WO2017190056A1 - Conversion of 1-carbon compounds to products - Google Patents

Abstract

Described herein are biotechnological methods for the production of industrially relevant chemicals. In particular, methods for the biological production of products containing at least one terminal alcohol, carboxyl group, amine or alkyl group directly by the assimilation of single carbon units using microorganisms are described.

Description

CONVERSION OF 1 -CARBON COMPOUNDS TO PRODUCTS

PRIOR RELATED APPLICATIONS

[0001] This invention claims priority to the following, each incorporated by reference in its entirety herein for all purposes: U. S. Serial No. 62/329,024, filed April 28, 2016; U. S. Serial No. 62/329,036, filed April 28, 2016; U.S. Serial No. 62/329,049, filed April 28, 2016; and U. S. Serial No. 62/329,056, filed April 28, 2016.

FEDERALLY SPONSORED RESEARCH STATEMENT

[0002] Not applicable.

FIELD OF THE DISCLOSURE [0003] The invention relates to biotechnological methods for the production of industrial ly relevant chemicals. In particular, methods for the biological production of products containing at least one terminal alcohol, carboxyl group, amine or alkyl group directly by the assimilation of single carbon units is described.

BACKGROUND OF THE DISCLOSURE [00041 In the past, much of our energy and chemicals have derived from fossilized carbon resources, such as petroleum and coal . However, concerns about climate change, political instability, and depletion and cost of petroleum resources have recently ignited interest in the establishment of a bio-based industry and a number of strategies are being pursued to achieve the sustainable production of fuels and chemicals. One of the most promising approaches is the use of microorganisms to convert biomass into the desired product. The use of these feedstocks, however, is hindered by the difficulty of accessing the resource, their high price, and limited availability. One-carbon compounds, including C0₂, CO, formate, methanol, and methane have recently emerged as alternative renewable feedstocks. Conversion of these substrates to fuels and chemicals not only offers an attractive alternativ e in terms of process economics, but also will further contribute to reducing the levels of greenhouse gases, such as C0₂ and methane. [00051 Microbes have been designed and engineered to synthesize products of interest using feedstocks as diverse as sugars, glycerol, carbon dioxide, carbon monoxide, formate, methanol, and methane. For the case of I -carbon feedstocks, such conversions are made possible by a general network of metabolic pathways that are organized as shown in FIG. 1 and include specialized pathways for carbon fixation, central metabolism, and product synthesis. This type of metabolic architecture has been exploited in all metabolic engineering efforts conducted to date to develop microbes for industrial applications. Thi s 'top-down' engineering strategy i s highly complex and suffers from inefficiencies ari sing from need to first produce common metabolic intermediates before eventually forming products. This architecture is also commonly limited to elongation of a carbon backbone by a minimum of two carbons per step, which is a result of the aforementioned use of common metabolic intermediates.

[0006] This disclosure describes an alternative platform for the byconversion of 1 -carbon substrates to products of interest, which consists of a single engineered metabolic pathway that allows for the direct assimilation of one-carbon compounds into chemical products containing at least one terminal alcohol, carboxyl group, amine or alkyl . Chemicals containing terminal alcohol, carboxyl , amine or alkyl groups have broad uses in the chemical industry including, but not limited to, applications such as fuels, cosmetics, and polymers. The engineered pathway, designed from the "bottom-up^", defines a new metabolic architecture that consolidates carbon fixation, central metaboli sm, and product synthesis into single pathway ( FIG. 2-7). The pathway uses single carbon extension units, which bypasses the need for the production of common metabolic intermediates and allows for elongation of a carbon backbone iteratively in single carbon increments.

SUMMARY OF THE DISCLOSURE [0007] The development of a synthetic, biological pathway that consolidates fixation of single carbon compounds, such as CO:, CO, formate, formaldehyde, methanol, and methane, into the synthesis of products containing at least one terminal alcohol is described. The pathway allows for direct synthesis of products from single carbon compounds, avoiding the need to produce the cel ls' typical intermediates and use of central metabolic pathways, as required by the prior approaches. This synthetic pathway is inspired by the catabolic a- oxidation pathway (one carbon shortening of fatty acids), and based on the key recent finding in our laboratory that the enzyme 2-bydroxyl-acyl-CoA lyase (HACL, part of the a-oxidation pathway) is reversible and able to catalyze C-C bond formation between formyl-CoA and an aldehyde. This mechanism for C-C bond formation had not been previously reported in the literature and allows for the novel carbon-chain elongation by one-carbon units.

[00081 The overall pathway accomplishes three main functions:

[0009] First: single carbon compounds are converted to an active ^"extender^" form that can be incorporated into a growing carbon skeleton. This is done by enzymatically conv erting a one-carbon unit into an activated Co A. deriv ative, and there are a variety of enzymes available to do this ( FIG. 2 ).

[0010] Second: the pathway allows for these single carbon extenders to iterativ ely elongate the carbon skeleton to desired length. An enzyme from the alpha oxidation pathway ( hy droxy 1 -acy 1 -Co A lyases or HACLs) is driven in the reverse direction and thus used to catalyze the key condensation (C-C bond formation) reaction. A series of dehydration and reduction reactions then converts the molecule to a form suitable for successiv e rounds of elongation (FIG. 3-4).

[0011] Third: intermediates of the abov e are diverted to or conv erted to products containing at least one terminal alcohol, carboxyl, amine or alky I once the desired carbon chain length is reached ( FIG 3- 13). [0012] The reactions of the pathway are enabled by prov iding enzymes that serve as biological catalysts. Enzymes are generally polypeptides, which are defined by an amino acid sequence, which i s in turn defined by a DNA or gene sequence. In one embodiment of the invention, the necessary gene sequences are provided in an engineered microbial host, such that the microorganism synthesizes the enzymes that comprise the pathway. In another embodiment, the enzymes that comprise the pathway are purified and combined in a reaction mixture, providing the ability to synthesize products containing at least one terminal alcohol, carboxyl group, amine or alkyl from single carbon molecules.

[0013] In one embodiment of the invention, products containing at least one terminal alcohol, carboxyl, amine or alkyl are derived solely from single carbon molecules. In another embodiment, however, products can be derived from a combination of single carbon molecules and multi -carbon molecules. One could provide any intermediate in the designed synthetic pathway as a starting compound, and need not make the entire molecule from scratch. Such embodiments may be useful where an intermediate is plentiful or a waste product from another process, and thus very inexpensive. These additions can also aid the throughput of the designed pathway and all ow for the production of more v aried products that contain at least one terminal alcohol, carboxyl, amine or alkyl .

[0014] Reactions and the enzymes that catalyze said reactions described herein are understood to operate both in the direction described or il lustrated, as well as in the reverse direction unless otherwise stated. [0015] The use of the word "a" or "an" when used in conj unction with the term "comprising^" in the claims or the specification means one or more than one, unless the context dictates otherwise.

[0016] The term "about^" means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated. [0017] The use of the term "or^" in the claims is used to mean "and/or^" unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.

[0018| The terms "compri se^", "have^", "include^" and "contain^" (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim. [0019] As used herein, the expressions "microorganism,^" "microbe,^"

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

[0020] As used herein, reference to a "cell,^" "microbe,^" etc. is generally understood to include a culture of such cell s, as the work described herein is done in cultures having 10^9"15 cells. [0021] As used herein, "growing" cells used it its art accepted manner, referring to exponential growth of a culture of cells, not the few cells that may not have completed their cell cycle at stationary phase or have not yet died in the death phase or after harvesting. [0022] As used in the claims, "homolog^" means an enzyme ith at least 40% identity to one of the listed sequences and also hav ing the same general catalytic activ ity, although of course Km, Kcat, and the like can vary. While higher identity (60%, 70%, 80%) and the like may be preferred, it i s typical for bacterial sequences to diverge significantly (40- 60%), yet still be identifiable as homologs, while mammalian species tend to diverge less (80-90%).

[00231 Reference to proteins herein can be understood to include reference to the gene encoding such protein. Thus, a claimed "permease^" protein can include the related gene encoding that permease. However, it is preferred herein to refer to the protein by standard name per ecoliwiki or HUGO since both enzymatic and gene names have varied widely, especially in the prokaryotic arts.

[00241 Once an exemplary protein i s obtained, many additional examples proteins of similar activity can be identified by BLAST search. Further, every protein record i s linked to a gene record, making it easy to design expression vectors. Many of the needed enzymes are already available in vectors, and can often be obtained from cell depositories or from the researchers who cloned them . But, if necessary, new clones can be prepared based on available sequence information using RT-PCR techniques. Thus, it should be easi ly possible to obtain all of the needed enzymes for expression or ov erexpression.

[0025] Another way of finding suitable enzymes/proteins for use in the invention i s to consider other enzymes with the same EC number, since these numbers are assigned based on the reactions performed by a given enzyme. An enzyme that thus be obtained, e.g., from AddGene or from the author of the work describing that enzyme, and tested for functionality as described herein.

[0026] Understanding the inherent degeneracy of the genetic code allows one of ordinary skill in the art to design multiple nucleotides that encode the same amino acid sequence. NCBI™ provides codon usage databases for optimizing DNA sequences for protein expression in various species. Using such databases, a gene or cDNA may be "optimized" for expression in /·„^'. coli, yeast, algal or other species using the codon bias for the species in which the gene will be expressed.

[0027] In calculating "% identity^" the unaligned terminal portions of the query sequence are not included in the calculation. The identity is calculated over the entire length of the reference sequence, thus short local alignments with a query sequence are not relevant (e.g., % identity = number of aligned residues in the query sequence/length of reference sequence). Alignments are performed using BLAST homology alignment as described by Tatusova TA & Madden TL (1999) FEMS Microbiol. Lett. 174:247-250, and available through the NCBI website. The default parameters were used, except the filters were turned OFF.

[0028] "Operably associated^" or "operably linked^", as used herein, refer to functionally coupled nucleic acid or amino acid sequences.

[0029] "Recombinant^" is relating to, derived from, or containing genetically engineered material . In other words, the genome was intentionally manipulated by the hand- of-man in some way.

[0030] "Reduced activity^" or "inactivation^" is defined herein to be at least a

75% reduction in protein activity, as compared with an appropriate control species (e.g., the wild type gene in the same host species). Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codon s, by frame shift mutation, and the like.

[0031] By "null" or "knockout^" what is meant is that the mutation produces undetectable active protein. A gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence. Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can al so completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All null mutants herein are signified by Δ.

[0032] "Overexpression^" or "overex pressed^" is defined herein to be at least 150%) of protein activity as compared with an appropriate control species, or any expression in a species that lacks the activ ity altogether. Preferably, the activity is increased 100-500%. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resi stant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like. All overexpressed genes or proteins are signified herein by "+".

[0001] In certain species it is possible to genetically engineer the endogenous protein to be overexpressed by changing the regulatory sequences or removing repressors, e.g., by homologous recombination, or CRISPR/CAS gene editing, and the like. However, ov erexpressing the gene by inclusion on selectable pi asm ids that exi st in hundreds of copies in the cell may be preferred due to its simplicity and ease of exerting external controls, although permanent modifications to the genome may be preferred in the long term for stability reasons.

