 8 of reaction volume) and the amount of protein added to each reaction was standardized with BL21 (DE3) to approximately 26 g / L protein (3 / 4 of reaction volume). Reactions were incubated at room temperature for 1 h unless otherwise specified. Reactions were terminated by adding 1 / 4 of the reaction volume of saturated ammonium sulfate acidified with 1% sulfuric acid. Samples were centrifuged at 20817 x g for 15 min and the supernatants were analyzed by HPLC or GC-MS as described below.

[0129] Quantification of product and substrate concentrations (formic acid, formaldehyde, and glycolic acid) was determined by HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) under operating conditions that optimized peak separation (0.3 ml / min flow rate, 30 mM H2SO4 mobile phase, column temperature 42°C). Compound identification and analysis were performed by GC-MS using an Agilent 7890B Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector Turbo EI Bundle (for identification) and an Agilent HP-5-ms capillary column (0.25 mm internal diameter, 0.25 μm film thickness, 30 m length).

[0130] We combined extracts of E. coli prepared as described in the previous example(s) expressing RuHACL and ACR1 variants and rapidly screened for the best performing combination. Of the ACRs tested, the Listeria monocytogenes derived variant (LmACR) was best adapted to enable the pathway. The combination of LmACR and RuHACL enabled the production of 6.2±0.6 mM glycolic acid in 1 hour (FIG. 3B).

[0131] Example 6: Methanol as a source for redox (NADH) and formyl COA production The purpose of this example is to illustrate the utilization of methanol as a source for the production of NADH and formyl-CoA demonstrated in vivo using both resting cells and growing cultures. +Methanol oxidation to formaldehyde, catalyzed by β-dependent methanol dehydrogenase, creates NADH, which can be used as reducing power for pathways or energy in the form of ATP when coupled with oxidative phosphorylation. Similarly, formaldehyde oxidation to formyl-CoA also generates one NADH. These two enzymes, combined with HACL (RuHACL) from the Rhodospirillales bacterium URHD0017, catalyze methanol to glycolyl-CoA, which is readily hydrolyzed to glycolate by an endogenously expressed thioesterase (Figure 4A).

[0132] To implement the methanol utilization pathway in vitro, we engineered vectors to express RuHACL and Listeria monocytogenes-derived acyl-CoA reductase (LmACR) and various methanol dehydrogenase (MDH) candidates. As hosts for these vectors, we used engineered strains of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhF ΔfdnG ΔfdoG) oxidation, as well as for glycolate utilization (ΔglcD), which we predicted might compete or interfere with our pathway analysis.

[0133] Resting cell prototyping was performed using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl) unless otherwise stated. Cells were initially grown in 125 mL baffled flasks (Wheaton, Millville, NJ) containing 25 mL of the above mentioned medium, further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (50 μg / mL carbenicillin, 50 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30°C, 250 rpm in a Lab Companion SI-600 rotary shaker (Jeio Tech, Seoul, South Korea) until they reached an OD550 of approximately 0.4, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. Flasks were incubated for a total of 24 h after inoculation.

[0134] Cells from the preculture described above were then centrifuged (5000×g, 22° C.), washed twice with the above minimal medium without carbon source, and resuspended to an optical density of approximately 10. Five mL of this cell suspension and the indicated amount of carbon source (e.g., formaldehyde) were added to a 25 mL Pyrex Erlenmeyer flask (Corning Inc., Corning, NY) and sealed with a foam plug filling the neck. 25 mM acetone and 5 mM formaldehyde were added at 0 hours, and additional 5 mM formaldehyde was added at 1, 2, and 3 hours, and the flasks were incubated at 30° C., 200 rpm in an NBS I24 benchtop incubator shaker (New Brunswick Scientific Co., Inc., Edison, NJ). After 24 hours of incubation at 30° C., the cells were pelleted by centrifugation and the medium was analyzed. The expected product, glycolic acid, was detected in the medium, indicating its production by the engineered organism.

[0135] For growing cell experiments, the growth medium used was M9 (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2, and 15 μM thiamine-HCl) further supplemented with 500 mM methanol, 10 g / L tryptone, 5 g / L yeast extract, and trace element elements from Neidhardt et al. Overnight LB cultures of each strain were used to inoculate 50 mL closed-cap conical tubes (Genesee Scientific Co.) containing 5 mL of the above-mentioned medium further supplemented with the appropriate antibiotics (50 μg / mL carbenicillin, 50 μg / mL spectinomycin) (1%). After approximately 3 h, gene expression was induced by the addition of 0.04 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and 0.04 mM cumate. The tubes were incubated at 30°C and 200 rpm in an NBS I24 benchtop incubator shaker (New Brunswick Scientific Co.). OD was measured as previously described. 600For measurement and HPLC analysis, samples (100 μL) were obtained every 24, 48, 72 and 96 hours after inoculation.

[0136] Resting cell experiments indicate that various MDH and LmACR combinations generate formyl-CoA as shown by the production of glycolate as a substitute. Among the variants tested, BmMDH MGA3 gave the best glycolate production up to 50 mg / L, and CnMDH at 30 mg / L in resting cell format. CT4-1 followed (Figure 4B).

[0137] The best two enzyme candidates were tested under growing cell experiments. The correlation between methanol consumption and cell growth indicates the utilization of methanol as a source of energy (ATP) via respiration with excess NADH production. The results with glycolate production are aligned with those from resting cell experiments, but CnMDH CT4-1 shows faster consumption of methanol and accumulation of formate (Figure 4C). MGA3 is a better choice for making formyl-CoA, and CnMDH CT4-1 is a better enzyme for energy production.

[0138] Example 7: Carboxylic Acid Platform Using Formic Acid This example shows the implementation of a carboxylic acid (CA) platform using formic acid (formate) as a CA via the C1 elongation pathway. Formate can be generated internally by cells via the enzyme pyruvate formate lyase (e.g., PFL1 from Chlamydomonas reinhardtii, pfl from Clostridium pasteurianum, pflB and tdcE from Escherichia coli, pfl from Streptococcus mutans: Nucleic Acids Research 42(1):D459-D471, 2014) and can be supplied as a carbon source generated from electrochemical or enzymatic reduction of CO2. Activation and reduction or amination of formate gives a (substituted) C1 aldehyde that serves as a substrate for a condensation reaction with formyl-CoA. The resulting 2-hydroxyacyl-CoA can be hydrolyzed to the corresponding acid, which can then be aminated to form an amino acid. Reduction of amino acids to aldehydes gives 2-aminoaldehydes, which can then be aminated to form diamines (catalyzed by diamine dehydrogenases or transaminases) or further reduced to form alkanoamines (2-aminoalcohols) (catalyzed by alcohol dehydrogenases). Alternatively, they can go through the α-reduction cycle to make 1,2-diols and alcohols (Figure 5).

[0139] Activation of formate to formyl-CoA has been demonstrated using a variety of natural and engineered acyl-CoA synthetases (Synth. Biol. 6:1-14, 2021), CoA transferases (J. Biol. Chem. 283: 6519-6529, 2008; ACS Catal. 11:5396-5404, 2021; Nat. Metab. 3:1385-1399, 2021) and carboxylate kinase-phosphotransacylases (J. Biol. Chem. 238:2639-2647, 1963). Further reduction of formyl-CoA to formaldehyde is catalyzed by CoA-acylated formaldehyde dehydrogenase (e.g., from Listeria monocytogenes: Synth. Biol. 6:1-14, 2021; Nat. Metab. 3:1385-1399, 2021).

[0140] When formaldehyde is made from formyl-CoA, HACL from the Rhodospirillales bacterium URHD0017 can be used to condense formaldehyde with formyl-CoA to form glycolyl-CoA. Glycolyl-CoA can be used as a CoA donor for activation of formate as shown using AbfT from Clostridium aminobutyricum or can be hydrolyzed to glycolate by the action of a thioesterase such as YciA from E. coli (ACS Catal. 11:5396-5404, 2021). It can also undergo a glycolyl-phosphate intermediate to make glycolate and ATP, similar to formate activation via a phosphate intermediate catalyzed by E. coli Pta and Ack or equivalent enzymes. Glycolate can be further converted to glycoxylate via GlcD from E. coli, and then alanine dehydrogenase reaction produces glycine, one of the essential amino acids for living organisms. Several alanine dehydrogenases have glyoxylate activity to produce glycine (J. Bacteriol. 194:1045-1054, 2012; Biochemistry 20:5650-5655, 1981). Glycine can be reduced to aminoacetaldehyde catalyzed by various aldehyde dehydrogenases / oxidases. The resulting aminoacetaldehyde can be further aminated to form ethylenediamine by diamine dehydrogenase or transaminase activity, or reduced to form ethanolamine.

[0141] Glycolyl-CoA can be further reduced to glycolaldehyde via acyl-CoA reductase. In this case, the same enzyme (LmACR) used to reduce formyl-CoA to formaldehyde can catalyze the reduction of glycolyl-CoA to glycolaldehyde (Nat. Metab. 3:1385-1399 (2021)). Glycolaldehyde reduction to ethylene glycol is catalyzed by E. coli fucO. Dehydration of ethylene glycol gives acetaldehyde, catalyzed by PddABC from Klebsiella oxytoca. The resulting acetaldehyde can feed into subsequent iterations of C1 elongation or can be reduced to ethanol, catalyzed by E. coli adhE (Figure 6 and Table 2). [Table 2]

[0142] Example 8: Conversion of formate to ethylene glycol This example demonstrates the implementation of a carboxylic acid (CA) platform that uses formic acid (formate) as a CA intermediate via the C1 elongation pathway. Activation of formate to formyl-CoA is demonstrated using a variety of natural and engineered acyl-CoA synthetases (Synth. Biol. 6:1-14, 2021) and CoA transferases (J. Biol. Chem. 283: 6519-6529, 2008; ACS Catal. 11:5396-5404, 2021; Nat. Metab. 3:1385-1399, 2021). Further reduction of formyl-CoA to formaldehyde is catalyzed by CoA-acylated formaldehyde dehydrogenase (e.g., from Listeria monocytogenes: Synth. Biol. 6:1-14, 2021; Nat. Metab. 3:1385-1399, 2021).

[0143] When formaldehyde is made from formyl-CoA, using HACL from the Rhodospirillales bacterium URHD0017 (RuHACL) or beach sand metagenome BsmHACS (UniProt accession: A0A3C0TX30), formaldehyde and formyl-CoA are condensed to form glycolyl-CoA. Glycolyl-CoA can be further reduced to glycolaldehyde via acyl-CoA reductase. In this case, the same enzyme (LmACR) used to reduce formyl-CoA to formaldehyde can catalyze the reduction of glycolyl-CoA to glycolaldehyde (Nat. Metab. 3:1385-1399, 2021). Glycolaldehyde reduction to ethylene glycol is catalyzed by E. coli fucO (Figure 7A).

