Hybrid polyketide synthase biosynthesis of alcohols and derivatives thereof

Abstract

The present invention provides for a hybrid polyketide synthase (PKS) capable of synthesizing an alcohol and / or a derivative thereof.

Description

Hybrid polyketide synthase biosynthesis of alcohols and derivatives thereofInventors: Qingyun Dan, Namil Lee, Yan Chiu, Jay D. KeaslingCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63 / 645,806, filed May 10, 2024, which is incorporated by reference in its entirety.STATEMENT OF GOVERNMENTAL SUPPORT

[0002] The invention was made with government support under Contract Nos. DE-AC02- 05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention is in the field of the production of alcohol and its derivatives.BACKGROUND OF THE INVENTION

[0004] Diols are industrially important commodity and specialty chemicals widely used today 1. Over the last decades, a few natural and artificial pathways have been established in Escherichia coli and other industrial microbes targeting different short-chain diols 2-5. The development and industrialization of microorganisms to convert renewable feedstocks into 1,3-propanediol, 1,4-butanediol, and 1,3 -butanediol (1,3-BDO or butylene glycol, used as a humectant and cosmetic solvent) has been a major advance for metabolic engineering 6,7. Besides short-chain diols, medium-chain and branched-chain diols are also important solvents, polymer building blocks, fragrances and cosmetics ingredients 1,8. A prime example is 2-ethyl-l,3-hexanediol (2-E-l,3-HDO or etohexadiol), an insect repellent, boron extractant, ink solvent and ingredient in cosmetics used for over half a century 9. Besides its own market use, its derivative 2-ethylhexanol is a major building block for polyvinyl chloride plasticizers, is produced in millions of metric tons annually, and had a six billion USD market size in 2022 10. Despite their importance, metabolic pathways to produce medium- and branched-chain diols are rare and have not been commercialized. Also, each molecule generally requires a distinct biosynthetic pathway that will not produce other analogs, making it impossible to extend learnings from synthesis of one molecule to others. In fact, thisbiosynthetic challenge is widely present in almost all medium- and branched-chain chemical bioproduction (alcohols, amines, amino alcohols, to list a few), presenting a major knowledge gap in metabolic engineering.SUMMARY OF THE INVENTION

[0005] The present invention provides for a system comprising a hybrid polyketide synthase (PKS) capable of producing an alcohol and / or a derivative thereof.

[0006] The present invention provides for a system comprising a hybrid polyketide synthase (PKS) capable of producing a P-hydroxy aldehyde (BHA). In some embodiments, the system further comprises: (a) a transaminase (TA) capable of converting the BHA into an amino alcohol, (b) an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, and / or (c) aldehyde dehydrogenase (ALDH) capable of converting the BHA into a 3-hydroxy acid (3 HA).

[0007] The present invention provides for a genetically modified host cell comprising a hybrid polyketide synthase (PKS) capable of producing a P-hydroxy aldehyde (BHA). In some embodiments, the host cell further comprises: (a) a transaminase (TA) capable of converting the BHA into an amino alcohol, (b) an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, and / or (c) aldehyde dehydrogenase (ALDH) capable of converting the BHA into a 3-hydroxy acid (3HA). In some embodiments, the (a) transaminase (TA), (b) alcohol dehydrogenase (ADH), and / or (c) aldehyde dehydrogenase (ALDH) are each independently endogenous to the host cell, or stably introduced into the host cell.