[0002] The term "endogenous^" or "native^" means that a gene originated from the species in question, without regard to subspecies or strain, although that gene may be naturally or intentionally mutated, or placed under the control of a promoter that results in overexpression or controlled expression of said gene. Thus, genes from Clostridia would not be endogenous to Escherichia, but a plasmid expressing a gene from E. coli or would be considered to be endogenous to any genus of Escherichia, even though it may now be ov erexpressed.

100031 "Expression vectors^" are used in accordance with the art accepted definition of a plasmid, virus or other propagatable sequence designed for protein expression in cells. There are thousands of such vectors commercially available, and typically each has an origin of replication (ori); a multiple cloning site; a selectable marker; ribosome binding sites; a promoter and often enhancers; and the needed termination sequences. Most expression vectors are inducible, although constitutive expression vectors also exist.

[0004] As used herein, "inducible^" means that gene expression can be controlled by the hand-of-man, by adding e.g., a ligand to induce expression from an inducible promoter. Exemplary inducible promoters include the lac operon inducible by IPTG, the yeast AOXl promoter inducible w ith methanol, the strong LAC4 promoter inducible with lactate, and the like. Low level of constitutive protein synthesi s may still occur even in expression vectors with tightly controlled promoters.

100051 As used herein, an "integrated sequence^" means the sequence has been integrated into the host genome, as opposed to being maintained on an expression vector. It will still be expressible, and preferably is inducible as well .

BRI E F DECRYPTION OF FIGURES

[0033] FIG. 1. Current 'top-down' metabolic engineering approach based on editing existing architecture of natural metabolism .

100341 FIG. 2. Single carbon manipulation reactions for the generation of formyl-CoA. Representative enzymes for each reaction are given in the legend.

10035] FIG. 3. A pathway for the assimilation of single carbon molecules into products containing at least one terminal alcohol . Referred to as Scheme 3 A . Representative enzymes for each reaction are given in the legend.

100361 FIG. 4. A pathway for assimilation of single carbon molecules into products containing at least one terminal alcohol . Referred to as Scheme 4B. Representative enzymes for each reaction are giv en in the legend.

10037] FIG. 5. A pathway for the assimilation of single carbon molecules into products containing at least one carboxyl group. Referred to as Scheme 5 A. Representative enzymes for each reaction are given in the legend.

100381 FIG. 6. A pathway for assimilation of single carbon molecules into products containing at least one carboxyl group. Referred to as Scheme 6B. Representative enzymes for each reaction are giv en in the legend.

10039] FIG. 7. A pathway for the assimilation of single carbon molecules into products containing at least one terminal amine. Referred to as Scheme 7 A. Representative enzymes for each reaction are given in the legend.

100401 FIG. 8. A pathway for assimilation of single carbon molecules into products containing at least one terminal amine. Referred to as Scheme 8B. Representativ e enzymes for each reaction are giv en in the legend. 100411 FIG. 9. A pathway for the assimilation of single carbon molecules into products containing at least one terminal alkyl group. Referred to as Scheme A. Representative enzymes for each reaction are given in the legend.

10042] FIG. 10. A pathway for assimilation of single carbon molecules into products containing at least one terminal alkyl group. Referred to as Scheme 10B. Representative enzymes for each reaction are given in the legend.

[0043] FIG. 1 1. A method for the production of products from solely single carbon molecules via Scheme 1 1 A ( FIG. 3).

[0044] FIG. 12. A method for the production of products from single carbon molecules and a supplied priming molecule (Scheme 12AB).

[0045] FIG. 13. A method for the production of products from single carbon molecules and an additionally supplied, unrelated multi-carbon substrate ( Scheme 3AB).

[00461 FIG. 14. Examples of functional groups ( R- groups) for the described products and primers. Groups can also be used in combinations. Not a comprehensive list. [0047] FIG. 15. Vector construct containing the gene encoding N-terminal

H IS-tagged acylating aldehyde reductase Lmo l 1 79 from hysteria monocytogenes for expression in E. coli.

[00481 FIG. 6. SDS-PAGE showing expression and purification of Lmo l 1 79 from /·,^". coli. [0049] FIG. 17. Time course of absorbance at 340 nm corresponding to the production of NADU in the assay of Lmol 179.

100501 FIG. 18. ESI-TOF MS data of the Co A content of Lmo l 1 79 reaction assay mixtures after solid phase extraction.

[00511 FIG. 19A S. cerevisiae and FIG. 19B E. coli expression vector constructs containing the gene encoding an amino-terminal 6X HIS-tagged hydroxyl-acyl- CoA lyase HACL 1 from Homo sapiens.

[0052] FIG. 20. SDS-PAGE showing the purification of hydroxy 1-acyl-Co A lyase HACL 1 from S. cerevisiae. Lane 1 corresponds to the protein ladder. Lane 2 is the S. cerevisiae cell extract. Lane 3 corresponds to purified HACL 1 . [0053] FIG. 21. GC-FID chromatograms of pentadecanal content in HACLl degradative reaction mixtures after extraction with hexane. Top: pentadecanal standard; Middle: HACLl assay sampled; Bottom: no enzyme control. In samples containing HACLl, a pentadecanal peak is seen, while there is no peak in the sample in which enzyme was omitted. [0054] FIG. 22. GC-FID chromatograms of HACLl synthetic reaction mixtures after hydrolysis of acyl-CoAs and denvatization. From top to bottom : no enzyme control; HACLl sample; 2-hydroxyhexadecanoyl-CoA standard; 2-hydroxyhexanoic acid standard. HACLl was incubated with pentadecanal and formyl-CoA and was capable of li gating the molecules to 2-hydroxyhexadecanoyl-CoA as indicated by the peaks corresponding to the standards. FIG. 18 and FIG. 19 prove that the enzyme can run in reverse, as required for the synthetic pathway.

[0055] FIG. 23. HPLC chromatogram of HACL 1 synthetic reaction samples incubated with acetal delu de and formyl-CoA. Solid line: HACLl sample; Dashed line: no enzyme control . The expected product of the ligation of acetal dehyde and formyl-CoA is lactoyl-CoA, which would be expected to be hydrolyzed to its acid form, lactate. A peak for lactate (26 min) was observed in samples containing purified HACLl and was not observed in samples that did not contain enzyme.

[0056] FIG. 24. HPLC chromatogram of HACLl synthetic reaction samples incubated with formaldehyde and formyl-CoA. Solid line: no enzyme control; Dashed line: HACL 1 sample; Dotted line: glycol ate standard. The expected product of the ligation of formaldehyde and formyl-CoA i s glycolyl-CoA, which would be expected to be hydrolyzed to its acid form, glycolate. A peak matching a glycolate standard (25.2 min) was observed in samples containing purified HACL 1 and was not observed in samples that did not contain enzyme. [0057] FIG. 25. NMR spectra of HACL 1 assay samples incubated with acetal dehyde and formyl-CoA. (A) HACL 1 sample. (B) No enzyme control . A peak corresponding to lactate was identified in the sample containing HACL 1 .

[0058] FIG. 26A-B. V ector constructs encoding oxaly -CoA decarboxylases from FIG. 20 A Oxalohacter formigenes and FIG. 20 B E. coli. [00591 FIG. 27. Vector construct encoding benzaldehyde lyase from

Pseudomonas fluorescens.

[0060] FIG. 28. Vector construct containing the gene encoding LcdABC from

Clostridium propionicam for expression in E. coli. LcdABC is an example of a 2- hydroxyacyl-CoA dehydratase.

[0061] FIG. 29. Eadie-Hofstee plot for the determination of Euglena gracilis

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

[0062] FIG. 30. Time course of absorbance at 340 nm corresponding to the consumption of N ADU in the assay of MhpF. [0063] FIG. 31. Time course of absorbance at 340 nm corresponding to the consumption of NADH in the assay of FucO.

[0064] FIG. 32. HPLC chromatogram of the in vitro assembly of the transfer) oyl -Co A reductase and acyl-CoA reductase steps of Scheme B. A peak corresponding to butyraldehyde (butyraldehyde standard in bold) was present in samples containing Treponema denticola TER (TdTER) and ./·,^". coli MhpF (solid, thin line), but not the control containing TdTER only (dashed line).

[0065] FIG. 33. Production of the 1 ,2-diol ethylene glycol in vivo using an engineered strain of E. coli.

[0066] FIG. 34. Production of 2-hydroxy acids and 1 ,2-diol s in the forms of gly colic acid and ethylene glycol using an assembled reaction mixture in vitro.

[0067] FIG. 35. Production of the 2-hydroxy acid gly colic acid in vivo using an engineered strain of E. coli.

[0068] FIG. 36. Production of 2-hydroxyacids glycolic acid and lactic acid using an assembled reaction mixture in vitro, demonstrating products of different carbon chain length.

[0069] FIG. 37. Assay of purified diol dehydratase KoPddABC coupled with aldehyde dehydrogenase AldB. The sample containing KoPddABC (blue) had observable reduction of NADP^' to NADPH measured by absorbance at 340 nm, whereas the sample without KoPddABC had no noticeable change in absorbance. 100701 FIG. 38. (A) SDS/'PAGE gel showing expression and purification of histidine tagged CV2025. (B) Thin-layer chromatogram showing the appearance of L-alanine when CV2025 (cell lysate or purified form) i s incubated with indicated amino-acid substrate and pyruvate (amino group donor and acceptor). L-alanine synthesis demonstrates functional activity in the reverse direction through the transfer of the amine group from the substrate to pyruvate, forming L-alanine.

100711 FIG. 39. Alkyl compound synthesis from aldehyde intermediates through the use of aldehyde decarboxylase ORF 1 593 from Synechococcus elongatus PCC7942. (A) Purification of n-terminal His-tagged ORF 1 593 from /·„^'. coli. Lane 1 : Protein ladder; Lane 2: Purified protein after dialysis; Lane 3 : Purified protein; Lane 4: Crude extract of MG I 655 expressing ORF 1 593 from pUCBB-pTrc-ntH6_orf 1 593. (B) GC-FID chromatogram demonstrating undecane production from dodecanal with purified ORF 1 593. Black: full reaction; Green: No enzyme control; Red: No substrate control; Blue: No PMS/NADH control .

[00721 Table 1. Reaction and enzyme list for the designed pathway (s). These are only a few of the enzymes that can be used, but prov ides a representative listing.

[0073] Table 2. Summary of enzymes that have been characterized in vitro.

DETAI LE D DESCRIPTION

[0074] Herein, our focus will be the design of a pathway that allows for the direct assimilation of single carbon molecules into products containing at least one terminal alcohol, carboxyl, amine or alkyl group. Such a pathway would allow for a carbon backbone to be elongated by one carbon units to give the desired product. The proposed invention performs three main functions, and the details describing how these functions are accomplished are provided below.