[0144] Expression of selected enzyme variants was achieved using plasmid-based gene expression by cloning the desired gene(s) into pETDuet-1 or pCDFDuet-1 (Novagen, Darmstadt, Germany) digested with appropriate restriction enzymes and utilizing In-Fusion cloning technology (Clontech Laboratories, Inc., Mountain View, CA). Linear DNA fragments for insertion were generated via PCR of the open reading frame of interest (for genes native to E. coli) or by gene synthesis for codon-optimized genes. Genes were synthesized by GeneArt (Life Technologies, Carlsbad, CA). The resulting In-Fusion reaction products were used to transform E. coli Stellar cells (Clontech Laboratories, Inc., Mountain View, CA) and clones identified by PCR screening were further confirmed by DNA sequencing.

[0145] Overnight cultures of expression strains were grown in LB and used to inoculate 25 mL TB medium in 250 mL baffled flasks at 1%. Cultures were grown at 30 °C and 250 rpm in a rotary shaker until the OD550 reached 0.4-0.6, at which point expression was induced with 0.1 mM IPTG. 24 h after inoculation, cells were harvested by centrifugation. Cell pellets were washed once with cold 9 g / L NaCl solution and stored at -80 °C until required. Antibiotics were included where appropriate at the following concentrations: ampicillin (100 μg / mL), carbenicillin (50 μg / mL) and spectinomycin (50 μg / mL).

[0146] For protein purification, E. coli cell pellets expressing the desired his-tagged enzymes were prepared as described above. Frozen cell pellets were resuspended in cold lysis buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.1% Triton-X 100) to an approximate OD550 of 40, to which 1 mg / mL lysozyme and 250 U of Benzonase nuclease were added. The mixture was further processed by sonication on ice using a Branson Sonifier 250 (5 min at 25% duty cycle and output control set at 3) and centrifuged at 7500×g for 15 min at 4° C. The supernatant was applied to a chromatography column containing 1 mL TALON metal affinity resin (Clontech Laboratories, Inc., Mountain View, CA) and pre-equilibrated with lysis buffer. The column was then washed first with 10 mL of lysis buffer, then twice with 20 mL of wash buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 20 mM imidazole). The his-tagged protein of interest was eluted by 1-2 applications of 4 mL elution buffer (50 mM NaPi pH 7.4, 300 mM NaCl, 250 mM imidazole). The eluate was collected and applied to a 10,000 MWCO Amicon ultrafiltration centrifugation device (Millipore, Billerica, MA) and the concentrate (approximately 100 μL) was washed twice with 4 mL of 50 mM KPi pH 7.4 for desalting. Protein concentration was estimated by the Bradford method. The purified protein was stored at -80 °C in 20 μL aliquots until needed.

[0147] SDS-PAGE was performed using NuPAGE 12% Bis-Tris Protein Gels with SDS running buffer and stained with SimplyBlue SafeStain (ThermoFisher Scientific, Waltham, MA) according to the manufacturer's protocol.

[0148] In vitro purified enzyme reactions for products derived from formate as the sole carbon source were performed in 200 mM KPi pH 8.0, 5 mM MgCl2, 0.2 mM TPP, 6 mM NADH, 2 mM succinyl-CoA, 2 μM BsmHACL or RuHACL. G390N , 2 μM OfFrc, 4 μM LmACR, 2 μM FucO, and 50 mM sodium formate. Unless otherwise specified, reactions were incubated at 30°C for 24 h. Reactions were terminated by adding 1 / 20 of the reaction volume of 10 M NaOH solution. After 30 min of hydrolysis, the pH was neutralized by adding 1 / 20 of the reaction volume of 10 N H2SO4. Samples were centrifuged at 20817 x g for 15 min and the supernatants were analyzed by HPLC or GC-MS as described below.

[0149] Quantification of product and substrate concentrations (formic acid, formaldehyde, glycolic acid, and ethylene glycol) was performed by HPLC using a Shimadzu Prominence SIL 20 system (Shimadzu Scientific Instruments, Inc., Columbia, MD) equipped with a refractive index detector and an HPX-87H organic acid column (Bio-Rad, Hercules, CA) under operating conditions that optimized peak separation (0.3 ml / min flow rate, 30 mM H2SO4 mobile phase, column temperature 42°C). Compound identification and analysis were performed by GC-MS using an Agilent 7890B Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector (for identification) and Agilent HP-INNOWax Columns (0.25 mm internal diameter, 0.25 μm film thickness, 30 m length).

[0150] By using the best identified ACR enzyme LmACR*, the ethylene glycol titer was improved by 85% compared to the wide type LmACR. By adding more LmACR*, more reducing equivalents NADH and more CoA donor were provided, and the ethylene glycol titer was further increased to 145.8 mg / L (Figure 7). In vitro experiments show that LmACR and NADH / reducing equivalents have a significant effect on formate conversion to ethylene glycol (Figure 7B).

[0151] Example 9: Formaldehyde-formyl-COA condensation in vivo The purpose of this example is to illustrate the aspects of the invention pertaining to C1-C1 (formaldehyde-formyl-CoA) condensation for product synthesis in vivo. To implement the C1 elongation pathway for glycolate production in vivo, we engineered vectors to independently control the expression of various HACS candidates and acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), where HACS was under the control of an IPTG-inducible T7 promoter in pETDuet-1 and CaAbfT was under the control of a cumate-inducible T5 promoter in pCDFDuet-1 (Figure 8C). As a host for these vectors, we used an engineered strain of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhF ΔfdnG ΔfdoG) oxidation, as well as for glycolate utilization (ΔglcD), which we predicted might compete or interfere with our pathway analysis (FIG. 8B).

[0152] In vivo product synthesis was performed using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl) unless otherwise stated. Cells were initially grown in 96-deep-well plates (USA Scientific, Ocala, FL) containing 0.2 mL of the above-mentioned medium further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an OD600 of approximately 0.4 was reached, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. Plates were incubated for a total of 24 hours after inoculation.

[0153] Cells from the above precultures were then centrifuged (4000 rpm, 22° C.), washed with the above minimal medium without carbon source, and resuspended in 1 mL of the above cell culture medium containing the indicated amount of carbon source. 5 mM formaldehyde and 20 mM formate were added at 0 hours and incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific). After 3 hours of incubation at 30° C., cells were pelleted by centrifugation and the medium was analyzed (FIG. 8A).

[0154] The results show that RuHACL and BsmHACL are the two best candidates under the given experimental conditions reaching up to 80 μM / h per OD600.

[0155] Example 10: Cell-free pathway prototyping using formaldehyde as the starting substrate The purpose of this example is to describe a cell-free method for prototyping a C1 elongation pathway for formaldehyde as a starting substrate that can be generated by activation and reduction of a carboxylic acid, formate. We prototyped the use of the enzymes identified in the previous example as part of a synthetic pathway for the conversion of C1 substrates to multicarbon products using formaldehyde as the sole carbon source (FIG. 9A). The most immediate two-carbon ligation product of the pathway is glycolic acid, which can be easily hydrolyzed from glycolyl-CoA by an endogenously expressed thioesterase. Thus, the synthesis of glycolic acid from formaldehyde using HACL requires only the further generation of formyl-CoA, which can be achieved by the use of acyl-CoA reductase (ACR).

[0156] Cell-free reactions for pathway prototyping were performed in 50 mM KPi pH 7.4, 4 mM MgCl2, 0.1 mM TPP, 2.5 mM CoASH, 5 mM NAD + and 50 mM formaldehyde. For time course experiments, 0.1 mM coenzyme B12 was added. Individual cell extract loadings were approximately 4.4 g / L protein (1 / 8 of reaction volume), and the amount of protein added to each reaction was standardized to approximately 26 g / L protein (3 / 4 of reaction volume) with BL21(DE3) extract. Reactions were incubated at room temperature for 1 h unless otherwise specified. Reactions were terminated by adding 1 / 4 of the reaction volume of saturated ammonium sulfate solution acidified with 1% sulfuric acid. Samples were centrifuged at 20817×g for 15 min, and the supernatants were analyzed by HPLC or GC-MS as previously described.

[0157] To demonstrate the utility of HACL-catalyzed elongation reactions to generate different chemical functionalities from the resulting 2-hydroxyacyl-CoA product, we included enzymes to extend the LmACR+RuHACL pathway (Figure 9B). Acyl-CoA reductase is required to reduce glycolyl-CoA to glycolaldehyde, and in screening the same set of ACRs for activity on glycolaldehyde, we found that LmACR could also act on glycolaldehyde. To minimize the complexity of the engineered system, we used LmACR in a bifunctional role, catalyzing both the oxidation of formaldehyde to formyl-CoA and the reduction of glycolyl-CoA to glycolaldehyde. As shown in Figure 9B, LmACR alone only produced the conversion of formaldehyde to formate. With the inclusion of RuHACL, glycolate was observed. However, glycolaldehyde was not significantly detected as a product, likely due to the presence of endogenous oxidoreductases in the cell extract system that catalyze the oxidation of glycolaldehyde to glycolic acid or, to a lesser extent, its reduction to ethylene glycol.

[0158] The synthesis of the next reduction product, ethylene glycol, was significantly increased by the addition of cell extracts of E. coli overexpressing E. coli FucO, a 1,2-diol oxidoreductase (2-fold increase from 1.37 ± 0.1 mM to 2.73 ± 0.03 mM) (Figure 9B). Ethylene glycol can be further dehydrated to acetaldehyde by diol dehydratase. Upon addition of E. coli cell extracts expressing diol dehydratase (DDR) from Klebsiella oxytoca, ethanol, the product of the reduction of acetaldehyde by endogenous aldehyde reductase, was detected with a corresponding drop in ethylene glycol (1.90 ± 0.03 mM at 1 h: Figure 9B). The synthesis of these different products (i.e. glycolate, ethylene glycol, ethanol) indicates the use of the 2-hydroxyacyl-CoA node to easily create products with different levels of reduction, chain length and functionality.

[0159] Example 11: Production of glycine from glycolate in a glycine auxotroph This example shows an alternative route of 2-hydroxy acids to amino acid production when formate is used as the starting carboxylic acid. Illustrating glycolate conversion to glycine was done by growth-coupled selection of two candidates for alanine dehydrogenase, which catalyzes glyoxylate reduction to glycine (FIG. 10A). As a host for the selection platform, we engineered a glycine auxotroph of E. coli based on MG1655(DE3) with a knockout for glycine production and utilization (ΔaceA Δkbl ΔltaE ΔglyA), and grew the strain only on glycine supplementation (FIG. 10A).