[0008] The present invention provides for a genetically modified host cell comprising a nucleic acid encoding a hybrid polyketide synthase (PKS) capable of producing a P-hydroxy aldehyde (BHA) operatively linked to a promoter. In some embodiments, the host cell further comprises: (a) a nucleic acid encoding a transaminase (TA), capable of converting the BHA into an amino alcohol, operatively linked to a promoter; (b) a nucleic acid encoding an alcohol dehydrogenase (ADH), capable of converting the BHA into a diol, operatively linked to a promoter; and / or (c) a nucleic acid encoding aldehyde dehydrogenase (ALDH), capable of converting the BHA into a 3-hydroxy acid (3HA), operatively linked to a promoter. In some embodiments, the (a) transaminase (TA), (b) alcohol dehydrogenase (ADH), and / or (c) aldehyde dehydrogenase (ALDH) are each independently endogenous to the host cell, or on avector stably introduced into the host cell, such as stably integrated into a genome, or on a vector capable of stable maintenance in the host cell.

[0009] The present invention provides for a hybrid polyketide synthase (PKS) comprising a NADPH-dependent terminal thioreductases. The hybrid PKS is capable of producing a P- hydroxy aldehyde (BHA). In some embodiments, the BHA has a chemical structure as follows:some embodiments, the host cell comprises a transaminase (TA) capable of converting the BHA into an amino alcohol. In some embodiments, the amino alcohol has a chemical structure as follows:some embodiments, the host cell comprises an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, such as a 1,3-diol. In some embodiments, the 1 ,3-diol has a chemical structure as follows:. In some embodiments, the host cell comprises an aldehyde dehydrogenase(ALDH) capable of converting the BHA into a 3-hydroxy acid (3HA). In some embodiments, the 3HA has a chemical structure as follows:. In some embodiments, Ri andR.2, each independently, — H or — alkyl. In some embodiments, Ri and R2, each independently, — H, — CH3, or — (CH2)nCH3. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, any number within a range of any 2 preceding values. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10, any number within a range of any 2 preceding values. In some embodiments, Ri and R2, each independently, — H, — CH3, — CH2CH3, or — (CH2)2CH3. In some embodiments, the BHA, 1,3-diol, amino alcohol, or 3HA is a compound described inFigure 1. In some embodiments, the host cell produces a medium-chain alcohol having n = 2,3, 4, 5, 6, 7, or 8, or a range of any two preceding values.

[0010] In some embodiments, the alcohol is a diol or amino alcohol. In some embodiments, the alcohol is a straight-chain or branched-chain alcohol diols and amino alcohols. In some embodiments, the alcohol is a medium- or branched-chain diol or amino alcohol. In some embodiments, the medium-chain alcohol has 6, 7, 8, 9, 10, 11, or 12, or a range of any two preceding values, carbon atoms.

[0011] In some embodiments, the hybrid PKS comprises a module or domain that is heterologous with another module or domain of the PKS, or the host cell.

[0012] In some embodiments, the hybrid PKS comprises a loading module from rimocidin PKS.

[0013] The reduction of the terminal aldehyde of the polyketide produced by the hybrid PKS with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specific transaminases enables production of various amino alcohols.

[0014] In some embodiments, the hybrid PKS comprises replacement of a malonyl- coenzyme A (CoA)-specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl-CoA-specific Ats, which enables the production of branched-chain diols and amino alcohols.

[0015] Herein is described the production of nine 1 ,3-diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl-l,3-hexanediol), six amino alcohols, and two carboxylic acids using a PKS platform in Streptomyces albus. In some embodiments, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g / L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform

[0016] The present invention provides for a nucleic acid encoding a hybrid polyketide synthase (PKS) capable of synthesizing the BHA, of the present invention.

[0017] The present invention provides for a genetically modified host cell comprising the nucleic acid of the present invention capable of expressing the hybrid PKS of the present invention and producing the BHA.

[0018] The present invention provides for a method of producing an alcohol and / or a derivative thereof, said method comprising: (a) providing a genetically modified host cell comprising the nucleic acid of the present invention capable of expressing the hybrid PKS of the present invention, and (b) culturing or growing the genetically modified host cell such that the genetically modified host cell expresses the hybrid PKS and produces the alcohol and / or a derivative thereof.