[0075] The first function of the pathway is to produce formyl-CoA. and is illustrated in FIG. 2. Single carbon molecules of various reduction lev els are intercon verted by the illustrated reactions to produce formyl-CoA, the single carbon unit used to extend a carbon backbone. Detail s regarding the reactions and exemplary enzymes that accompli sh the first function can be found in TABLE 1. Methane can be oxidized to methanol ( FIG. 2, reaction 1 ) by a suitable methane monooxygenase. Methanol can be oxidized to formaldehyde ( FIG. 2, reaction 2) by a suitable methanol dehydrogenase. Formaldehyde can be oxidized to form \ -Co A ( FIG. 2, reaction 3) by an acylating aldehyde dehydrogenase. Carbon dioxide can be reduced to formate ( FIG. 2, reaction 4) by a carbon dioxide reductase or by electrochemical methods. Formate can be converted to formyl-CoA either directly ( FIG. 2, reaction 7) by a suitable acetyl -Co A synthetase or through the intermediate formyl- phosphate ( FIG. 2, reaction 5-6) by a suitable formate kinase and phosphate acetyl- transferase.

[0076] At a minimum, the provision of formyl-CoA can be accomplished from either formaldehyde, by the expression of an acylating aldehyde dehydrogenase, or from formate, by a suitable acetyl -Co A synthetase or combined formate kinsane and phosphate acetyl-transferase. Combinations of the above reactions can be used to generate formyl-CoA from other single carbon molecules. For example, an implementation that makes use of methane would include the expression of a methane monooxygenase, a methanol dehydrogenase, and an acylating aldehyde dehydrogenase. Even more combinations of the described reactions and accompanying enzymes can be used to allow for implementations that use a mixture of single carbon units, for example a combination of methane and carbon dioxide through all of the described reactions.

[0077] The second function of the pathway is the iterative elongation of a carbon backbone by the single carbon unit formyl-CoA, known as an "extender unit" herein. This is illustrated in FIG. 3- 10. Details regarding the reactions and exemplary enzymes that accomplish the second function can be found in TABLE 1.

[0078] In one embodiment, referred to as Scheme A, formyl-CoA i s condensed with an aldehyde to give a 2 - h y d rox y a cy 1 -C o A that is one carbon longer than the initial aldehyde (e.g., FIG. 3, reaction 1) by a suitable 2-hydroxyacy -CoA lyase. The 2- hy droxy acyl -Co A is then reduced to a 2 -hydroxy aldehyde (e.g., FIG. 3, reaction 2) by a suitable acyl-CoA reductase. The 2-hy droxy al dehy de is further reduced to a 1 ,2-diol (e.g., FIG. 3, reaction 3) by a suitable 1 ,2-diol oxidoreductase. The diol can be dehydrated to give an aldehyde (e.g., FIG. 3, reaction 4) by a suitable diol dehydratase, which is capable of further elongation . [00791 In another embodiment, referred to as Scheme B, formyl-CoA is condensed with an aldehyde to give a 2-hydroxyacyl-CoA that is one carbon longer than the initial aldehyde (e.g., FIG. 4, reaction 1) by a suitable 2-hydroxyacyl-CoA lyase. The 2- hydroxyacyl-CoA is then dehydrated to give a tran s-2-enoy 1 -C o A (e.g., FIG. 4, reaction 2) by a suitable 2-hydroxyacyl-CoA dehydratase. The trans-2-enoyl-CoA is then reduced to an acyl- CoA (e.g., FIG. 4, reaction 3) by a suitable trans-2-enoyl-CoA reductase. Finally, the acyl- CoA can be reduced to give an aldehyde (e.g., FIG. 4, reaction 4) by an acyl-CoA reductase, which is capable of further elongation.

[0080] The final function of the pathway is the formation of the products that contain at least one terminal alcohol, carboxyl, amine or alkyl . Detail s regarding the reactions and exemplary enzymes that accomplish the third function can be found in TABLE 1.

[00811 With regards to Scheme 3 A, terminal alcohols can be derived from the aldehyde intermediate by reduction of the aldehyde group to an alcohol group ( FIG. 3-4, reaction 5) by a suitable alcohol dehydrogenase. The 1 ,2-diol also already contains a terminal alcohol group, and is thus a representative product. For Scheme 4B, terminal alcohol s can be derived from the aldehyde intermediate by reduction of the aldehyde group to an alcohol group ( FIG. 3-4, reaction 5) by a suitable alcohol dehydrogenase. The acyl-CoA. intermediates can also be reduced to a terminal alcohol ( FIG. 4, reaction 6) by a suitable alcohol-forming coenzyme- A. thioester reductase, where reduction of the 2-hydroxyacy -CoA gives a 1 ,2-diol, reduction of the tran s-2-enoy 1 -Co A gives an unsaturated alcohol, and reduction of the acyl- CoA gives an alcohol .

[0082] With regards to Scheme 5 A, a carboxyl group can be derived from the aldehyde intermediates by oxidation of the aldehyde group to a carboxyl group (FIG. 5/6, reaction 5) by a suitable aldehyde dehydrogenase. A 2-hydroxycarboxylic acid can be obtained by oxidation of the 2-hydroxy aldehyde. For Scheme 6B, a carboxyl group can be derived from the aldehyde intermediate by oxidation of the aldehyde group ( FIG. 5/6, reaction 5) by a suitable aldehyde dehydrogenase. The acyl-CoA. intermediates can al so be converted to a carboxylic acid (FIG. 5/6, reaction 6) by a suitable thioesterase, where a 2-hydroxyacyl- CoA gives a 2-hydroxy carboxy i c acid, the trans-2-enoyl-CoA. gives an unsaturated carboxylic acid, and the acyl-CoA gives a carboxylic acid. [00831 With regards to Scheme 7 A, terminal amines can be derived from the aldehyde intermediates by the transfer of an amine group ( FIG. 7/8, reaction 5) by a suitable transaminase. For Scheme 8B, terminal amines can be derived from the aldehyde intermediate by the transfer of an amine group ( FIG. 7/8, reaction 5) by a suitable transaminase. The acyl- Co A intermediates can also be reduced to aldehydes ( FIG. 8, reaction 4) by a suitable acyl- CoA reductase, where, in combination with a suitable transaminase, results in 2- hydroxyamines from 2-hydroxyacyl-CoA or unsaturated amines from tran s-2-enoy 1 -Co A .

[0084] With regards to Scheme 9 A, terminal alkyl groups can be derived from the aldehyde intermediates decarbonylation ( FIG. 9/10, reaction 5) by a suitable aldehyde decarbonylase. For Scheme 10B, terminal alkyl s can be derived from the aldehyde intermediate by decarbonylation ( FIG. 9/10, reaction 5) by a suitable aldehyde decarbonylase. The acy -CoA intermediates can also be reduced to aldehydes ( FIG. 10, reaction 4) by a suitable acyl-CoA reductase, followed by decarbonylation resulting in a 2-hydroxy alkyl from a 2-hydroxyacyl-CoA or an alkene from trans-2-enoy -CoA. [00851 In one embodiment of the invention, the described pathway is provided within the context of a microbial host. The pathway in a living system is generally created by transforming the microbe with one or more expression vector(s) containing a gene encoding one or more of the needed enzymes, but the genes can also be added to the chromosome by recombineering, homologous recombination, and similar techniques. Where the needed protein is endogenous, as is the case in some instances, it may suffice as is, but i s usual ly overexpressed for better functionality and control over the level of active enzyme. Preferable, one or more, or all, such genes are under the control of an inducible promoter.

[0086] Initial cloning experiments have proceeded in E. coli for convenience since most of the required genes are already available in pi asm ids suitable for bacterial expression, but the addition of genes to bacteria is of nearly universal applicability. Indeed, since recombinant methods were invented in the 70' s and are now so commonplace, even school children perform genetic engineering experiments using bacteria. Such species include e.g.. Bacillus, Streptomyces, Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, Streptococcus, Paracoccus, Methanosarcina, and Methylococcus, or any of the completely sequenced bacterial species. Indeed, hundreds of bacterial genomes have been completely sequenced, and this information greatly simplifies both the generation of vectors encoding the needed genes, as well as the planning of a recombinant engineering protocol . Such species are listed along with links at enAvi kipedia.org/vviki/List_of_sequenced_bacterial_genomes.

[0087] Additionally, yeasts are a common species used for microbial manufacturing, and many species can be successfully transformed. In fact, the alpha oxidation pathway is present in yeast and the alpha-oxidation enzyme hy drox yl-acyl -C o A lyases (HACL), which is a key part of this inv ention was successfully expressed in yeast Saccharomyces. Other species include but are not limited to Candida, Aspergillus, Arxida adeninivorans, Candida boidinii, Hansenida polymorpha (Pichia angnsta), Kluyveromyces lactis, Pichia pastoris, and Yarrow ia lipolytica, to name a few.

[00881 It is also possible to genetically modify many species of algae, including e.g., Spirulina, Ape rgi litis, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica, and the like. Indeed, the microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii i s the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas. [0089] Furthermore, a number of databases include vector information and/or a repository of vectors and can be used to choose expression v ectors suitable for the chosen host species. See e.g., AddGene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See al so PI asm id Information Database (PlasmID) and DNASU having over 191,000 plasmids. A collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community. Furthermore, vectors (including particular ORFS therein) are usually available from colleagues.

100901 The enzymes can be added to the genome or via expression vectors, as desired. Preferably, multiple enzymes are expressed in one vector or multiple enzymes can be combined into one operon by adding the needed signals between coding regions. Further improvements can be had by overexpressing one or more, or even all of the enzymes, e.g., by adding extra copies to the cell via pi asm id or other vector. Initial experiments may employ expression plasmids hosting 3 or more ORFs for convenience, but it may be preferred to insert operons or individual genes into the genome for stability reasons.

[00911 Still further improvements in yield can be had by reducing competing pathways, such as those pathways for making e.g., acetate, formate, ethanol, and lactate, and it is already well known in the art how to reduce or knockout these pathways. See e.g., the Rice patent portfolio by Ka-Yiu San and George Bennett (US7569380, US7262046, US8962272, U S879599 1 ) and patent by the current inventors (US8 129 1 57 and US8691552) (each incorporated by reference herein in its entirety for all purposes). Many others have worked in this area as well .

[00921 Following the construction of a suitable strain containing the engineered pathway, culturing of the developed strains can be performed to evaluate the effectiv eness of the pathway at its intended goal— the production of products from single carbon compounds. The organism can be cultured in a suitable grow th medium, and can be evaluated for product formation on single carbon substrates, from methane to O:, either alone or in combination with multi-carbon molecules. The products produced by the organism can be measured by HP I or GC, and indicators of performance such as growth rate, productivity, titer, yield, or carbon efficiency can be determined.

[0093] Further evaluation of the interaction of the heterologously expressed pathway enzymes with each other and with the host system can allow for the optimization of pathway performance and minimization of deleterious effects. Because the pathway i s under synthetic control, rather than under the organism' s natively evolved regulatory mechanisms, the expression of the pathway is usually manually tuned to av oid potential issues that slow cell growth or production and to optimize production of desired compounds. [0094] Additionally, an imbalance in relative enzyme activities might restrict overall carbon flux throughout the pathway, leading to suboptimal production rates and the buildup of pathway intermediates, which can inhibit pathway enzymes or be cytotoxic. Analysis of the cell cultures by I I PLC or GC can reveal the metabolic intermediates produced by the constructed strains. This information can point to potential pathway issues. [00951 As an alternative to the in vivo expression of the pathway, a cell free in vitro version of the pathway can be constructed. By purifying the relevant enzyme for each reaction step, the overall pathway can be assembled by combining the necessary enzymes in a reaction mixture. With the addition of the relevant cofactors and substrates, the pathway can be assessed for its performance independently of a host.