[0160] For gene deletion, CRISPR is used based on the method developed in Appl. Environ. Microbiol. 81:2506-2514, 2015). First, the host strain is transformed with the vector for expression of plasmid pCas, Cas9 and λ-red recombinase. The resulting strain is grown with L-arabinose for induction of λ-red recombinase expression at less than 30°C, and when OD reaches about 0.6, competent cells are prepared and transformed with pTargetF (AddGene 62226) expressing sgRNA and N20 spacer targeting locus and template for insertion of target gene. The template is the deleted gene with about 500bp sequence homologous to the upstream and downstream of the insertion locus, constructed by overlap PCR using Phusion polymerase or synthesized by GenScript (Piscataway, NJ). The method for replacing the N20 spacer of the pTargetF plasmid is inverse PCR with a modified N20 spacer dangling at the 5' end of the primer using Phusion polymerase, followed by self-ligation using T4 DNA ligase and T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA). Transformants growing below 30°C on solid medium (LB+agar) supplemented with spectinomycin and kanamycin (or other appropriate antibiotics) are isolated and screened for chromosomal gene inserts by PCR. The sequences of gene inserts amplified from genomic DNA by PCR using Phusion polymerase are further confirmed by DNA sequencing. pTargetF can then be cured by IPTG induction and pCas can be cured by growth at higher temperatures such as 37-42°C.

[0161] The resulting glycine auxotrophs were transformed with vectors constitutively expressing alanine dehydrogenase from Mycobacterium tuberculosis (MtAld) or Bacillus subtilis (BsAld). Of the two candidates, the strain carrying BsAld started growing on glycolate instead of glycine supplementation (Figure S10B), indicating that glycolate was successfully converted to glycine by the native glcD and heterologously expressed BsAld genes.

[0162] Example 12: Formamide as a substrate for condensation with formyl-COA This example shows the implementation of a carboxylic acid (CA) platform using formic acid (formate) as a CA intermediate via the C1 elongation pathway. Formamidase-catalyzed amination of formate produces formamide, which can also be used as a substituted C1 aldehyde substrate for the 2-hydroxyacyl-CoA synthase (HACS) reaction. Functional expression and characterization of formamidase from Methylophilus methylotrophus is shown in Eur. J. Biochem. 240:314-322 (1996).

[0163] Subsequent C1 elongation of formamide catalyzed by HACS forms 2-aminolactoyl-CoA, which can be utilized as a CoA donor for activation of formate using CoA transferases such as AbfT from Clostridium aminobutyricum or can be hydrolyzed via the action of thioesterases such as YciA from E. coli to give 2-hydroxyglycine. This can also undergo a 2-aminolactoyl-phosphate intermediate to give 2-hydroxyglycine and ATP, similar to formate activation via a phosphate intermediate catalyzed by E. coli Pta and Ack or equivalent enzymes. 2-Hydroxyglycine can be further converted to oxamate via GlcD from E. coli or similar enzymes, followed by an amino acid dehydrogenase reaction to give diaminoacetic acid. Diaminoacetic acid can be reduced to diaminoethanal catalyzed by various aldehyde dehydrogenases / oxidases. The resulting diaminoethanal can be further aminated via diamine dehydrogenase or transaminase activity to form 1,1,2-ethanetriamine or reduced to form 2,2-diaminoethanol.

[0164] 2-Aminolactoyl-CoA can be further reduced to 2-aminolactaldehyde via acyl-CoA reductase activity. 2-Aminolactaldehyde reduction to glycolamine is catalyzed by E. coli fucO or a similar enzyme. Dehydration of glycolamine produces 2-aminoacetaldehyde, catalyzed by diol dehydratase. The resulting 2-aminoacetaldehyde can feed into subsequent iterations of C1 elongation or can be reduced to ethanolamine, catalyzed by alcohol dehydrogenase (Figure 11 and Table 3). [Table 3]

[0165] Example 13: Construction of formamidase expression vector This example describes the design and construction of a vector used to express Methylophilus methylotrophus formamidase (FmdA).

[0166] The FmdA gene is codon-optimized and synthesized by Twist Biosciences. The gene is then amplified by PCR using appropriate primers to add homology to each end for recombination into the vector backbone, for example, by Phusion polymerase (Thermo Scientific, Waltham, MA) to serve as the gene insert. The plasmid is linearized with appropriate restriction enzymes (New England Biolabs, Ipswich, MA, USA) and recombined with the gene insert using the In-Fusion HD Eco-Dry cloning system. The mixture is then transformed into Stellar competent cells. Transformants growing on solid medium (LB+agar) supplemented with the appropriate antibiotic are isolated and screened for gene insert by PCR. Plasmids from verified transformants are isolated and the sequence of the gene insert is further confirmed by DNA sequencing. The sequence-confirmed plasmid is then introduced into the host strain by electroporation (Figure 12).

[0167] Example 14: Carboxylic Acid Platform Using C2+ Carboxylic Acids as CA Intermediates via the C1 Elongation Pathway This example describes the implementation of a carboxylic acid (CA) platform using a C2+ carboxylic acid as a CA intermediate via the C1 elongation pathway. A C2+ carboxylic acid can be supplied as a carbon source. Activated and reduced C2+ carboxylic acid produces a C2+ aldehyde that serves as a substrate for a condensation reaction with formyl-CoA. The resulting 2-hydroxyacyl-CoA can be hydrolyzed to the corresponding 2-hydroxy acid, which can then be aminated to form 2-amino acids, alkanolamines, and diamines. Alternatively, 2-hydroxy acids can go through an α-reduction cycle to produce 1,2-diols, methyl ketones, alcohols, 3-hydroxy acids, and α,β-unsaturated acids (Figure 13). [Table 4] [Table 5-1] [Table 5-2]

[0168] Example 15: Carboxylic Acid Platform Using Acetaldehyde Derived from Acetic Acid as a Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using acetate (R=H) as a CA intermediate via the C1 elongation pathway (FIG. 14A). Acetate can be supplied as a carbon source or produced from electrochemical reduction or microbial conversion from acetogenic bacteria using CO2 as a carbon source. Conversion of CO2 to acetate has been demonstrated using a CO2 electrolysis system with carbon selectivity up to 57% (Hann et al. Nat. Food 3:461-471 (2022)).

[0169] The activation of acetate to acetyl-CoA and then reduction to acetaldehyde has been thoroughly examined in the literature.Acetate can be activated to acetyl-CoA via acetyl-phosphate.First, acetate is phosphorylated to acetylphosphate by acetate kinase from E. coli (Skarstedt et al. J Biol Chem. 251(21):6775-6783 (1976)), and then converted to acetyl-CoA by phosphate acetyltransferase from E. coli (Campos-Bermudez et al. FEBS J. 277(8):1957-1966 (2010)). Other pathways for activating acetate are acetyl-CoA synthetase from Escherichia coli (Biochem Biophys Res Commun. 449(3):272-277 (2014)) or succinyl-CoA transferase from Clostridium kluyveri (Soehling et al. Eur J Biochem. 212(1):121-127 (1993)). Finally, acetyl-CoA is reduced to acetaldehyde by acetaldehyde dehydrogenase from Escherichia coli (Song et al. Metab Eng. 35:38-45 (2016)).

[0170] C1 elongation is initiated by the energy- and formyl-CoA-generating step to generate formyl-CoA. 2-Hydroxyacyl-CoA synthase (HACS) condenses acetaldehyde and formyl-CoA to generate lactoyl-CoA. Lactoyl-CoA is converted to lactate by lactoyl-CoA transferase from Megasphaera elsdenii (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)).

[0171] Lactate can be further oxidized to pyruvate by lactate dehydrogenase from Bacillus casei (Gordon et al. Eur J Biochem. 67(2):543-555 (1976)). Pyruvate is reduced to alanine by alanine dehydrogenase from Bacillus subtilis (Yoshida et al. Methods in enzymology 17:176-181 (1970)).

[0172] Lactoyl-CoA, as the product of condensation, can be reduced to 1,2-propanediol (1,2-PDO), acetone, and 1-propanol. First, lactoyl-CoA is reduced to lactaldehyde by lactaldehyde dehydrogenase from Salmonella enterica (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)), which can be further reduced to 1,2-PDO by lactaldehyde reductase from Escherichia coli (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)). Dehydration of 1,2-PDO by propanediol dehydratase from Roseburia inulinivorans (LaMattina et al. J Biol Chem. 291(30):15515-15526 (2016)) gives acetone. Furthermore, dehydration of 1,2-PDO by diol dehydratase from Salmonella enterica (Bibok et al. J Bacteriol. 179(21):6633-6639(1997)) gives propionaldehyde. The resulting propionaldehyde can feed subsequent rounds of C1 elongation or can be reduced to 1-propanol by aldehyde reductase from Escherichia coli (Pick et al. Appl Microbiol Biotechnol. 97(13):5815-5824(2013)).

[0173] Unsaturated acids can be made using lactoyl-CoA, which is dehydrated to acryloyl-CoA by lactoyl-CoA dehydratase from Anaerotignum propionicum (Kandasamy et al. Appl Microbiol Biotechnol. 97(3):1191-1200(2013)). Acryloyl-CoA is converted to acrylic acid by CoA transferase from Halomonas sp. HTNK1 (Todd et al. Environ Microbiol. 12(2):327-343 (2010)). Additionally, acryloyl-CoA can be hydrolyzed to 3-hydroxypropionyl-CoA and further oxidized to 3-hydroxypropionic acid by acryloyl-CoA hydratase from Halomonas sp. HTNK1 (Todd et al. Environ Microbiol. 12(2):327-343 (2010)). An alternative pathway for acryloyl-CoA is converted to propionyl-CoA by propionyl-CoA synthase from Chloroflexus aurantiacus (Alber et al. J Biol Chem. 277(14):12137-12143 (2002)). Propionyl-CoA is further reduced to propionaldehyde by propanal dehydrogenase from Salmonella enterica (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)).

[0174] To implement the C1 elongation pathway for lactate production in vivo, we engineered vectors to independently control the expression of various HACS candidates and acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), with HACS under the control of an IPTG-inducible T7 promoter in pCDFDuet-1 and CaAbfT under the control of a cumate-inducible T5 promoter in pETDuet-1 (Figure 15B). As hosts for these vectors, we used engineered strains of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhFΔfdnGΔfdoG) oxidation and for glycolate utilization (ΔglcD).

[0175] In vivo product synthesis was carried out using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl) unless otherwise stated. Cells were initially grown in 96-deep-well plates (USA Scientific, Ocala, FL) containing 0.2 mL of the above medium, further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until a D600 of approximately 0.40 was achieved, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. Plates were incubated for a total of 24 hours after inoculation.

[0176] Cells from the precultures above were then centrifuged (4000 rpm, 22° C.), washed with the above minimal medium without carbon source, and resuspended in 1 mL of the above minimal medium with the indicated amount of carbon source. 5 mM acetaldehyde and 20 mM formate were added at 0 hours and incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific). After 1 hour incubation at 30° C., cells were pelleted by centrifugation and the medium was analyzed using HPLC ( FIG. 15A ).