[00961 In one embodiment of the invention, single carbon molecules, such as carbon dioxide, formate, formaldehyde, methanol, methane, and carbon monoxide are solely used in the production of products containing at least one terminal alcohol, carboxyl, amine, alkyl or derivatives thereof. This is illustrated in FIG. 11. In this embodiment, formyl-CoA is produced from single carbon molecules as usual, but formaldehyde, a one carbon aldehyde, serves as the initial aldehyde that is elongated. This initial aldehyde will be referred to as the "primer^" or "priming aldehyde.^" In this embodiment, formaldehyde is already provided when molecules at the same reduction level or more reduced (formaldehyde, methanol, methane) are used. From molecules that are more oxidized, such as formate and carbon dioxide, some of the produced formyl-CoA. can be reduced to give formaldehyde (reverse of the reaction shown). When using formaldehyde as the primer. Scheme A must be used for elongation, because Scheme B requires a third carbon to proceed with further rounds of elongation.

[0097] In another embodiment of the invention, the priming aldehyde can be provided along with single carbon molecules for elongation. This is illustrated in FIG. 12. In providing a specific priming aldehyde, the product containing at least one terminal alcohol can be made to also contain other interesting functional groups. To give an example, a ω-phenyl aldehyde can serve as the priming aldehyde, which i s converted to an elongated ω-phenyl alcohol through the described invention. Some exemplary functional groups are given in FIG. 14. In this embodiment, it is not necessary that the priming aldehyde is exogenously added. As an example, the 2-hy droxy al dehy de intermediate in Scheme A can serve as a priming aldehyde for further rounds of elongation to giv e polyols. The trans-2-enoyl-CoA. can be converted to a trans-2-enaldehyde and used for further rounds of elongation to give polyunsaturated products.

[0098] In another embodiment of the inv ention, the priming aldehyde can be derived from provided unrelated multi-carbon molecules or substrates, which allow for product formation along with provided single carbon molecules. This is illustrated in FIG. 13. These unrelated multi-carbon molecules, i .e. multi-carbon molecules that do not contain at least one aldehyde group, must first be converted to a suitable priming aldehyde. In this embodiment, the pathway will be implemented in an engineered microbial host that i s engineered to be able to convert the multi-carbon molecules into a priming aldehyde. Some exemplary substrates include glucose and other sugars or glycerol and other sugar alcohols, which may be converted to priming aldehydes via a pathway such as glycolysis, resulting in the production of acetaldehyde or succinic semialdehyde. This embodiment can further increase the diversity of products produced by the pathway. In addition, the unrelated multi- carbon substrates may provide additional carbon and energy for microbial survival . GENERAL METHODS

[0099] The following description of experiments provides additional details, any one of which can be subject to patenting in combination with any other. The specification in its entirety is to be treated as providing a variety of details that can be used interchangeably with other detail s, as it would be of inordinate length if one were to li st every possible combination of genes/vectors/enzymes/hosts that can be made to implement the engineered pathway(s).

[001001 Enzymes of interest are expressed from vectors such as pCDFDuet- 1

(Merck KGaA, Germany), which makes use of the DE3 expression system. Genes can be codon optimized according to the codon usage frequencies of the host organism and synthesized by a commercial vendor or in-house. However, thousands of expression vectors and hosts are available, and this is a matter of convenience.

[001011 The genes can be amplified by PCR using primers designed with 15-25 base pairs of homology for the appropriate vector cut site. For enzymes that will not require a 6X-histadine tag fusion for purification, pCDFDuet- 1 can be linearized with Ncol and EcoRI. Enzymes that wi ll be purified by Ni-NTA column w il l make use of the 6X-HIS tag in pCDFDuet- 1 . The vector can be linearized using only EcoRI in this case.

[001021 The PCR product can be inserted into the vector using e.g., the In-

Fusion HD EcoDry Cloning System and the vector transformed by heat shock into competent E. coli cells. Transformants can be selected on solid media containing the appropriate antibiotic. Plasmid DNA can be isolated using any suitable method, including QIAprep Spin Miniprep Kit (QIAGEN, Limburg), and the construct confirmed by PCR and sequencing. Confirmed constructs can be transformed by e.g., electroporation into a host strain such as E. coli for expression, but other host species can be used with suitable expression vectors and possible codon optimization for that host species. [00103| Expression of the desired enzymes from the constructed strain can be conducted in liquid culture, e.g., shaking flasks, bioreactors, chemostats, fermentation tanks and the like. Gene expression is typically induced by the addition of a suitable inducer, when the culture reaches an OD550 of approximately 0.5-0.8. Induced cells can be grown for about 4-8 hours, at which point the cells can be pelleted and saved to -20°C. Expression of the desired protein can be con finned by running cell pellet samples on SDS-PAGE.

[001041 The expressed enzyme can be directly assayed in crude cell lysates, simply by breaking the cells by chemical, enzymatic, heat or mechanical means. Depending on the expression level and activity of the enzyme, however, purification may be required to be able to measure enzyme activity over background levels. Purified enzymes can also allow for the in vitro assembly of the pathway, allowing for its controlled characterization . N- terminal or C-terminal HIS-tagged proteins can be purified using e.g., a Ni-NTA Spin Kit (Qiagen, Venlo, Limburg) following the manufacturer's protocol, or other methods could be used. The H I S-tag system was chosen for convenience only, and other tags are available for purification uses. Further, the proteins in the final assembled pathway need not be tagged if they are for in vivo use. Tagging was conv enient, however, for the enzyme characterization work performed herein.

[00105] The reaction conditions for enzyme assays can vary greatly with the type of enzyme to be tested. In general, howev er, enzyme assays follow a similar general protocol . Purified enzyme or crude lysate is added to suitable reaction buffer. Reaction buffers typically contain salts, necessary enzyme cofactors, and are at the proper pH. Buffer compositions often change depending on the enzyme or reaction type. The reaction is initiated by the addition of substrate, and some aspect of the reaction related either to the consumption of a substrate or the production of a product is monitored.

[00106] Choice of the appropriate monitoring method depends on the compound to be measured. Spectrophotometri c assays are convenient because they allow for the real time determination of enzyme activ ity by measuring the concentration dependent absorbance of a compound at a certain wavelength. Unfortunately, there are not always compounds with a measureable absorbance at convenient wavelengths in the reaction. In these situations, other methods of chemical analysis may be necessary to determine the concentration of the involved compounds. [00107| Gas chromatography (GC) is convenient for the quantification of volati le substances, of which fatty acids and aldehydes are of particular relevance. Internal standards, typically one or more molecules of similar type not involved in the reaction, is added to the reaction mixture, and the reaction mixture i s extracted with an organic solvent, such as hexane. Fatty acid samples, for example, can be dried under a stream of nitrogen and converted to their tri methyl si lyl derivatives using BSTFA and pyridine in a 1 : 1 ratio. After 30 minutes incubation, the samples are once again dried and re-suspended in hexane to be applied to the GC. Aldehyde samples do not need to be derivatized. Samples can be run e.g., on a Varian CP-3800 gas chromatograph (VARIAN ASSOCIATES, INC., Palo Alto, CA) equipped with a flame ionization detector and 1IP-5 capillary column (AGILENT TECHNOLOGIES, CA).

[001081 Once the pathway has been fully studied in vitro, the pathway can be constructed in vivo with greater confidence. The strain construction for the in vivo pathway operation should allow for the ell-defined, controlled expression of the enzymes of the pathway. As before, E. coli, B. subtilus or yeast will be a host of choice for the in vivo pathway, but other hosts could be used. The Duet system (MERCK KGaA, Germany ), allows for the simultaneous expression of up to eight proteins by induction with IPTG in E. coli, and initial experiments will use this host.

[00109| Pathway enzymes can al so be inserted into the host chromosome, allowing for the maintenance of the pathway without requiring antibiotics to ensure the continued upkeep of pi asm ids. There are also, theoretically, an infinite number of genes that can be placed on the chromosome, as chromosomal expression does not require separate origins of replication as is the case with pi asm id expression.

[001 10| DNA constructs for chromosomal integration usual ly include an antibiotic resistance marker with flanking FRT sites for removal, as described by Datsenko and Wanner, a well characterized promoter, a ribosome binding site, the gene of interest, and a transcriptional terminator. The overal 1 product i s a linear DNA fragment with 50 base pairs of homology for the target site on the chromosome flanking each side of the construct.

[00111] However, the Flp-FRT recombination method i s only one system for adding genes to a chromosome, and other systems are available, such as the RecBCD pathway, the RecF pathway, RecA recombinase, non-homologous end joining (NHEJ), Cre- Lox recombination, TYR recombinases and integrases, SER resolvases/invertases, SER integrases, P C31 Integrase, and the like. Chromosomal modifications in E. coli can also achieved by the method of recombineering, as originally described by Datsenko and Wanner, or using new gene editing tools, such as CRISPR/CAS. [00112] In a recombineering method, for example, the cells are prepared for electroporation following standard techniques, and the cells transformed with linear DNA that contains flanking 50 base pair targeting homology for the desired modification site. For seamless integration of a DNA construct, a two-step approach can be taken using a cassette that contains both positive and negative selection markers, such as the combination of cat and sacB. In the first round of recombineering, the cat-sacB cassette with targeting homology for the desired modification site is introduced to the cells. The cat gene provides resistance to chloramphenicol, which allows for positive recombinants to be selected for on solid media containing chloramphenicol. A positive isolate can be subjected to a second round of recombineering introducing the desired DNA construct with targeting homology for sites that correspond to the removal of the cat-sac B cassette. The sacB gene encodes for an enzyme that provides sensitivity to sucrose. Thus, growth on media containing sucrose allows for the selection of recombinants in which the cat-sacB construct was removed. P 1 phage ly sates can be made from isolates confirmed by PGR and sequencing. The lysates can be used to transduce the modification into desired strains, as described prev iously. [00113] Engineered strains expressing the designed pathway can be cultured under the following or similar conditions. Overnight cultures started from a single colony can be used to inoculate flasks containing appropriate media. Cultures are grown for a set period of time, and the culture media analyzed. The conditions will be highly dependent on the specifications of the actual pathway and what exactly is to be tested. For example, the ability for the pathway to be used for autotrophic grow th can be tested by the use of formate or formaldehyde as a substrate in MOPS minimal media, as described by Neidhardt, supplemented with appropriate antibiotics, and inducers. Mixotrophic growth can be characterized by the addition of both single carbon compounds and glucose or glycerol .

[001 14| Analysis of culture media after fermentation provides insight into the performance of the engineered pathway. GC can perform quantification of longer chain products. Other metabolites, such as short chain organic acids and substrates such as glucose or glycerol can be analyzed by HPLC. Once the pathway is ful ly functional, the cultures can be grown in chemostat, providing continuous uninterrupted production of product in a living, growing system if desired.

[00115] Various -omics techniques, such as microarray or 2D-PAGE can give information about gene expression or protein expression, respectively. Genome scale modeling allows for the identification of additional modifications to the host strain that might lead to improved performance. Deletion of competing pathways, for example, might increase carbon flux through the engineered pathway for product production.