[0177] The results show that BsmHACS is the best candidate, reaching up to 95 μM / hr per OD600 under the given experimental conditions (Figure 14B).

[0178] Example 16: Carboxylic Acid Platform Using Propionic Acid-Derived Propionaldehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using propionic acid (R = CH3) as a CA intermediate via the C1 elongation pathway (Figure 16A).

[0179] The activation of propionate to propionyl-CoA and its subsequent reduction to propionaldehyde has been thoroughly examined in the literature. Propionate can be activated to propionyl-CoA via propionyl-phosphate. First, propionate is phosphorylated to propionyl-phosphate by propionate kinase from E. coli (Hesslinger et al. Mol Microbiol. 27(2):477-492(1998)), and then converted to propionyl-CoA by phosphate acetyltransferase from E. coli (Hesslinger et al. Mol Microbiol. 27(2):477-492(1998)). Other pathways for activating propionate are propionyl-CoA synthetase from Escherichia coli (Guo et al. Prikl Biokhim Mikrobiol. 48(3):289-293(2012)) or succinyl-CoA transferase from Escherichia coli (Haller et al. Biochemistry. 39(16):4622-4629(2000)). Finally, propionyl-CoA is reduced to propionaldehyde by propanal dehydrogenase from Salmonella enterica (Niu et al. ACS Synth Biol. 4(4):378-382(2015)).

[0180] C1 elongation is initiated by the energy- and formyl-CoA-generating step to generate formyl-CoA. 2-Hydroxyacyl-CoA synthase (HACS) condenses propionaldehyde and formyl-CoA to generate 2-hydroxybutyryl-CoA. 2-Hydroxybutyryl-CoA is converted to 2-hydroxybutyrate by acyl-CoA thioesterase from Escherichia coli (Lee et al. Biochem Biophys Res Commun. 231(2):452-456(1997)).

[0181] 2-Hydroxybutyrate can be further oxidized to 2-oxobutyrate by lactate dehydrogenase from Desulfovibrio vulgaris (Ogata et al. J Biochem. 89(5):1423-1431 (1981)). 2-Oxobutyrate is reduced to 2-aminobutyrate by phenylalanine dehydrogenase from Thermoactinomyces intermedius (Kataoka et al. J Biochem. 116(4):931-936 (1994)).

[0182] 2-Hydroxybutyryl-CoA, as the product of condensation, can be reduced to 1,2-butanediol (1,2-BDO), 2-butanone, and 1-butanol. First, 2-hydroxybutyryl-CoA is reduced to 2-hydroxybutyraldehyde by aldehyde dehydrogenase from Salmonella enterica (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)), which can be further reduced to 1,2-BDO by aldehyde reductase from Escherichia coli (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)). Dehydration of 1,2-BDO by diol dehydratase gives 2-butanone. Furthermore, dehydration of 1,2-BDO by diol dehydratase from Klebsiella oxytoca (Toraya et al. J Biochem. 144(4):437-446 (2008)) yields butyraldehyde, which can feed into subsequent rounds of C1 elongation or be reduced to 1-butanol by aldehyde reductase from Escherichia coli (Pick et al. Appl Microbiol Biotechnol. 97(13):5815-5824(2013)).

[0183] Unsaturated acids can be made using 2-hydroxybutyryl-CoA, which is dehydrated to crotonyl-CoA by acyl-CoA dehydratase from Anaerotignum propionicum (Kandasamy et al. Appl Microbiol Biotechnol. 97(3):1191-1200(2013)). Crotonyl-CoA is converted to crotonic acid by crotonyl-CoA thioesterase from Emergencia timonensis (Buffa et al. Nat Microbiol. 7(1):73-86 (2022)). Additionally, crotonyl-CoA can be hydrolyzed to 3-hydroxybutyryl-CoA by acyl-CoA dehydratase from Clostridium acetobutylicum (Waterson et al. Methods Enzymol. 71 Pt C:421-430 (1981)) and further oxidized to 3-hydroxybutyrate by acyl-CoA thioesterase from Escherichia coli (Tseng et al. Appl Environ Microbiol. 75(10):3137-3145 (2009)). An alternative pathway for crotonyl-CoA is its conversion to butyryl-CoA by butyryl-CoA dehydrogenase from Clostridium kluyveri (Li et al. J Bacteriol. 190(3):843-850 (2008)). Butyryl-CoA is further reduced to butyraldehyde by aldehyde dehydrogenase from Clostridium beijerinckii (Yan et al. Appl Environ Microbiol. 56(9):2591-2599 (1990)).

[0184] To implement the C1 elongation pathway for 2-hydroxybutyrate production in vivo, we engineered vectors to independently control the expression of various HACS candidates and acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), where HACS was under the control of an IPTG-inducible T7 promoter in pCDFDuet-1 and CaAbfT was under the control of a cumate-inducible T5 promoter in pETDuet-1 (Figure 15B). As a host for these vectors, we used an engineered strain of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhF ΔfdnG ΔfdoG) oxidation and for glycolate utilization (ΔglcD).

[0185] In vivo product synthesis was performed using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl) unless otherwise stated. Cells were initially grown in 96-deep-well plates (USA Scientific, Ocala, FL) containing 0.2 mL of the above-mentioned medium further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an OD600 of approximately 0.4 was reached, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. After inoculation, plates were incubated for a total of 24 hours.

[0186] Cells from the precultures described above were then centrifuged (4000 rpm, 22° C.), washed with the above minimal medium without carbon source, and resuspended in 1 mL of the above minimal medium with the indicated amount of carbon source. 5 mM propionaldehyde and 20 mM formate were added at 0 hours and incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific). After 1 hour incubation at 30° C., cells were pelleted by centrifugation and the medium was analyzed using HPLC ( FIG. 15A ).

[0187] The results show that BsmHACS is the best candidate under the given experimental conditions, achieving up to 20 μM / hr per OD600 (Figure 16B).

[0188] Example 17: Carboxylic Acid Platform Using Butyric Acid-Derived Butyraldehyde as a Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using butyric acid (R = CH2CH3) as a CA intermediate via the C1 elongation pathway (Figure 17).

[0189] The activation of butyrate to butyryl-CoA and subsequent reduction to butyraldehyde has been well examined in the literature. Butyrate can be activated to butyryl-CoA via butyryl-phosphate. First, butyrate is phosphorylated to butyryl-phosphate by butyrate kinase from Clostridium acetobutylicum (Cary et al. J Bacteriol. 170(10):4613-4618 (1988)), and then converted to butyryl-CoA by phosphotransbutyrylase from Clostridium acetobutylicum (Wiesenborn et al. Appl Environ Microbiol. 55(2):317-322 (1989)). Other pathways for activating butyrate are the CoA synthetase from Pseudomonas putida (Rand et al. Nat Microbiol. 2(12):1624-1634 (2017)) or the CoA transferase from Clostridium kluyveri (Seedorf et al. Proc Natl Acad Sci US A. 105(6):2128-2133 (2008)). Finally, butyryl-CoA is reduced to butyraldehyde by aldehyde dehydrogenase from Clostridium acetobutylicum (Fontaine et al. J Bacteriol. 184(3):821-830 (2002)).

[0190] C1 elongation is initiated by the energy- and formyl-CoA-generating step to make formyl-CoA. 2-Hydroxyacyl-CoA synthase (HACS) condenses butyraldehyde and formyl-CoA to make 2-hydroxypentanoyl-CoA. 2-Hydroxypentanoyl-CoA is converted to 2-hydroxyvalerate by acyl-CoA thioesterase.

[0191] 2-Hydroxyvalerate can be further oxidized to 2-oxovalerate by 2-hydroxyacid dehydrogenase, which is reduced to 2-aminovalerate by aminodehydrogenase / transaminase.

[0192] 2-Hydroxypentanoyl-CoA as the product of condensation can be reduced to 1,2-pentanediol, 2-pentanone and valeraldehyde. First, 2-hydroxypentanoyl-CoA can be reduced to 2-hydroxypentanal via aldehyde dehydrogenase and further reduced to 1,2-pentanediol by aldehyde reductase. Dehydration of 1,2-pentanediol by diol dehydratase gives 2-pentanone. Further, dehydration of 1,2-pentanediol by diol dehydratase gives valeraldehyde. The resulting valeraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to 1-pentanol by aldehyde reductase.

[0193] Using 2-hydroxypentanoyl-CoA, unsaturated acids can be made. 2-Hydroxypentanoyl-CoA is dehydrated to 2-pentanoyl-CoA by acyl-CoA dehydratase. 2-Pentanoyl-CoA is converted to 2-pentenoic acid by CoA thioesterase. Furthermore, 2-pentanoyl-CoA can be hydrolyzed to 3-hydroxypentanoyl-CoA by acyl-CoA dehydratase from Pseudomonas putida (Rand et al. Nat Microbiol. 2(12):1624-1634 (2017)), which can be further oxidized to 3-hydroxyvaleric acid by acyl-CoA thioesterase. An alternative pathway for 2-pentanoyl-CoA is its conversion to pentanoyl-CoA by acyl-CoA dehydrogenase from Escherichia coli (Campbell et al. J Bacteriol. 184(13):3759-3764 (2002)). Pentanoyl-CoA is further reduced to valeraldehyde by aldehyde dehydrogenase.

[0194] Example 18: Carboxylic Acid Platform Using Glycolic Acid-Derived Glycolaldehyde as a Substrate for Condensation with Formyl-COA The purpose of this example is to illustrate the implementation of a carboxylic acid (CA) platform using glycolic acid (R=OH) as a CA intermediate via the C1 elongation pathway (FIG. 18A).

[0195] Glycolic acid can be activated to glycolyl-CoA via glycolyl-phosphate. First, glycolic acid is phosphorylated to glycolyl-phosphate by kinase, then converted to glycolyl-CoA by phosphotransacylase. Another pathway for activating glycolic acid is CoA synthetase or CoA transferase. Finally, glycolyl-CoA is reduced to glycolaldehyde by aldehyde dehydrogenase.

[0196] C1 elongation is initiated by the energy- and formyl-CoA-generating step to make formyl-CoA. 2-Hydroxyacyl-CoA synthase (HACS) condenses glycolaldehyde and formyl-CoA to make glyceryl-CoA. Glyceryl-CoA is converted to glyceric acid by acyl-CoA thioesterase.

[0197] Glyceric acid can be further oxidized to hydroxypyruvic acid by 2-hydroxyacid dehydrogenase. Hydroxypyruvic acid is reduced to serine by aminodehydrogenase / transaminase. Dehydration of serine gives 2-amino-3-hydroxypropanal, which can be further reduced to serinol or aminated to 2,3-diamino-1-propanol.