[001 16| Standard molecular biology techniques ere used for gene cloning, pi asm id isolation, and E. coli transformation. Native E. coli genes were amplified from E. coli MG1655 genomic DNA using primers to append 15 bp of homology on each end of the gene insert for recombination into the vector backbone. Genes from other organisms were codon optimized and synthesized by either GeneArt (LIFE TECH., CA or GENSCRIPT, NJ). Plasmids were linearized by the appropriate restriction enzymes and reconibined with the gene inserts using the In-Fusion HD Eco-Dry Cloning system (CLONTECH LAB., CA). The mixture was subsequently transformed into Stellar competent cells (CLONTECH LAB ).

[001 17| Transformants that grew on solid media (LB + Agar) supplemented with the appropriate antibiotic were isolated and screened for the gene insert by PGR. PI asm id was isolated from the verified transformants and the sequence of the gene insert was further confirmed by DNA sequencing (LONE STAR LABS, TX). Plasmids (also referred to as vectors) in each case contain at least one promoter, a ribosome binding site for each gene, the gene(s) of interest, at least one terminator, an origin of replication, and an antibiotic resistance marker. Exemplary plasmids are shown in FIG. 15, 19, and 16-28.

[001 18| The genes that encode the enzymes of the engineered pathway were cloned and expressed as described above. The purified enzymes were assessed for their ability to catalyze the proposed reactions as summarized in Table 2. Here, we describe testing in vitro some key steps that comprise a portion of an exemplary pathway.

[00119] hysteria monocytogenes Lmo l 1 79 was cloned (FIG. 15), expressed, and purified (FIG. 16) in E. coli as described above. The purified enzyme was evaluated for its ability to convert formaldehyde into the extender unit formyl-CoA. Enzyme assays were performed in 23 niM potassium phosphate buffer pH 7.0, 1 inM CoASH, 0.5 mM N AD^", 20 mM 2-mercaptoethanol, and 50 mM formaldehyde. The reaction was monitored by measuring absorbance at 340 nm, corresponding to the production of NADU. Co A compounds were extracted from the reaction mixture by solid phase extraction (SPE) using a CI 8 column, and the mass of the extracted Co As were determined by ESI -TOP MS.

[001201 The inclusion of Lmo l 1 79 in the reaction mixture resulted in the conversion of formaldehyde to formyl-CoA as indicated by the coproduction of NADH ( FIG. 17). Mass spectrometry analysis con finned the production of formy -CoA by Lmo l 1 79. A peak at the expected mass of formyl-CoA (796) was identified in the sample incubated with enzyme, as shown in FIG. 18. The peak was not present in the no-enzyme control, indicating that Lmo l 1 79 produced form \ -Co A.

[00121] A pi asm id containing the codon optimized gene encoding human HIS- tagged HACL 1 was constructed as described. The resulting construct, FIG. 19, was transformed into S. cerevisiae InvSC 1 (Life Tech. ). The resulting strain was cultured in 50 mL of SC-URA media containing 2% glucose at 30°C for 24 hours. The cells were pelleted and the required amount of cell s were used to inoculate a 250 mL culture volume of SC-URA media containing 0.2% galactose, 1 mM MgCl₂, and 0.1 mM thiamine to 0.4 OD600. After 20 hours incubation with shaking at 30°C, the cells were pelleted and saved. In addition, a pi asm id containing the codon optimized gene encoding 6X HIS-tagged HACL 1 from Homo sapiens was constructed as described above. The resulting construct was transformed into E. coli BL21(DE3) for expression. The resulting strain was cultured in LB media containing 50 ug/mL spectinomycin. When the culture reached an OD550 of approximately 0.6, expression was induced by addition of 0.4 mM IPTG. Cells were harvested by centrifugation after overnight incubation at room temperature. [001221 When needed, the cell pellets from S. cerevisiae were resuspended to an OD600 of approximately 100 in a buffer containing 50 mM potassium phosphate pH 7.4, 0.1 mM thiamine pyrophosphate, 1 mM MgC , 0.5 mM AEBSF, 10 inM imidazole, and 250 units of Benzonase nuclease. To the cell suspension, approximately equal volumes of 425-600 μηι glass beads were added. Cells were broken in four cycles of 30 seconds of vortexing at 3000 rpm followed by 30 seconds on ice. The glass beads and cell debris were pelleted by centrifugation and supernatant containing the cell extract was collected.

[00123] The HIS-tagged HACL 1 was purified from the cell extract using Talon

Metal Affinity Resin as described above, with the only modification being the resin bed volume and all subsequent washes were halved. The eluate was collected in two 500 uL fractions. Cell pellets from E. coli were resuspended in a buffer containing 50 mM Tris-HCl pH 7.4. Cells were di srupted by sonication and cell debris w as pel leted by centrifugation. HIS- tagged HACL 1 was purified from the supernatant using Talon Metal Affinity Resin as described above. A sample purification of HACL 1 is shown in FIG. 20.

[00124| To catalyze the ligation step, human HACL 1 was purified and tested for its native catabolic activity by assessing its ability to cleave 2-hydroxyhexadecanoyl-CoA to pentadecanal and formyl-CoA. Enzyme assays were performed in 50 mM tris-HCl pH 7.5, 0.8 mM MgCL, 0.02 mM TPP, 6.6 μΜ BSA, and 0.3 mM 2-hydroxyhexadecanoyl-CoA . The assay mixtures were incubated for one hour at 37°C, after which the presence of pentadecanal was assessed by extraction with hexane and analysis by GC-FID. As shown in FIG. 21 pentadecanal was produced in the sample containing HACL 1 , but not in the control sample, which did not contain HACL 1 , indicating that the protein was expressed and purified in an active form.

[00125] The ability of purified HACL 1 to run in the anabolic direction (reverse from the physiological direction ) was also determined. An aldehyde and formyl-CoA were tested for ligation in a buffer comprised of 60 mM potassium phosphate pH 5.4, 2.5 mM MgCl₂, 0.1 mM TPP, 6.6 μΜ BSA, 5 mM aldehyde, 20% DMSO, approximately 1 mM freshly prepared formyl-CoA, and approximately 0.5 nig/mL purified HACL 1 . The reaction was allowed to take place at room temperature for 16 hours, after which acy -CoAs were hydrolyzed to their corresponding acids by adjusting to pH > 12.0. For instances in which a short carbon chain product was expected, for example lactate production from acetaldehyde, samples were analyzed by I I PLC. In the case of longer products, for example the production of 2-hydroxyhexadecanoic acid from pentadecanal, samples were acidified with HC1 and extracted with diethyl ether. The extracted diethyl ether was evaporated to dryness under a stream of nitrogen and derivatized by the addition of 1 : 1 BSTFA:pyridine. After incubation at 70°C for 30 min, these samples were analyzed by GC-FID.

[00126] When the purified enzyme was supplied with pentadecanal and formyl-CoA, as in FIG. 22, HACL 1 was shown to catalyze the ligation of these molecules to 2-hydroxyhexadecanoyl-CoA as hypothesized. After hydrolysi s of acyl-CoAs, the chromatogram of the sample containing enzyme shows similar peaks to the 2- hydroxyhexadecanoyl-CoA spiked standard, which are absent from the no-enzyme control .

[00127| The purified HACL I w as further tested for activity on shorter aldehydes, such as the ligation of acetaldehyde or formaldehyde with formyl-CoA to produce lactoyl-CoA or glycolyl-CoA, respectively. After hydrolysi s of acyl-CoAs to their acid forms, these samples were analyzed by HPLC. The presence of lactate from elongation of acetaldehyde and formyl-CoA was identified in the sample containing HACL 1 , but not in the no-enzyme control as shown in FIG. 23. Similar results were observed for glycolate from formaldehyde and formyl-CoA as shown in FIG. 24 The presence of lactate in the relevant samples was confirmed by NMR, as shown in FI G. 25. This demonstrates that HACL 1 is capable of catalyzing the ligation of aldehydes with chain lengths ranging at least from C 1 - C 1 5 with formyl-CoA, making it suitable for the engineered iterative pathway.

[00128] 2-hydroxyhexadecanoyl-CoA was prepared by the n- hydroxy succi ni m i de method (Blecher, 1981). In summary, the n-hydroxysuccinimide ester of 2-hydroxyhexadecanoic acid is prepared by reacting n-hydroxysuccinimide with the acid in the presence of dicyclohexylcarbodiimide. The product was filtered and puri ied by recrystallization from methanol to give pure n-hydroxysuccinimide ester of 2- hydroxyhexadecanoic acid. The ester was reacted with CoA-SH in presence of thioglycolic acid to give 2-hydroxyhexadecanoyl-CoA . The 2-hydroxyhexadecanoyl-CoA was purified by precipitation using perchloric acid, filtration, and washing the filtrate with perchloric acid, diethyl ether, and acetone.

[00129] Formyl-CoA was prepared by first forming formic ethylcarbonic anhydride as previously described (Parasaran & Tarbell, 1964). Briefly, formic acid (0.4 mmol ) and ethyl chloroformate (0.4 mmol ) were combined in 4 ml., anhydrous diethyl ether and cooled to -20°C. 0.4 mmol tri ethyl amine was added to the mixture and the reaction was allowed to proceed at -2Q°C for 30 minutes. The reaction mixture was filtered over glass wool to give a solution containing formic ethyl carbonic anhydride in diethyl ether. To obtain formyl-CoA, 7 μηιοΐ Co ASH was dissolved in 5 niL 3 :2 watentetrahydrofuran, to which 10 mg of sodium bicarbonate were added. The solution of formic ethyl carbonic anhydride was added dropwise to the CoASH solution with vigorous agitation, after which the organic phase was evaporated under a stream of nitrogen. The mixture was kept at 4°C for two hours, after which any remaining diethyl ether was evaporated under nitrogen. Solid phase extraction using a C I 8 column was used to purify formyl-CoA. from the reaction mixture. Formyl-CoA was eluted from the C 1 8 column in methanol and stored in 2: 1 m et h an ol : am m on i u m acetate pH 5.5.

SCHEME A: ALCOHOLS

[00130] The purpose of this example is to demonstrate the synthesis of products containing a primary alcohol group using the pathway referred to as Scheme 3 A. 1 ,2- diols are obtained from this scheme out of the reaction step catalyzed by a suitable 1 ,2-diol oxidoreductase. In one embodiment, the pathway is assembled in vitro. Lmo l 1 79, an acyl- CoA reductase from hysteria monocytogenes, HACL1 , a 2-hydroxyacyl-CoA lyase from Homo sapiens, and FucO, a 1 ,2-diol oxidoreductase from Escherichia coli, were cloned, expressed, and purified as described above. A reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCl₂, 0.1 mM TPP, 2.5 mM CoASH, 5 mM NAD ^", 50 mM formaldehyde, 0.5 mM DTT, 0.1 g/L HACLl, 0.5 g/L Lmol 179, and 0.5 g/L FucO. After overnight incubation at room temperature, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pelleted by centrifugation and the supernatant was analyzed by HPLC. Nearly 5 mM (0.31 g/L ) 1 ,2-ethanediol (ethylene glycol ) was produced, demonstrating the production of diols from the pathway ( FIG. 34). [001311 In another embodiment, the pathway is assembled in vivo using E. coli as the host organism. Lmo l 1 79, HACL 1 , and FucO were cloned as described above to give the plasmids pCDFDuet- 1 -P 1 -ntH6-HACL 1 and pETDuet- 1 -P 1 -Lmo 1 1 79-FucO. A strain of /·,^'. coli MG1655(DE3) was engineered with knockouts of the genes glcD, frmA , fdhF, fdnG, fdoG using standard methods described above. This MG1655(DE3) AglcD AfrmA AfdhF fdnG AfdoG is hereby referred to as AC440. AC440 was transformed with the plasmids pCDFDuet- 1 -P 1 -ntH6-FIACL 1 and pETDuet- 1 -P 1 -Lmo 1 1 79-FucO using standard methods described above to give the strain AC440 pCDFDuet- 1 -P I -ntH6-H ACL 1 -P2-AMA pETDuet- 1-P l-Lmol 179, hereby referred to as AC529.

[00132] MOPS medium (Neidhardt et al ., 1974) with 125 mM MOPS and

Na2HP04 in place of K₂HP0 (2.8 mM ), supplemented with 20 g/L glycerol, 10 g/L tryptone, 5 g L yeast extract, 5 mM (NH₄)j>S04, 30 mM H₄CI, and 1 5 uM thiamine, hereby referred to as MOPS-LB-glycerol, was used to grow AC 29 at 30°C. When OD550 reached 0.4-0.6, pathway expression was induced by the addition of 0.1 mM IPTG. After 24 hours, the cells were pelleted and washed twice with MOPS medium (Neidhardt et al ., 1 974) with 1 25 mM MOPS and Na₂HP0₄ in place of K₂HP0₄ (2.8 mM), 5 mM ( H₄)₂S0₄, 30 mM NH₄C1 and 15 uM thiamine, hereby referred to as MOPS medium with no carbon source. The cells were resuspended to 25 OD in the MOPS medium with no carbon source, to which 50 mM formaldehyde was added. After 24 hours incubation at room temperature, cells were pelleted and the supernatant was analyzed by HPLC. Around 0.31 mM (20 mg/L) ethylene glycol was detected (FIG. 33), demonstrating that the pathway can be used to produce 1 ,2-diols in vivo. [00133] Normal alcohols, such as ethanol, or 1 -propanol, can also be produced through Scheme 3 A through the reaction step catalyzed by diol dehydratase by adding a suitable alcohol dehydrogenase. Lmo l 1 79, HACL 1 and FucO were cloned as above. As described above, the pathway consisting of Lmo l 1 79, HACL 1 , and FucO allows for the production of 1 ,2-diols. KoPddABC, a diol dehydratase from Klebsiella oxytoca, and YqhD, an alcohol dehydrogenase from E. coli, were cloned, expressed, and purified. Cell extracts of E. coli expressing KoPddABC were prepared by resuspending a pellet of said E. coli to an OD 550 of 40 in 60 mM potassium phosphate buffer pFI 7.4 with 200 mM 1 ,2-ethanediol . 1 ml., of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor (Scientific Industries, Bohemia, NY, USA). The cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays. The cell extract was incubated at 30°C for 3 hours in the presence of 10 μΜ coenzyme B 12. The reaction w as terminated by the addition of 1% sulfuric acid, and the precipitant was pelleted by centrifugation. The supernatant was analyzed by HPLC.

[00134] Acetaldehyde was detected in extracts of cells expressing KoPddABC, indicating that the dehydratase can convert 1 ,2-diols to their corresponding aldehydes. /·,^'. coli YqhD and FucO were expressed from A SKA collection strains and the cell extracts were assayed for activity for acetaldehyde reduction to ethanol by monitoring NAD(P)H oxidation at 340 nni. Both cell extracts of YqhD and FucO were capable of reducing acetaldehyde with specific activities of 0.1 10 ± 0.001 μηιοΐ/mg/min and 0.381 ± 0.000 μηιοΐ/mg/min, respectively. FucO was also tested for its ability to reduce longer chain aldehydes propionaldehyde and butyraldehyde to 1 -propanol and 1-butanol, resulting in specific activities of 3.35 ± 0.07 μηιοΐ/rng/min and 3.864 ± 0.008 μηιοΐ/rng/min, respectively. These results demonstrate that normal alcohols can be produced via this reaction pathway.

SCHEME A: CARBOXYLIC ACIDS

[00135] The purpose of this example is to demonstrate the synthesis of products containing a carboxylic acid group from one carbon molecules using the pathway referred to as Scheme 5 A . 2-hydroxyacids are obtained by prov iding a suitable aldehyde dehydrogenase. In one embodiment, the pathway is assembled in vitro. Lmol l79, HACLl, and AldA, an aldehyde dehydrogenase from E. coli, were cloned, expressed, and purified as described above. A reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCl₂, 0.1 mM TPP, 2.5 mM CoASH, 15 mM NAD^÷, 50 mM formaldehyde, 0.5 mM DTT, 0.2 g/L HACL l , 0.5 g/L Lmo l 1 79, 0.5 g/L AldA . After overnight incubation, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pelleted by centrifugation and the supernatant was analyzed by HPLC. Glycolic acid (2- hydroxyacetic acid) was detected at concentration of 4.6 mM (0.35 g/L ), demonstrating 2- hydroxy acid production from the pathway ( FIG. 34).

[001361 The ability of the pathway to generate products of varying chain length was demonstrated in an in vitro embodiment of the pathway. ACS, an acetyl -Co A synthetase from E. coli, Lmo l 1 79, HACL 1 and AldA were cloned, expressed, and purified. A reaction mixture was assembled comprised of 60 mM potassium phosphate pH 7.4, 2.5 mM MgCL, 0.1 mM TPP, 2.5 mM CoASH, 12.5 mM AT P, 5 mM NADH, 5 mM acetaldehyde, 50 mM formic acid, 0.5 mM DT T , 0.5 g/L ACS, 0.2 g/L HACL l , 0.5 g/L Lmo l 1 79, 0.5 g/L AldA. After overnight incubation at room temperature, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pel leted by centrifugation and the supernatant was analyzed by HPLC. Lactic acid ( 2-h droxy propi oni c acid) was detected at nearly 1 mM (90 mg/L ), demonstrating that the pathway can generate products of varying chain length ( FIG. 36). [00137] In another embodiment, the pathway is assembled in vivo using E. coli as the host organism. Lmo l 1 79, HACL 1 , and Aid A, were cloned and AC440 was transformed w ith the pi asm ids pCDFDuet- 1 -P 1 -ntH6-H ACL 1 -P2-AldA and pETDuet- l -P l - Lmo l 1 79 using standard methods described above to give the strain AC440 pCDFDuet- 1 - P 1 -ntH6-H ACL 1 -P2-AldA pETDuet-l -Pl-Lmol 179, hereby referred to as AC453. An additional strain was constructed by transfomiing AC440 with the pi asm ids pCDFDuet- 1 -P2- AldA and pETDuet- 1 -P 1 -Lmo 1 1 79, hereby referred to as AC 542 to serve as a control.

1001381 MOPS-LB-glycerol was used to grow AC453 and AC542 at 30°C.

When OD550 reached 0.4 to 0.6, expression of the pathway enzymes was induced by addition of 0.1 m M IPTG. After 24 hours, the cells were pel leted by centrifugation and washed twice with MOPS medium with no carbon source. The cells were resuspended to 25 OD in the MOPS medium with no carbon source, to which 50 niM formaldehyde was added. After 24 hours incubation at room temperature, cells were pelleted and the supernatant was analyzed by HPLC. AC453 produced 0. 12 g/L gly colic acid, whereas the AC542 produced only 0.015 g L gly colic acid (FIG. 35), indicating that the pathway can be used to produce carboxylic acids in vivo.

[00139] Non-functionalized carboxylic acids, such as acetic acid, can also be produced from Scheme 5 A through the reaction step catalyzed by diol dehydratase by supplying a suitable aldehyde dehydrogenase. Lmo l 1 79, HACL 1 , fucO, KoPddABC, and AldB, an aldehyde dehydrogenase from E. coli, were cloned, expressed, and purified. A reaction mixture containing 60 mM potassium phosphate pH 7.4, 0.5 niM NADP^" 200 niM 1 ,2-ethanediol, 1 5 uM B 1 2, 0.1 g/L AldB, and 40 mg L KoPddABC was prepared and acetaldehyde oxidation to acetate was monitored by corresponding NADP^" reduction to NADPH at 340 nm. Reduction of NADP was obser ed in a sample containing KoPddABC but was not observed in a sample where KoPddABC was omitted, indicating that the diol could be converted to the aldehyde and further oxidized to the acid form (FIG. 37).

SCHEME A: AMINES

[00140] The purpose of this example is to demonstrate the synthesis of compounds containing terminal amines from one carbon molecules through the pathway referred to as Scheme 7 A. 2-hydroxy amines can be produced from the 2-hydroxy aldehyde resulting from the reaction step catalyzed by a suitable acyl-CoA reductase acting on 2- hydroxy acyl -Co A . Purified Lnio l 1 79 and HACLl were included in a reaction mixture comprising 60 mM potassium phosphate pH 6.8, 2.5 mM MgCl:, 0.1 mM TPP, 2.5 mM CoASH, 5 mM NAD^', 50 mM formaldehyde, 0.1 g/L Lmo l 1 79, and 0.03 g/L HACLl . After overnight incubation at room temperature, the reaction was terminated by addition of 1% sulfuric acid. Precipitant was pelleted by centrifugation and the supernatant was analyzed by HPLC. Glycolaldehyde (2-hydroxyacetaldehyde) was detected as the product of these reactions. Non-functionalized terminal amines can be produced from n-aldehydes resulting from the reaction step catalyzed by diol dehydratase. It has been demonstrated in above examples that KoPddABC can act on 1 ,2-diols to give said aldehydes. [001411 Transaminase activity was determined by incubating the purified enzyme with methylbenzylamine (MBA) (amino donor) with and without the addition of pyruvate as the amino group acceptor in a standard assay mixture (Enzyme and Microbial Technology 4 1 , 628-637, 2007). Fol lowing incubation, thin layer chromatography was utilized to detect the presence of L-alanine, the compound resulting from amino group transfer from the donor to pyruvate. Using this procedure, the omega-transaminase CV2025 (AAQ59697.1) from Chromobacterium violaceum was shown to possess functional activity for amine transfer with various chain length intermediates (FIG. 38).

SCHEME A: ALKYLS

[00142] The purpose of this example is to demonstrate the synthesis of compounds containing alkyl groups from one carbon molecules through the pathway referred to as Scheme 9 A. Alkyl compounds can be produced from aldehyde intermediates such as those resulting from the reaction step catalyzed by diol dehydratase. It has been demonstrated in above examples that KoPddABC can act on 1 ,2-diol s to give said aldehydes.