[0198] Glyceryl-CoA as the product of condensation can be reduced to glycerol, hydroxyacetone and 1,3-propanediol. First, glyceryl-CoA is reduced by aldehyde dehydrogenase to 2,3-dihydroxypropionaldehyde, which can be further reduced to glycerol by aldehyde reductase. Dehydration of glycerol by diol dehydratase produces hydroxyacetone. Further, dehydration of glycerol by diol dehydratase produces 3-hydroxypropionaldehyde. The resulting 3-hydroxypropionaldehyde can be fed to the subsequent iteration of C1 elongation or reduced to 1,3-propanediol by aldehyde reductase.

[0199] Glyceryl-CoA can be used to make unsaturated acids. Glyceryl-CoA is dehydrated to 3-hydroxyacryloyl-CoA by acyl-CoA dehydratase. 3-hydroxyacryloyl-CoA is converted to 3-hydroxyacrylic acid by CoA thioesterase. In addition, 3-hydroxyacryloyl-CoA can be hydrolyzed to 3,3-dihydroxypropionyl-CoA by acyl-CoA dehydratase and further oxidized to 3,3-dihydroxypropionic acid by acyl-CoA thioesterase. Another pathway for 3-hydroxyacryloyl-CoA is to be converted to 3-hydroxypropionyl-CoA by acyl-CoA dehydrogenase. 3-hydroxypropionyl-CoA is further reduced to 3-hydroxypropionaldehyde by aldehyde dehydrogenase.

[0200] To implement the C1 elongation pathway for glycerate production in vivo, we engineered vectors to independently control the expression of various HACS candidates and acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), where HACS was under the control of an IPTG-inducible T7 promoter in pCDFDuet-1 and CaAbfT was under the control of a cumate-inducible T5 promoter in pETDuet-1 (Figure 15B). As a host for these vectors, we used an engineered strain of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhF ΔfdnG ΔfdoG) oxidation and for glycolate utilization (ΔglcD).

[0201] Unless otherwise stated, in vivo product synthesis was performed using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl). Cells were initially grown in 96-deep-well plates (USA Scientific, Ocala, FL) containing 0.2 mL of the above medium further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an OD600 of approximately 0.4 was reached, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. After inoculation, plates were incubated for a total of 24 hours.

[0202] Cells from the precultures above were then centrifuged (4000 rpm, 22° C.), washed with the above minimal medium without carbon source, and resuspended in 1 mL of the above minimal medium with the indicated amount of carbon source. 5 mM glycolaldehyde and 20 mM formate were added at 0 hours and incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific). After 1 hour incubation at 30° C., cells were pelleted by centrifugation and the medium was analyzed using HPLC (FIG. 15A).

[0203] The results show that JGI15 is the best candidate under the given experimental conditions, achieving up to 39 μM / hr per OD600 (FIG. 18B).

[0204] Example 19: Carboxylic Acid Platform Using Lactic Acid-Derived Lactaldehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to illustrate the implementation of a carboxylic acid (CA) platform using lactate (R = CH3OH) as a CA intermediate via the C1 elongation pathway (Figure 19).

[0205] Lactate can be activated to lactoyl-CoA via lactoyl-phosphate. First, lactate is phosphorylated to lactoyl-phosphate by kinases and then converted to lactoyl-CoA by phosphotransacylase. Other pathways for activating lactate are CoA synthetase or CoA transferase from Megasphaera elsdenii (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)). Finally, lactoyl-CoA is reduced to lactaldehyde by aldehyde dehydrogenase from Salmonella enterica (Niu et al. ACS Synth Biol. 4(4):378-382 (2015)).

[0206] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-Hydroxyacyl-CoA synthase (HACS) condenses lactaldehyde and formyl-CoA to produce 2,3-dihydroxybutyryl-CoA. 2,3-Dihydroxybutyryl-CoA is converted to 2,3-dihydroxybutyric acid by acyl-CoA thioesterase.

[0207] 2,3-Dihydroxybutyrate can be further oxidized to 2-oxo-3-hydroxybutyrate by 2-hydroxyacid dehydrogenase. 2-oxo-3-hydroxybutyrate is reduced to 2-amino-3-hydroxybutyrate by aminodehydrogenase / transaminase. Dehydration of 2-amino-3-hydroxybutyrate produces 2-amino-3-hydroxybutanal, which can be further reduced to 2-amino-1,3-butanediol or aminated to 3,4-diamino-2-butanol.

[0208] 2,3-dihydroxybutyryl-CoA as the product of condensation can be reduced to 1,2,3-butanetriol, acetion and 1,3-butanediol. First, 2,3-dihydroxybutyryl-CoA is reduced to 2,3-dihydroxybutyraldehyde by aldehyde dehydrogenase, which can be further reduced to 1,2,3-butanetriol by aldehyde reductase. Dehydration of 1,2,3-butanetriol by diol dehydratase produces acetion. Further, dehydration of 1,2,3-butanetriol by diol dehydratase produces 3-hydroxybutyraldehyde. The resulting 3-hydroxybutyraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to 1,3-butanediol by aldehyde reductase.

[0209] 2,3-Dihydroxybutyryl-CoA can be used to make unsaturated acids. 2,3-Dihydroxybutyryl-CoA is dehydrated to 3-hydroxycrotonyl-CoA by acyl-CoA dehydratase. 3-Hydroxycrotonyl-CoA is converted to 3-hydroxycrotonic acid by CoA thioesterase. In addition, 3-hydroxycrotonyl-CoA can be hydrolyzed to 3,3-dihydroxybutyryl-CoA by acyl-CoA dehydratase and further oxidized to 3,3-dihydroxybutyric acid by acyl-CoA thioesterase. Another pathway for 3-hydroxycrotonyl-CoA is to be converted to 3-hydroxybutyryl-CoA by acyl-CoA dehydrogenase. 3-Hydroxybutyryl-CoA is further reduced to 3-hydroxybutyraldehyde by aldehyde dehydrogenase.

[0210] Example 20: Carboxylic Acid Platform Using Glyceric Acid-Derived Glyceraldehyde as a Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using glyceric acid (R = OHCH2OH) as a CA intermediate via the C1 elongation pathway (Figure 20).

[0211] Glyceric acid can be activated to glyceryl-CoA via glyceryl-phosphate. First, glyceric acid is phosphorylated to glyceryl-phosphate by kinase, and then converted to glyceryl-CoA by phosphotransacylase. Another pathway for activating glyceric acid is CoA synthetase or CoA transferase. Finally, glyceryl-CoA is reduced to glyceraldehyde by aldehyde dehydrogenase.

[0212] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-Hydroxyacyl-CoA synthase (HACS) condenses glyceraldehyde and formyl-CoA to produce 2,3,4-trihydroxybutyryl-CoA. 2,3,4-Trihydroxybutyryl-CoA is converted to 2,3,4-trihydroxybutyric acid by acyl-CoA thioesterase.

[0213] 2,3,4-trihydroxybutyrate can be further oxidized to 2-oxo-3,4-dihydroxybutyrate by 2-hydroxyacid dehydrogenase. 2-oxo-3,4-dihydroxybutyrate is reduced to 2-amino-3,4-dihydroxybutyrate by aminodehydrogenase / transaminase. Dehydration of 2-amino-3,4-dihydroxybutyrate produces 2-amino-3,4-dihydroxybutanal, which can be further reduced to 3-amino-1,2,4-butanetriol or aminated to 3,4-diamino-1,2-butanediol.

[0214] 2,3,4-Trihydroxybutyryl-CoA as the product of condensation can be reduced to threitol, 3,4-dihydroxy-2-butanone and 1,2,4-butanetriol. First, 2,3,4-trihydroxybutyryl-CoA is reduced to 2,3,4-trihydroxybutyraldehyde by aldehyde dehydrogenase, which can be further reduced to threitol by aldehyde reductase. Dehydration of threitol by diol dehydratase produces 3,4-dihydroxy-2-butanone. Further, dehydration of threitol by diol dehydratase produces 3,4-dihydroxybutyraldehyde. The resulting 3,4-dihydroxybutyraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to 1,2,4-butanetriol by aldehyde reductase.

[0215] 2,3,4-trihydroxybutyryl-CoA can be used to make unsaturated acids. 2,3,4-trihydroxybutyryl-CoA is dehydrated to 3,4-dihydroxycrotonyl-CoA by acyl-CoA dehydratase. 3,4-dihydroxycrotonyl-CoA is converted to 3,4-dihydroxycrotonic acid by CoA thioesterase. In addition, 3,4-dihydroxycrotonyl-CoA can be hydrolyzed to 3,3,4-trihydroxybutyryl-CoA by acyl-CoA dehydratase and further oxidized to 3,3,4-trihydroxybutyric acid by acyl-CoA thioesterase. Another pathway for 3,4-dihydroxycrotonyl-CoA is converted to 3,4-dihydroxybutyryl-CoA by acyl-CoA dehydrogenase. 3,4-Dihydroxybutyryl-CoA is further reduced to 3,4-dihydroxybutyraldehyde by aldehyde dehydrogenase.

[0216] Example 21: Carboxylic Acid Platform Using 3-Hydroxypropionic Acid-Derived 3-Hydroxypropionaldehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using 3-hydroxypropionic acid (R = CH2OH) as a CA intermediate via the C1 elongation pathway (Figure 21).

[0217] 3-hydroxypropionate can be activated to 3-hydroxypropionyl-CoA via 3-hydroxypropionyl-phosphate. First, 3-hydroxypropionate is phosphorylated to 3-hydroxypropionyl-phosphate by kinase, then converted to 3-hydroxypropionyl-CoA by phosphotransacylase. Another pathway for activating 3-hydroxypropionate is CoA synthetase or CoA transferase. Finally, 3-hydroxypropionyl-CoA is reduced to 3-hydroxypropionaldehyde by aldehyde dehydrogenase.

[0218] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-Hydroxyacyl-CoA synthase (HACS) condenses 3-hydroxypropionaldehyde and formyl-CoA to produce 2,4-dihydroxybutyryl-CoA. 2,4-Dihydroxybutyryl-CoA is converted to 2,4-dihydroxybutyrate by acyl-CoA thioesterase.

[0219] 2,4-Dihydroxybutyrate can be further oxidized to 2-oxo-4-hydroxybutyrate by 2-hydroxyacid dehydrogenase. 2-oxo-4-hydroxybutyrate is reduced to 2-amino-4-hydroxybutyrate by aminodehydrogenase / transaminase. Dehydration of 2-amino-4-hydroxybutyrate produces 2-amino-4-hydroxybutanal, which can be further reduced to 2-amino-1,4-butanediol or aminated to 3,4-diamino-1-butanol.

[0220] 2,4-dihydroxybutyryl-CoA as the product of condensation can be reduced to 1,2,4-butanetriol, 4-hydroxy-2-butanone and 1,4-butanediol. First, 2,4-dihydroxybutyryl-CoA is reduced to 2,4-dihydroxybutyraldehyde by aldehyde dehydrogenase, which can be further reduced to 1,2,4-butanetriol by aldehyde reductase. Dehydration of 1,2,4-butanetriol by diol dehydratase produces 4-hydroxy-2-butanone. Furthermore, dehydration of 1,2,4-butanetriol by diol dehydratase produces 4-hydroxybutyraldehyde. The resulting 4-hydroxybutyraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to 1,4-butanediol by aldehyde reductase.

[0221] 2,4-Dihydroxybutyryl-CoA can be used to make unsaturated acids. 2,4-Dihydroxybutyryl-CoA is dehydrated to 4-hydroxycrotonyl-CoA by acyl-CoA dehydratase. 4-Hydroxycrotonyl-CoA is converted to 4-hydroxycrotonic acid by CoA thioesterase. Furthermore, 4-hydroxycrotonyl-CoA can be hydrolyzed to 3,4-dihydroxybutyryl-CoA by acyl-CoA dehydratase and further oxidized to 3,4-dihydroxybutyric acid by acyl-CoA thioesterase. Another pathway for 4-hydroxycrotonyl-CoA is to be converted to 4-hydroxybutyryl-CoA by acyl-CoA dehydrogenase. 4-Hydroxybutyryl-CoA is further reduced to 4-hydroxybutyraldehyde by aldehyde dehydrogenase.

[0222] Example 22: Carboxylic Acid Platform Using Oxalic Acid-Derived Oxalic Semialdehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to illustrate the implementation of a carboxylic acid (CA) platform using oxalic acid (R=OOH) as a CA intermediate via the C1 elongation pathway (Figure 22A).

[0223] Oxalate can be activated to oxalyl-CoA via oxalyl-phosphate. First, oxalate is phosphorylated to oxalyl-phosphate by kinase, then converted to oxalyl-CoA by phosphotransacylase. Other pathways for activating oxalate are oxalate-CoA ligase from Arabidopsis (Foster et al. Plant Cell. 24(3):1217-1229 (2012)) or CoA transferase from Escherichia coli (Mullins et al. PLoS One. 8(7):e67901 (2013)). Finally, oxalyl-CoA is reduced to glyoxylate by aldehyde dehydrogenase.

[0224] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-hydroxyacyl-CoA synthase (HACS) condenses glyoxylate and formyl-CoA to produce tartronyl-CoA. Tartronyl-CoA is converted to tartronic acid by acyl-CoA thioesterase.

[0225] Tartronic acid can be further oxidized to mesoxalic acid by 2-hydroxyacid dehydrogenase. Mesoxalic acid is reduced to aspartic acid by aminodehydrogenase / transaminase. Dehydration of aminomalonic acid gives 2-amino-3-oxopropionic acid, which can be further reduced to serine or aminated to 3-aminoalanine.

[0226] Tartronyl-CoA as the product of condensation can be reduced to glyceric acid, pyruvate and 3-hydroxypropionic acid. First, tartronyl-CoA is reduced to 2-hydroxy-3-oxopropionic acid by aldehyde dehydrogenase, which can be further reduced to glyceric acid by aldehyde reductase. Dehydration of glyceric acid by diol dehydratase produces pyruvate. Further, dehydration of glyceric acid by diol dehydratase produces 3-oxopropionic acid. The resulting 3-oxopropionic acid can be fed to the subsequent iteration of C1 elongation or reduced to 3-hydroxypropionic acid by aldehyde reductase.

[0227] To implement the C1 elongation pathway for tartronic acid production in vivo, we engineered vectors to independently control the expression of various HACS candidates and acyl-CoA transferase from Clostridium aminobutyricum (CaAbfT), where HACS was under the control of an IPTG-inducible T7 promoter in pCDFDuet-1, and CaAbfT was under the control of a cumate-inducible T5 promoter in pETDuet-1 (Figure 15B). As hosts for these vectors, we used engineered strains of E. coli based on MG1655(DE3) with knockouts for formaldehyde (ΔfrmA) and formate (ΔfdhFΔfdnGΔfdoG) oxidation, as well as for glycolate utilization (ΔglcD).

[0228] Unless otherwise stated, in vivo product synthesis was performed using M9 minimal medium (6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2 and 15 μM thiamine-HCl). Cells were initially grown in 96-deep-well plates (USA Scientific, Ocala, FL) containing 0.2 mL of the above medium further supplemented with 20 g / L glycerol, 10 g / L tryptone and 5 g / L yeast extract. A single colony of the desired strain was cultured overnight (14-16 h) in LB medium with the appropriate antibiotic and used as inoculum (1%). Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. Cultures were then incubated at 30°C, 1000 rpm in a Digital Microplate Shaker (Fisher Scientific) until an OD600 of approximately 0.4 was reached, at which point the appropriate amount of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) was added. After inoculation, plates were incubated for a total of 24 hours.

[0229] Cells from the precultures above were then centrifuged (4000 rpm, 22° C.), washed with the above minimal medium without carbon source, and resuspended in 1 mL of the above minimal medium with the indicated amount of carbon source. 5 mM glyoxylic acid and 20 mM formate were added at 0 hours and incubated at 30° C., 1000 rpm in a Digital Microplate Shaker (Fisher Scientific). After 1 hour incubation at 30° C., cells were pelleted by centrifugation and the medium was analyzed using HPLC ( FIG. 15A ).

[0230] The results show that RuHACL is the best candidate under the given experimental conditions achieving up to 13 μM / hr per OD600 (Figure 22B).

[0231] Example 23: Carboxylic Acid Platform Using Malonic Acid-Derived Malonic Semialdehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using malonic acid (R=COOH) as a CA intermediate via the C1 elongation pathway (FIG. 23).

[0232] Malonate can be activated to malonyl-CoA via malonyl-phosphate. First, malonate is phosphorylated to malonyl-phosphate by kinase, then converted to malonyl-CoA by phosphotransacylase. Another pathway for activating malonate is CoA synthetase or CoA transferase. Finally, malonyl-CoA is reduced to malonate semialdehyde by aldehyde dehydrogenase.

[0233] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-hydroxyacyl-CoA synthase (HACS) condenses malonate semialdehyde and formyl-CoA to yield malyl-CoA. Malyl-CoA is converted to malate by acyl-CoA thioesterase.

[0234] Malate can be further oxidized to 2-oxosuccinate (oxalacetate) by 2-hydroxyacid dehydrogenase. 2-oxosuccinate is reduced to aspartate by aminodehydrogenase / transaminase. Dehydration of aspartate produces 3-amino-4-oxobutyrate, which can be further reduced to 3-amino-4-hydroxybutyrate or aminated to 3,4-diaminobutyrate.

[0235] Malyl-CoA as the product of condensation can be reduced to 3,4-dihydroxybutyrate, acetoacetate and 4-hydroxybutyrate. First, malyl-CoA is reduced to 3-hydroxy-4-oxobutyrate by aldehyde dehydrogenase, which can be further reduced to 3,4-dihydroxybutyrate by aldehyde reductase. Dehydration of 3,4-dihydroxybutyrate by diol dehydratase produces acetoacetate. Further, dehydration of 3,4-dihydroxybutyrate by diol dehydratase produces 4-oxobutyrate. The resulting 4-oxobutyrate can be fed to the subsequent iteration of C1 elongation or reduced to 4-hydroxybutyrate by aldehyde reductase.

[0236] Malyl-CoA can be used to make unsaturated acids. Malyl-CoA is dehydrated to fumaryl-CoA by acyl-CoA dehydratase. Fumaryl-CoA is converted to fumaric acid by CoA thioesterase. Furthermore, fumaryl-CoA can be hydrolyzed to 2-hydroxysuccinyl-CoA by acyl-CoA dehydratase and further oxidized to 2-hydroxysuccinic acid by acyl-CoA thioesterase. Another route for fumaryl-CoA is to be converted to succinyl-CoA by acyl-CoA dehydrogenase. Succinyl-CoA is further reduced to 4-oxobutyric acid by aldehyde dehydrogenase.

[0237] Example 24: Carboxylic Acid Platform Using Succinic Acid-Derived Succinic Semialdehyde as a Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using succinic acid (R = CH2COOH) as a CA intermediate via the C1 elongation pathway (Figure 24).

[0238] The activation of succinate to succinyl-CoA and then reduction to succinic semialdehyde has been well examined in the literature. Succinate can be activated to succinyl-CoA via succinyl-phosphate. First, succinate is phosphorylated to succinyl-phosphate by a kinase and then converted to succinyl-CoA by a phosphotransacylase. Other pathways for activating succinate are CoA synthetase from Escherichia coli (Yim et al. Nat Chem Biol. 7(7):445-452 (2011)) or CoA transferase. Finally, succinyl-CoA is reduced to succinic semialdehyde by aldehyde dehydrogenase from Clostridium kluyveri (Schwander et al. Science. 354(6314):900-904 (2016)).

[0239] C1 elongation is initiated by the energy- and formyl-CoA-generating step to generate formyl-CoA. 2-Hydroxyacyl-CoA synthase (HACS) condenses succinic semialdehyde and formyl-CoA to produce 2-hydroxyglutaryl-CoA. 2-Hydroxyglutaryl-CoA is converted to 2-hydroxyglutaric acid by acyl-CoA thioesterase.

[0240] 2-Hydroxyglutarate can be further oxidized to 2-oxoglutarate by 2-hydroxyacid dehydrogenase. 2-oxoglutarate is reduced to glutamate by aminodehydrogenase / transaminase. Dehydration of glutamate produces 4-amino-5-oxopentanoic acid, which can be further reduced to 4-amino-5-hydroxypentanoic acid or aminated to 4,5-diaminopentanoic acid.

[0241] 2-Hydroxyglutaryl-CoA as the product of condensation can be reduced to 4,5-dihydroxypentanoic acid, levulinic acid and 5-hydroxypentanoic acid. First, 2-hydroxyglutaryl-CoA is reduced to 4-hydroxy-5-oxopentanoic acid by aldehyde dehydrogenase, which can be further reduced to 4,5-dihydroxypentanoic acid by aldehyde reductase. Dehydration of 4,5-dihydroxypentanoic acid by diol dehydratase gives levulinic acid. Furthermore, dehydration of 4,5-dihydroxypentanoic acid by diol dehydratase gives 5-oxopentanoic acid. The resulting 5-oxopentanoic acid can be fed to the subsequent iteration of C1 elongation or reduced to 5-hydroxypentanoic acid by aldehyde reductase.