[00143] Conversion of aldehyde intermediates to alkyl compounds was demonstrated in vitro through the use of aldehyde decarbonylase enzymes. The His-tagged protein was purified using Ni-NTA-spin kit (Qiagen) according manufacturer's protocol with following exceptions: The lysis buffer and wash buffer contains 20 mM and 40 mM imidazole, respectiv ely. The purification was carried out under reducing conditions (ImM tris( hydroxy propyl )phosphine was added to all buffers. After elution, the buffer was changed to 50 mM HEPES (pH 7.2), 100 mM NaCl, 10% glycerol, and 1 m M TUP at 4°C for 4 hours. The in vitro aldehyde decarbonylase assay contained 50 mM HEPES (pH7.2), 100 mM KG, 10% glycerol, 1 mM THP, 80 μΜ ammonium iron sulfate, Phenazine methosulfate 160 μ,Μ, NADH 1 .6 mM, BSA 1 mg/mL, 5 mM aldehyde (Dodecanal ) and purified protein in a final volume of 400 μ]_. Reactions were carried out at 37°C, 200 rpm, 2 hours and were stopped by adding 400 μΐ, of ethyl acetate containing 50 mg/L tridecane as internal standard. Samples were analyzed by GC using the following parameters: run time: 27 min; column: Innowax (length 30 m, I D. 0.25 mm ID, film 0.25 μηι); inlet: 250°C splitless; carrier gas: Helium, 1.0 niL/min flow; oven temp: 45°C hold 5 min, 250°C at 25°C/min, 250°C hold 10 min.

[00144] Using the above method, an activity of 0. 1 nmol undecane/min/mg protein was measured with the purified aldehyde decarbonylase ORF 1 593 from S. elongatus PCC7942. This activity was confirmed through GC-FID analysis of the reaction mixture, in which undecane production was only observed in the presence of dodecanal and PMS/NADH with purified ORF 1593 (FIG. 39).

SCHEME B

[00145] The purpose of this example is to demonstrate the use of Scheme B for the production of alcohol s, carboxylic acids, amines, and alkyls. In Scheme B, the 2- hydroxyacyl-CoA resulting from carboligation of formaldehyde and formyl-CoA catalyzed by HACL1 then undergoes dehydration to its corresponding trans-2-enoyl-CoA. A 2- hy droxy acyl -Co A dehydratase was identified as LcdABC from C. propionicum. LcdABC has been characterized by Hofmeister and Buckel for the dehydration of 2-hydroxybutyryl-CoA to crotonyl-CoA, with specific activity corresponding to 1.21 ± 0.08 μηιοΐ/min/mg protein (Hofmeister & Buckel, 1992). The genes encoding LcdABC were cloned as described above (FIG. 28).

[00146] The trans-2-enoyl-CoA is then reduced to the saturated acyl -Co A by a trans-2-enoyl-CoA reductase. A variant from E. gracilis, EgTER, was identified and cloned. After expression and purification of EgTER, in vitro assays were performed by monitoring the loss of NADH absorbance in the presence of 100 mM Tris HCL pH 7.5 and 0.2 mM NADH in a final volume of 200 μΕ at 25°C. This revealed that the enzyme i s capable of catalyzing the conversion of crotonyl-CoA. to butyryl-CoA with specific activity corresponding to 1 .2 1 ± 0.08 μηιοΐ/min/mg protein (FIG. 29). [001471 The saturated acyl-CoA i s finally converted to its corresponding aldehyde by an acyl-CoA reductase. Native E. coli acyl-CoA reductase MhpF was tested for its ability to conv ert butyryl-CoA to butyraldehyde (FIG. 30). E. coli MhpF was expressed from an A SKA collection strain (Kitagavva et al., 2005). Strains were grown anaerobically in 10 ml.. LB with 100 inM Tris pH 8.0, 10 g/L glucose, 50 μΜ FeS0₄, 5 μΜ NaH₂Se0₃, and 5 μΜ (ΝΗ₄)₆Μθ7θ24· The cells were grown to 0.4 OD550 at which point protein expression was induced by the addition of 0.1 mM 1PTG.

[00148] After 3 hours growth, cells were pelleted and the pellet was resuspended to 40 OD in 100 mM MOPS pH 7.5. 1 niL of the cell suspension was added to 0.75 g of glass beads and the cells were disrupted for 3 minutes using a cell disruptor. The cell debris and glass beads were pelleted by centrifugation and the supernatant comprising the cell extract was used for assays. The assay mixture consisted of 100 mM MOPS pH 7.5, 6 mM DTT, 5 mM MgSO.,, 0.3 mM Fe(NH₄)₂(S0₄)₂, 0.3 mM NADH, and 0.2 mM butyryl-CoA. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of N ADH, as shown in FIG. 30. MhpF was capable of catalyzing the conv ersion with a specific activ ity of 0.009 ± 0.003 μηιοΐ/min/mg protein.

[00149] A portion of Scheme B was assembled in vitro to demonstrate the functionality of the combined pathway steps. The tran s-2-en oy 1 -C o A reductase and acyl-CoA reductase steps were combined to assess the ov erall conversion of crotony -CoA to butyraldehyde. Experiments were carried out carried out in 50 mM Tris buffer, pH 7.5 containing 1 mM DTT at 37°C. The reaction with MhpF contained 0. 1 5 g/L Treponema denticola Ter (TdTer), 0.1 g/L, of MhpF, 7.5 mM NADH and 1 .7 mM crotony -CoA was added to the media for use as a primer in the new reaction pathway. Reaction with Lmo l 1 79 contained 0. 1 5 g/L TdTer, 0.9 g/L Lmo l 1 79, 7.5 mM NADH and 1 .7 mM crotonyi-CoA. The assay was monitored by measuring production of butyraldehyde with HPLC. When assayed under the conditions described abov e, the combination of trans-2-enoyl-CoA reductase from T. denticola and E. coli enzyme MhpF was shown to produce butyraldehyde from crotonyi-CoA. to a final concentration of 0.55 mM ( FIG. 32).

[00150] Alcohols can be produced in this scheme from aldehyde intermediates using a suitable alcohol dehydrogenase. FucO was expressed from an A SKA collection strain and purified as described abov e. Purified FucO was assayed in a buffer containing 100 mM Tris-HCl pH 7.5, 0.3 niM NADH, and 10 mM butyraldehyde. The reaction was monitored by loss of absorbance at 340 nm corresponding to the consumption of N ADH, as shown in FIG. 31. FucO was capable of catalyzing the reduction of butyraldehyde to butanol with a specific activity of 5.08 ± 0.08 μηιοΐ/min/mg protein. Amines and alky Is can also be produced in this scheme from aldehyde intermediates. These have been demonstrated in previous examples.

[00151] The above experiments are repeated in Bacillus subtilis. The same genes can be used, especially since Bacillus has no significant codon bias. A protease- deficient strain like WB800N i s preferably used for greater stability of heterologous protein . The E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability can be used to move the genes easi ly to a more suitable vector for Bacillus. Alternatively, two vectors pHTO 1 and pHT43 allow high-level expression of recombinant proteins within the cytoplasm. As yet another alternative, plasmids using the theta-mode of replication such as those derived from the natural plasmids ρΑΜβΙ and pBS72 can be used. Several other suitable expression systems are av ailable. Since the FAS and BOX-R enzymes are ubiquitous, the inv ention is predicted to function in Bacillus.

[00152] The abov e experiments are repeated in yeast. The same genes can be used, but it may be preferred to accommodate codon bias. Sev eral yeast E.

shuttle vectors are av ailable for ease of the experiments. Since the HACL enzymes are found in yeast, the inv ention is predicted to function in yeast.

[00153] The following references are incorporated by reference in their entirety herein for all purposes.

[00154] US Serial No. 61/440, 192, filed February 7, 20 1 1

[00155] US serial No. 61/531/91 1, filed Sept. 7, 20 1 1

[00156] PCT/US 12/2405 1 , filed February 7, 20 12

1001571 US20 1303 164 13, US20 140273 1 1 0.

1001581 PCT/US 1 5/58 1 2 1 , filed October 29, 20 1 5

[00159] Binstock, IF. & Schulz, H. ( 198 1 ) Fatty acid oxidation complex from

Escherichia coli . Methods in Enzymology, 7 1 Pt C ( 1967), 403-4 1 1 . 1001601 Blecher, M. (1981) Synthesis of long-chain fatty acyl-CoA thioesters using N-hydroxysuccinimide esters. Methods in Enzymology, 72( 1967), 404-408.

[00161] Cintolesi, A., et al . (2014) In silico assessment of the metabolic capabi lities of an engineered functional reversal of the β-oxidation cycle for the synthesis of longer-chain (C>4) products. Metabolic Engineering, 23, 100- 1 5.

[00162] Datsenko, K.A. & Wanner, B.L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K- 12 using PGR products. Proceedings of the National Academy of Sciences of the United States of America, 97( 12), 6640-5.

[00163] Hofmeister, A. & Buckel, W. (1992) (R)-Lactyl-CoA dehydratase from Clostridium propionicum. European J. Biochem., 552, 547-552.

[00164] Kitagawa, M., et al. (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Research : An International Journal for Rapid Publication of Reports on Genes and Genomes, 12(5 ), 29 1 -9. [00165] Neidhardt, F. C, Bloch, P. L., & Smith, D. F. (1974) Culture Medium for Enterobacteria. Journal Of Bacteriology, 1 19(3), 736-747. Retriev ed from pubmedcentral .nih.gov7articlerender.fcgi'^,artid^:::245675&tool ^:::pmcentrez&rendertype^:::abstra ct

[00166] Orth, J. D., Conrad, T. M., Na, J., Lerman, J. a. Nam, H., Feist, A. M., & Palsson, B. 0. (201 1) A comprehensive genome-scale reconstruction of Escherichia coli metabolism— 201 1. Molecular Systems Biology, 7(535),

[00167] Parasaran, T., & Tarbell, D. S. (1964) Formic Ethyl carbonic

Anhydride. The Journal of Organic Chemistry, 29(1 1), 3422-3423.

[00168] Schellenberger, J. et al . (201 1). Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat. Protoc. 6, 1290- 307.

[00169] Schuchmann, K., & Miiller, V. (20 1 3 ) Direct and rev ersible hydrogenation of C02 to formate by a bacterial carbon dioxide reductase. Science (New York, N.Y.), 342(6 164), 1382-5. [00170] Tatusova TA & Madden TL (1999) BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174:247-250

[00171] Tobimatsu, T., et al., (1997) Heterologous Expression, Purification, and Properties of Diol Dehydratase, an Adenosylcobalamin-Dependent Enzyme of Klebsiella oxytoca. Archives of Biochemistry and Biophysics, 347(1), 132-140.

[00172] Zhu, H , et al., (2011) Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli. Applied and Environmental Microbiology, 77( 18), 6441-50.