[0242] 2-Hydroxyglutaryl-CoA can be used to make unsaturated acids. 2-Hydroxyglutaryl-CoA is dehydrated to glutaconyl-CoA by acyl-CoA dehydratase. Glutaconyl-CoA is converted to glutaconic acid by CoA thioesterase. Furthermore, glutaconyl-CoA can be hydrolyzed to 3-hydroxyglutaryl-CoA by acyl-CoA dehydratase and further oxidized to 3-hydroxyglutaric acid by acyl-CoA thioesterase. Another route for glutaconyl-CoA is to be converted to glutaryl-CoA by acyl-CoA dehydrogenase. Glutaryl-CoA is further reduced to 5-oxopentanoic acid by aldehyde dehydrogenase.

[0243] Example 25: Carboxylic Acid Platform Using Isobutyric Acid-Derived Isobutyraldehyde as Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using isobutyric acid (R = CH3CH3) as a CA intermediate via the C1 elongation pathway (Figure 25).

[0244] Isobutyric acid can be activated to isobutyryl-CoA via isobutyryl-phosphate. First, isobutyric acid is phosphorylated to isobutyryl-phosphate by kinase, then converted to isobutyryl-CoA by phosphotransacylase. Another pathway for activating isobutyric acid is CoA synthetase or CoA transferase. Finally, isobutyryl-CoA is reduced to isobutyraldehyde by aldehyde dehydrogenase.

[0245] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-Hydroxyacyl-CoA synthase (HACS) condenses isobutyraldehyde and formyl-CoA to yield 2-hydroxy-3-methylbutyryl-CoA. 2-Hydroxy-3-methylbutyryl-CoA is converted to 2-hydroxyisovaleric acid by acyl-CoA thioesterase.

[0246] 2-Hydroxyisovaleric acid can be further oxidized to 2-oxoisovaleric acid by 2-hydroxyacid dehydrogenase. 2-Oxoisovaleric acid is reduced to valine by aminodehydrogenase / transaminase. Dehydration of valine gives 2-amino-3-methylbutanal, which can be further reduced to valinol or aminated to 3-methylbutane-1,2-diamine.

[0247] 2-Hydroxy-3-methylbutyryl-CoA as the product of condensation can be reduced to 3-methylbutane-1,2-diol, 3-methyl-2-butanone and isoamyl alcohol. First, 2-hydroxy-3-methylbutyryl-CoA is reduced to 2-hydroxy-3-methylbutanal by aldehyde dehydrogenase, which can be further reduced to 3-methylbutane-1,2-diol by aldehyde reductase. Dehydration of 3-methylbutane-1,2-diol by diol dehydratase gives 3-methyl-2-butanone. Furthermore, dehydration of 3-methylbutane-1,2-diol by diol dehydratase gives isovaleraldehyde. The resulting isovaleraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to isoamyl alcohol by aldehyde reductase.

[0248] 2-Hydroxy-3-methylbutyryl-CoA can be used to make unsaturated acids. 2-Hydroxy-3-methylbutyryl-CoA is dehydrated to 3-methylcrotonyl-CoA by acyl-CoA dehydratase. 3-Methylcrotonyl-CoA is converted to 3-methylcrotonyl acid by CoA thioesterase. Furthermore, 3-methylcrotonyl-CoA can be hydrolyzed to 3-hydroxy-3-methylbutyryl-CoA by acyl-CoA dehydratase and further oxidized to 3-hydroxyisovaleric acid by acyl-CoA thioesterase. Another pathway for 3-methylcrotonyl-CoA is to be converted to 3-methylbutyryl-CoA by acyl-CoA dehydrogenase. 3-Methylbutyryl-CoA is further reduced to isovaleraldehyde by aldehyde dehydrogenase.

[0249] Example 26: Carboxylic Acid Platform Using Isovaleric Acid-Derived Isovaleraldehyde as a Substrate for Condensation with Formyl-COA The purpose of this example is to demonstrate the implementation of a carboxylic acid (CA) platform using isovaleric acid (R = CHCH3CH3) as a CA intermediate via the C1 elongation pathway (Figure 26).

[0250] Isovaleric acid can be activated to isopentanoyl-CoA via isopentanoyl-phosphate. First, isovaleric acid is phosphorylated to isopentanoyl-phosphate by kinase, then converted to isopentanoyl-CoA by phosphotransacylase. Another pathway for activating isovaleric acid is CoA synthetase or CoA transferase. Finally, isopentanoyl-CoA is reduced to isovaleraldehyde by aldehyde dehydrogenase.

[0251] C1 elongation is initiated by the generation of formyl-CoA through an energy- and formyl-CoA-generating step. 2-Hydroxyacyl-CoA synthase (HACS) condenses isovaleraldehyde and formyl-CoA to yield 2-hydroxy-4-methylpentanoyl-CoA. 2-Hydroxy-4-methylpentanoyl-CoA is converted to leucic acid by acyl-CoA thioesterase.

[0252] Leucine acid can be further oxidized to ketroucine by 2-hydroxyacid dehydrogenase. Ketroucine is reduced to leucine by aminodehydrogenase / transaminase. Dehydration of leucine produces 2-amino-4-methylpentanal, which can be further reduced to leucinol or aminated to 4-methylpentane-1,2-diamine.

[0253] 2-Hydroxy-4-methylpentanoyl-CoA as the product of condensation can be reduced to 4-methylpentane-1,2-diol, 4-methyl-2-pentanone and isohexanol. First, 2-hydroxy-4-methylpentanoyl-CoA is reduced to 2-hydroxy-4-methylpentanal by aldehyde dehydrogenase, which can be further reduced to 4-methylpentane-1,2-diol by aldehyde reductase. Dehydration of 4-methylpentane-1,2-diol by diol dehydratase gives 4-methyl-2-pentanone. Furthermore, dehydration of 4-methylpentane-1,2-diol by diol dehydratase gives 4-methylvaleraldehyde. The resulting 4-methylvaleraldehyde can be fed to the subsequent iteration of C1 elongation or reduced to isohexanol by aldehyde reductase.

[0254] 2-Hydroxy-4-methylpentanoyl-CoA can be used to make unsaturated acids. 2-Hydroxy-4-methylpentanoyl-CoA is dehydrated to 4-methyl-2-pentanoyl-CoA by acyl-CoA dehydratase. 4-Methyl-2-pentanoyl-CoA is converted to 4-methyl-2-pentanoic acid by CoA thioesterase. In addition, 4-methyl-2-pentanoyl-CoA can be hydrolyzed to 3-hydroxy-4-methylpentanoyl-CoA by acyl-CoA dehydratase and further oxidized to 3-hydroxy-4-methylpentanoic acid by acyl-CoA thioesterase. Another pathway for 4-methyl-2-pentanoyl-CoA is to be converted to 4-methylpentanoyl-CoA by acyl-CoA dehydrogenase. 4-Methylpentanoyl-CoA is further reduced to 4-methylvaleraldehyde by aldehyde dehydrogenase.

[0255] Example 27: Carboxylic Acid Platform Using 2-Hydroxy Acid-Derived Methyl Ketones as Substrates for Condensation with Formyl-COA This example shows the implementation of condensation of methyl ketone with formyl-CoA using purified enzyme. The production of formyl-CoA catalyzed by CoA transferase and the condensation catalyzed by HACS are the same as the above example. Methyl ketone can be produced from the above 2-hydroxy acid or through fatty acid synthesis and β-oxidation pathways shown in the literature (Appl Environ Microbiol 78:70-80, 2012; Metab Eng 62:84-94, 2020).

[0256] The enzymes acyl-CoA transferase and HACS were overexpressed and purified as described above. In vitro purified enzyme reactions for the condensation of methyl ketones and formyl-CoA consisted of 100 mM KPi pH 6.9, 10 mM MgCl2, 0.15 mM TPP, 2 mM acetyl-CoA, 1 μM BsmHACL, 2 μM CaAbfT, 20 mM formate and 50 mM of the tested methyl ketone. Reactions were incubated at 30°C for 24 hours unless otherwise specified.

[0257] For this analysis, samples containing acyl-CoA were first treated with 1 / 20 of the reaction volume of 10M NaOH solution, which was added to terminate the reaction. After 30 min of hydrolysis, 1 / 20 of the reaction volume of 10N H2SO4 was added to improve the efficiency of the acid extraction. The resulting samples were extracted into 4 mL ethyl acetate by vortexing vigorously for 90 s. The organic phase was separated and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 μL pyridine and 50 μL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and incubated at 60°C for 15 min. Compound identification and analysis were performed by GC-MS using an Agilent 7890B Series Custom gas chromatography system equipped with a 5977B Inert Plus Mass Selective Detector Turbo EI Bundle (for identification) and an Agilent HP-5-ms capillary column (0.25 mm internal diameter, 0.25 μm film thickness, 30 m length). Samples were analyzed by GC (1 μL injection with a separation ratio of 20:1) using helium as the carrier gas at a flow rate of 1.5 mL / min and the following temperature profile: start, 90°C for 3 min; ramp to 170°C at 15°C / min; ramp to 300°C at 20°C / min and hold for 8 min. Injector and detector temperatures were 250°C and 350°C, respectively.

[0258] Methyl ketones that are good carbonyl-containing substrates for the acyloin condensation reaction with formyl-CoA include, but are not limited to, acetone, methyl ethyl ketone (e.g., C n -ketones, n>3, butanone, pentanone and heptanone), hydroxylated ketones (hydroxyacetone) and other functional ketones (acetylacetone, branched chain ketones, methylglyoxal), etc. BsmHACS was able to catalyze the condensation of all tested ketones with formyl-CoA, as shown in Figure 36B.

[0259] Example 28: Carboxylic Acid Platform Using Lactic Acid-Derived Acetone as a Substrate for Condensation with Formyl-COA This example demonstrates the condensation of methyl ketones with formyl-CoA (made from formate) using cells growing in vivo, with acetone used as a representative methyl ketone. As mentioned above, this process requires only two enzymes, formyl-CoA generating enzyme (FAE) and condensing enzyme (HACS). BsmHACS was cloned under the control of an IPTG-inducible T7 promoter in pCDFduet-1, and FAE was cloned under the control of a cumate-inducible T5 promoter in pETDuet-1. Meanwhile, a HACL variant recently identified in Actinomycetospora chiangmaiensis DSM 45062 (Frontiers in microbiology 11:691, 2020) was also named AcHACL.

[0260] Condensation of acetone with formate-derived formyl-CoA in actively growing cells was achieved using a medium containing 6.78 g / L Na2HPO4, 3 g / L KH2PO4, 1 g / L NH4Cl, 0.5 g / L NaCl, 2 mM MgSO4, 100 μM CaCl2, 15 μM thiamine-HCl, 10 g / L tryptone, and 5 g / L yeast extract, in addition to the Neidhardt 68The assay was performed using M9-LB medium supplemented with a trace element solution of 100 μg / mL. A single colony of the desired strain was grown overnight (14–16 h) in LB medium with the appropriate antibiotic and used as an inoculum (1%) into a 50 mL centrifuge tube containing 5 mL of M9-LB medium. Antibiotics (100 μg / mL carbenicillin, 100 μg / mL spectinomycin) were included when appropriate. The culture was then incubated at 30 °C, 250 rpm in a Lab Companion SI-600 rotary shaker (Jeio Tech, Seoul, South Korea) until it reached an OD550 of approximately 0.4, at which point appropriate amounts of inducer(s) (isopropyl β-D-1-thiogalactopyranoside and cumate) and substrate (acetone and formate) were added. The tube was clamped and incubated for a total of 48 h after inoculation. The cells were pelleted by centrifugation and the medium was analyzed by HPLC or GC-MS as described above.

[0261] Growing cell experiments show that HACS catalyzed the condensation of acetone and formyl-CoA, but BsmHACS performed better, with 2HIB up to 2.8 mM (291 mg / L) produced after 2 days of fermentation (Figure 37B). [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16] [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6] [Table 18-1] [Table 18-2]

[0262] The experimental method was similar to that described in the above examples.

[0263] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The documents cited herein and the materials for which they are cited are specifically incorporated by reference.

[0264] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

**Claim 1** A genetically modified microorganism that converts a carboxylic acid into a product, comprising: a. a first set of nucleic acids encoding an enzyme for activating the carboxylic acid into a corresponding acyl-CoA intermediate; b. a second set of nucleic acids encoding an enzyme for converting the acyl-CoA intermediate into a product; and c. a third set of nucleic acids encoding an enzyme for generating reducing equivalents and ATP from an externally supplied energy source A microorganism comprising the same. **Claim 2** The microorganism according to claim 1, wherein the first set of metabolic enzymes comprises acyl-CoA synthetase, or acyl-CoA transferase, or carboxylate kinase and phosphotransacylase, or carboxylic acid reductase and acyl-CoA reductase, or aldehyde dehydrogenase and acyl-CoA reductase that converts the carboxylic acid into a corresponding acyl-CoA intermediate. **Claim 3** The microorganism according to claim 1, wherein the second set of metabolic enzymes comprises aldehyde-forming acyl-CoA reductase that converts the acyl-CoA into an aldehyde; or The microorganism according to claim 1, wherein the second set of metabolic enzymes comprises aldehyde-forming acyl-CoA reductase and alcohol dehydrogenase, or alcohol-forming acyl-CoA reductase that converts the acyl-CoA into an alcohol. **Claim 4** The microorganism according to claim 3, wherein the carboxylic acid is 2-hydroxy acid and the aldehyde is 2-hydroxy aldehyde; or The microorganism according to claim 3, wherein the carboxylic acid is 2-hydroxy acid and the alcohol is 1,2-diol. **Claim 5** The microorganism according to claim 4, wherein the second set of metabolic enzymes further comprises diol dehydratase that converts the 1,2-diol into a ketone; or The microorganism according to claim 4, wherein the second set of metabolic enzymes further comprises diol dehydratase that converts the 1,2-diol into an aldehyde. **Claim 6** The microorganism according to claim 5, wherein the second set of metabolic enzymes further comprises alcohol dehydrogenase that converts the aldehyde into a primary alcohol; or The microorganism according to claim 5, wherein the second set of metabolic enzymes further comprises acylating aldehyde dehydrogenase and thioesterase that converts the aldehyde into acyl-CoA, or acyl-CoA transferase, or phosphotransacylase and carboxylate kinase that converts the acyl-CoA into a carboxylic acid. **Claim 7** (i) The second set of metabolic enzymes further comprises an enzyme that converts a one-carbon substrate to formyl-CoA and 2-hydroxyacyl-CoA lyase or oxalyl-CoA decarboxylase that condenses the aldehyde and the formyl-CoA to form 2-hydroxyacyl-CoA that is one carbon longer than the aldehyde; or (ii) the carboxylic acid is a 2-hydroxy acid and the aldehyde is a 2-hydroxy aldehyde; or the carboxylic acid is a 2-hydroxy acid and the alcohol is a 1,2-diol, and the second set of metabolic enzymes further comprises an enzyme for converting a one-carbon substrate to formyl-CoA and 2-hydroxyacyl-CoA lyase or oxalyl-CoA decarboxylase that condenses the 2-hydroxy aldehyde and the formyl-CoA to form 2,3-dihydroxyacyl-CoA that is one carbon longer than the 2-hydroxy aldehyde, the microorganism according to claim 3. **Claim 8** the second set of metabolic enzymes further comprises a thioesterase, or an acyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase that converts the 2-hydroxyacyl-CoA to a 2-hydroxy acid; or the second set of metabolic enzymes further comprises an acyl-CoA reductase that converts the 2-hydroxyacyl-CoA to a 2-hydroxy aldehyde; or the second set of metabolic enzymes further comprises an alcohol-forming acyl-CoA reductase that converts the 2-hydroxyacyl-CoA to a 1,2-diol, the microorganism according to claim 7. **Claim 9** the second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the 2-hydroxy aldehyde to a 1,2-diol, the microorganism according to claim 8. **Claim 10** the second set of metabolic enzymes further comprises a diol dehydratase that converts the 1,2-diol to an aldehyde, the microorganism according to claim 9. **Claim 11** the second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the aldehyde to a primary alcohol, the microorganism according to claim 10. **Claim 12** the second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the 2,3-dihydroxyacyl-CoA to a 1,2,3-triol; or The second set of metabolic enzymes further comprises a thioesterase, or an acyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase that converts the 2,3-dihydroxyacyl-CoA into 2,3-dihydroxy acid; or The second set of metabolic enzymes further comprises an acyl-CoA reductase that converts the 2,3-dihydroxyacyl-CoA into 2,3-dihydroxy aldehyde, the microorganism according to claim 7.

13. The second set of metabolic enzymes further comprises an alcohol-forming acyl-CoA reductase that converts the 2,3-dihydroxyacyl-CoA into 1,2-diol, the microorganism according to claim 7.

14. The second set of metabolic enzymes further comprises a diol dehydratase that converts the 1,2,3-triol into 3-hydroxy aldehyde, the microorganism according to claim 12.

15. The second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the 3-hydroxy aldehyde into 1,3-diol, the microorganism according to claim 14.

16. The second set of metabolic enzymes further comprises an enzyme for converting a one-carbon substrate into formyl-CoA and a 2-hydroxyacyl-CoA lyase or an oxalyl-CoA decarboxylase that condenses the ketone and the formyl-CoA to form 2-methyl-2-hydroxyacyl-CoA that is one carbon longer than the ketone; or The second set of metabolic enzymes further comprises an enzyme for converting a one-carbon substrate into formyl-CoA and a 2-hydroxyacyl-CoA lyase or an oxalyl-CoA decarboxylase that condenses the aldehyde and the formyl-CoA to form 2-hydroxyacyl-CoA that is one carbon longer than the aldehyde, the microorganism according to claim 5.

17. The second set of metabolic enzymes further comprises a thioesterase, or an acyl-CoA transferase, or a phosphotransacylase and a carboxylate kinase that converts the 2-methyl-2-hydroxyacyl-CoA into 2-methyl-2-hydroxy acid; or The second set of metabolic enzymes further comprises an acyl-CoA reductase that converts the 2-methyl-2-hydroxyacyl-CoA into 2-methyl-2-hydroxy aldehyde; or The microorganism according to claim 16, wherein the second set of metabolic enzymes further comprises an alcohol-forming acyl-CoA reductase that converts the 2-methyl-2-hydroxyacyl-CoA into 2-methyl-1,2-diol.

18. The microorganism according to claim 17, wherein the second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the 2-methyl-2-hydroxyaldehyde into 2-methyl-1,2-diol.

19. The microorganism according to claim 18, wherein the second set of metabolic enzymes further comprises a diol dehydratase that converts the 2-methyl-1,2-diol into 2-methyl-aldehyde.

20. The microorganism according to claim 19, wherein the second set of metabolic enzymes further comprises an alcohol dehydrogenase that converts the 2-methyl-aldehyde into 2-methyl primary alcohol.

21. The enzyme for converting a one-carbon substrate into formyl-CoA comprises a methanol dehydrogenase that converts methanol into formaldehyde and an acyl-CoA reductase that converts formaldehyde into formyl-CoA, and the one-carbon substrate is methanol; or The enzyme for converting a one-carbon substrate into formyl-CoA comprises an acyl-CoA reductase that converts formaldehyde into formyl-CoA, and the one-carbon substrate is formaldehyde; or The enzyme for converting a one-carbon substrate into formyl-CoA comprises an acyl-CoA synthetase, or an acyl-CoA transferase, or a carboxylate kinase and a phosphotransacylase, or a carboxylic acid reductase and an acyl-CoA reductase, or an aldehyde dehydrogenase and an acyl-CoA reductase that converts formate into formyl-CoA, and the one-carbon substrate is formate, the microorganism according to claim 7.

22. The microorganism according to claim 1, wherein the externally supplied energy source is a reduced one-carbon substrate.

23. The microorganism according to claim 22, wherein the reduced one-carbon substrate is selected from the group consisting of methanol and formaldehyde.

24. The third set of metabolic enzymes includes methanol dehydrogenase that converts methanol to formaldehyde, formaldehyde dehydrogenase that converts formaldehyde to formate, and formate dehydrogenase that converts formate to CO 2 ; or The third set of metabolic enzymes comprises a formaldehyde dehydrogenase that converts formaldehyde into formate and a formate dehydrogenase that converts formate into CO2; or The microorganism according to claim 23, further comprising an acylating formaldehyde dehydrogenase that converts formaldehyde to formyl-CoA, a phosphate formyltransferase that converts formyl-CoA to formyl-phosphate, and a formate kinase that converts formyl-phosphate to formate as the third set of metabolic enzymes.

25. The microorganism according to claim 22, further comprising an enzyme that generates ATP from NADH as the third set of metabolic enzymes.

26. The microorganism according to claim 1, further comprising a metabolic enzyme that enables the production of carboxylic acids from a carbon source, wherein the carbon source is arbitrarily selected from the group consisting of glucose, xylose, arabinose, glycerol, methane, CO2, methanol, formate, formaldehyde, and similar substances.

27. The microorganism according to claim 1, which is a bacterium or yeast.

28. A method for culturing the microorganism according to claim 1, comprising incubating the microorganism, a carboxylic acid, and an external energy source under appropriate conditions such that the carboxylic acid is converted to the desired reduction product, and optionally further comprising isolating the desired product from the microorganism culture.

29. A method for culturing the microorganism according to claim 1, comprising incubating the microorganism, a carbon source, and an external energy source under appropriate conditions such that the carbon source is converted to the desired reduction product, and optionally further comprising isolating the desired product from the microorganism culture.

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