Claims

etically engineered microorganism, comprising:

overexpressed polypeptide(s) catalyzing a pathway for the conversion of a single-carbon substrate to formyl-coA, said pathway comprising i and ii and iii; or iv and v and vi; or vii; wherein i-vii are:

i. a methane monooxygenase catalyzing the conversion of methane to methanol

ii. a methanol dehydrogenase catalyzing the conversion of methanol to formaldehyde

iii. an acylating aldehyde dehydrogenase catalyzing the conversion of formaldehyde to formyl-CoA;

iv. a carbon dioxide reductase catalyzing the conversion of carbon dioxide to formate;

v. an acetate kinase or formate kinase catalyzing the conversion of

formate to formyl-phosphate;

vi. a phosphate acetyl-transferase catalyzing the conversion of formyl- phosphate to formyl-CoA;

vii. an acetyl-CoA synthetase catalyzing the conversion of formate to formyl-CoA;

overexpressed polypeptides catalyzing a pathway converting formyl-CoA and an C(n)-aldehyde to a C(n+l)-aldehyde, said pathway selected from i and ii and iii and iv; or i and v and vi and vii; wherein i-vii are:

i. a 2-hydroxyacyl-CoA lyase catalyzing the conversion of formyl-CoA and a C(n)-aldehyde to a 2-hydroxy-C(n+l)-acyl-CoA;

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

iii. a 1,2-diol oxidoreductase or alcohol dehydrogenase catalyzing the conversion of a2-hydroxy-C(n+l)-aldehyde to a l,2-C(n+l)-diol; iv. a diol dehydratase catalyzing the conversion of a l,2-C(n+l)-diol to a C(n+l)-aldehyde;

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

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

c. a termination pathway for converting an intermediate of a pathway in step b to a product selected from an alcohol, a carboxylic acid, an amine, or an alkyl, said pathway selected from:

i. an alcohol forming coenzyme- A thioester reductase catalyzing the conversion of an acyl-CoA to an alcohol;

ii. an alcohol dehydrogenase catalyzing the conversion of an aldehyde to an alcohol (or vice versa);

iii. a thioesterase catalyzing the conversion of an acyl-CoA to a carboxylic acid;

iv. an aldehyde dehydrogenase catalyzing the conversion of an aldehyde to a carboxylic acid;

v. an acyl-CoA reductase catalyzing the conversion of an acyl-CoA to an aldehyde;

vi. a transaminase catalyzing the conversion of an aldehyde to an amine; and

vii. an aldehyde decarbonylase catalyzing the conversion of an aldehyde to an alkyl;

wherein n refers to the number of carbons, and

wherein said microorganism converts single-carbon molecules to an alcohol, a carboxylic acid, an amine, or an alkyl containing at least two carbons.

The microorganism of claim 1, wherein said acylating aldehyde dehydrogenase is an amino acid sequence selected from E. coli mhpF ( P 414885.1), Pseudomonas sp. CF600 dmpF (Q52060), or homologs thereof.

The microorganism of claim 1, wherein said methanol dehydrogenase is an amino acid sequence selected from Bacillus methanolicus mdh (P31005), Mycobacterium sp. DSM 3803 mdo (C5MRT8), Methylobacterium extorquens moxl, moxF (P14775, PI 6027), or homologs thereof.

4. The microorganism of claim 1, wherein said methane monooxygenase is an amino acid sequence selected from Methylosinus trichosporium OB3b mmoXYZBC, or/Y (P27353, P27354, P27355, Q53563, P27356, Q53562), Methylococcus capsulatus Bath mmoXYZBC, orfl (P22869, PI 8798, PI 1987, PI 8797, 22868, P22867), or homologs thereof.

5. The microorganism of claim 1, wherein said acetyl-CoA synthetase is an amino acid sequence of Escherichia coli acs (NP 418493.1), or homologs thereof.

6. The microorganism of claim 1, wherein said formate kinase is an amino acid

sequence selected from Salmonella typhimurium stAckA (P6341 1), E. coli AckA (NP_416799.1), or homologs thereof.

7. The microorganism of claim 1, wherein said phosphate formyl-transferase is an

amino acid sequence selected from . coli eutD ( P 416953.1,), E. coli pta

( P 416800.1), Salmonella typhimurium PduL (Q9XDN5), or homologs thereof.

8. The microorganism of claim 1, wherein said carbon dioxide reductase is an amino acid sequence selected from Acetobacterium woodiifdhF2, hycB2, hycB3, hydA2 (YP_005268502.1, YP_005268503.1, YP_005268505.1, YP_005268506.1), or homologs thereof.

9. The microorganism of claim 1, wherein the 2-hydroxyacyl-CoA lyase is an amino acid sequence selected from Homo sapiens hacll (Q9UJ83), Rattus norvegicus hacll (Q8CHM7), Dictyostelium discoideum hacll (Q54DA9), Mus musculus hacll (Q9QXE0), or homologs thereof.

10. The microorganism of claim 1, wherein the 1,2-diol oxidoreductase or alcohol

dehydrogenase is an amino acid sequence selected from E. coli betA ( P 414845.1), E. coli dkgA ( P_417485.4), E. coli eutG ( P_416948.4), E. colifucO

( P_417279.2), E. coli ucpA ( P_416921.4), E. coli yahK (NP_4\4859 A), E. coli ybbO (NP_415026.1), E. coli ybdH NP_4\ 5132 A), E. coliyiaY (YP_026233.1), E. coliyjgB ( P 418690.4), or homologs thereof.

1 1. The microorganism of claim 1, wherein the diol dehydratase is an amino acid

sequence selected from Klebsiella ocytoca pddABC (Q59470, Q59471, Q59472), E. colipduCDE (CAS09680, CAS09681, CAS09682), S. enterica pduCDE

( P_456590, P_456591, NP_456592), or homologs thereof.

12. The microorganism of claim 1, wherein the 2-hydroxyacyl-CoA dehydratase is an amino acid sequence selected from Clostridium propionicum IcdABC (G3KTM3, G3KIM4, G3KIM5), Clostridium difficile hadBCI (AAY40M8 A, AAV40819.1, AAV40820.1), or homologs thereof.

13. The microorganism of claim 1, wherein the trans-2-enoyl-CoA reductase is an amino acid sequence selected from Rhodobacter sphaeroides acul (Q3 J6K9.1), E. coli yhdH (NP_417719.1), E. colifabl ( P_415804.1), Enterococcus faecalis fabK ( P 816503.1), Bacillus subtilis fabL (KFK80655.1), Euglena gracilis egTER (Q5EU90.1), Treponema denticola tdTER ( P 971211.1), Vibrio cholera fabV (B1P0R8), or homologs thereof.

14. The microorganism of claim 1, wherein the acyl-CoA reductase is an amino acid sequence selected from Acinetobacter calcoaceticus acrl (AAC45217.1),

Acinetobacter sp Strain M-l acrM (BAB85476.1), Clostridium beijerinckii aid (AAT66436.1), E. coli eutE ( P_416950.1), Salmonella enterica eutE

(AAA80209.1), E. coli mhpF ( P_414885.1), or homologs thereof.

15. The microbe of claim 1, wherein said overexpressed alcohol -forming coenzyme- A thioester reductase is an amino acid sequence selected from Clostridium

acetobutylicum adhE2 (YP 009076789.1), Arabidopsis thaliana At3gl 1980

(AEE75132.1), Arabidopsis thaliana At3g44560 (AEE77915.1), Arabidopsis thaliana At3g56700 (AEE79553.1), Arabidopsis thaliana At5g22500

(AED93034.1), Arabidopsis thaliana CER4 (AEE86278.1), Marinobacter aquaeolei VT8 maqu_2220 (YP_959486.1), Marinobacter aquaeolei VT8 maqu_2507

(YP 959769.1), or homologs thereof.

16. The microorganism of claim 1, wherein said overexpressed alcohol dehydrogenase is an amino acid sequence selected from E. coli betA ( P_414845.1), E. coli dkgA

( P_417485.4), E. coli eutG ( P_416948.4), E. colifucO ( P_417279.2), E. coli ucpA ( P_416921.4), E. coliyahK ( P_414859.1), E. coliybbO ( P_415026.1), E. coliybdH ( P_415132.1), £. coliyiaY (YP_026233.1), E. coliyjgB ( P_418690.4), or homologs thereof.

The microorganism of claim 1, wherein said overexpressed thioesterase is an amino acid sequence encoded by a gene selected from E. coli tesA ( P_415027.1), E. coli tesB ( P_414986.1), E. coliyciA ( P_415769.1), E. coli fadM (NP_414977.1), E. coli ydil (NP_416201.1), E. coliybgC ( P_415264.1), Alcanivorax borkumensis tesB2 (YP_692749.1), Fibrobacter succinogenes Fs2108 (YP_005822012.1), Prevotella ruminicola Pr655 (YP 003574018.1), Prevotella ruminicola Prl687 (YP_003574982.1), or homologs thereof.

The microorganism of claim 1, wherein said overexpressed aldehyde dehydrogenase is an amino acid sequence encoded by a gene selected from Rhodococcus ruber SCI cddD (AAL14238.1), Acinetobacter sp. SE19 chnE (AAG10022.1), E. coli adhA ( P_415933), E. colipaoABC ( P_414820), E. coli aldB ( P_418045), E. coli yqhD (NP_418045), Saccharomyces cerevisiae ALD2 ( P_013893), Saccharomyces cerevisiae ALD3 ( P_013892), Saccharomyces cerevisiae ALD6 ( P_015264), or homologs thereof.

17. The microorganism of claim 1, wherein said overexpressed transaminase is an amino acid sequence encoded by a gene selected from Arabidopsis thaliana At3g22200 ( P_001189947.1), Alcaligenes denitrificans AptA (AAP92672.1), Bordetella bronchiseptica BB0869 (WP_015041039.1), Bordetella parapertussis BPP0784 (WP 010927683.1), Brucella melitensis BAWG 0478 (EEW88370.1), Burkholderia pseudomallei BP1026B I0669 (AFI65333.1), Chromobacterium violaceum CV2025 (AAQ59697.1), Oceanicola granulosus OG2516_07293 (WP_007254984.1), Paracoccus denitrificans PD 1222 Pden_3984 (ABL72050.1), Pseudogulbenkiania ferrooxidans ω-ΤΑ (WP 008952788.1), Pseudomonas putida ω-ΤΑ (P28269.1), Ralstonia solanacearum ω-ΤΑ (YP 002258353.1), Rhizobium meliloti SMc01534 ( P 386510.1), and Vibrio fluvialis ω-ΤΑ (AEA39183.1), Mus musculus abaT (AAH58521.1), £. coli gabT (YP 490877.1), or homologs thereof.

18. The microbe of claim 1, wherein said aldehyde decarbonylase is an amino acid

sequence encoded by a gene selected from Synechococcus elongatus PCC7942 orfl593 (Q54764.1), Nostoc punctiforme PCC73102 npun_R1711 (B2J1M1.1), Prochlorococcus marinus MIT9313 pmtl231 (Q7V6D4.1), or homologs thereof.

19. A method for the production of alcohols, carboxylic acids, amines, or alkyls from single carbon molecules comprising:

a. isolating polypeptides from a microorganism of claims 1-20;

b. providing a reaction mixture comprising said isolated polypeptides and buffers, minerals, vitamins, and cof actors; c. contacting said reaction mixture with single-carbon molecules under conditions whereby said single-carbon molecules are converted into products containing at least one alcohol, carboxylic acid, amine, or alkyl group; and

d. isolating said product from said reaction mixture.

20. A method for the production of alcohols, carboxylic acids, amines, or alkyls from single-carbon molecules comprising:

a. providing the microorganism of claim 1-20;

b. growing said microorganism in a culture medium under conditions whereby the microorganism assimilates single-carbon molecules to produce a product containing at least one alcohol, carboxylic acid, amine, or alkyl group; and

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