[0019] In some embodiments, the alcohol is a diol, amino alcohol, or carboxylic acid. Diols, amino alcohols and carboxylic acids are important plastic monomers, plasticizer building blocks, solvents, cosmetics and pharmaceutical ingredients, and the like.

[0020] In some embodiments, the host cell is a Streptomyces species, such as Streptomyces albus. Herein is described a series of Streptomyces albus hosts comprising engineered PKSs for bioproduction of at least seventeen target molecules included in this invention.

[0021] Modular polyketide synthases (PKSs) are megasynthases that produce secondary metabolites with great structural diversity from simple coenzyme A (CoA) substrates in an assembly-line manner, and are utilized in the present invention for programmed access of precise chemistry control. PKS terminal thioreductases (TRs) are identified and engineered to terminate PKSs with various polyketide aldehydes which are key precursor molecules.

[0022] Host engineering strategy for diol, amino alcohol and carboxylic acid production. Streptomyces are one of the main PKS carriers in nature and have been genetically modified for enhanced production of polyketide-based secondary metabolites, thus this invention focuses on engineering Streptomyces for the production of diols, amino alcohols and carboxylic acids. Streptomyces albus (S. albus) JI 074 was chosen as the host microbe because it is well characterized and widely used for PKS genetic integration and heterologous expression. For gene integration, bacteriophage integrase-based method was chosen in couple with proper antibiotic selection for successful integration selection, with nearly 100% success rate. Four pairs of phase integrases and selection markers were selected for constructing integration plasmids: 1. PhiC31 integrase gene and spectinomycin resistance gene; 2. VWB integrase gene and apramycin resistance gene; 3. PhiBTl integrase gene and kanamycin resistance gene; 4. pSAM2 integrase gene and hygromycin resistance gene. Corresponding attP sites were also built in the four integration plasmids, which can be integrated onto natural attB sites in S. albus JI 074. In particular, we used a triparental conjugation method forgenetic integration: the integration plasmid was carried by Escherichia coli (E. coli) DH1O0 cell as the donor cell, E. coli ET12567 cell harboring pUB307 plasmid is the helper cell, and S. albus J1074 or any engineered S. albus is the acceptor cell. This combined host engineering strategy enable in maximum four rounds of genetic integration in S. albus JI 074. The base strain information is listed in Table 1.

[0023] Table 1. Base strain information.

[0024] Pathway engineering strategy for diol, amino alcohol and carboxylic acid production. To produce a wide array of diols, amino alcohols and acids, we designed a rimocidin PKS- based biosynthetic platform with four aspects of focus: (1) a versatile loading module which accepts multiple CoA starter units; (2) an extension module with exchangeable domains; (3) terminal TRs for polyketide aldehyde production; (4) post-PKS enzymes to diversify the terminal chemistry. The representative pathway scheme and product summary are shown in Figure 1.

[0025] (1) Rimocidin loading module: Rimocidin (Rim) PKS loading module (M0) accepts acetyl-CoA, propionyl-CoA or butyryl-CoA as its natural starter unit, and its product profile can be tuned by regulating its CoA substrate pools. This unique versatility of its loading module can be utilized for production of both odd-chain and even-chain molecules, as well as both short-chain and medium-chain products. Crotonyl-CoA reductases (CCRs) fromrimocidin pathway (Rim J) and FK-520 pathway (FkbS), which convert crotonyl-CoA to butyryl-CoA and ethylmalonyl-CoA, were optionally added to our designs when we need to prioritize butyryl-CoA initiation.

[0026] (2) Exchangeable extension module: Rimocidin module 1 (Rim Ml) contains a “ketosynthase-acyltransferase-ketoreductase-acyl carrier protein” (KS-AT-KR-ACP) architecture, and both its AT and KR can be exchanged with domains from other suitable PKS pathways to alter the chemical outcome. We successfully exchanged Rim Ml malonyl- CoA specific AT to a series of methylmalonyl-CoA loading ATs from Rim M7 or pimaricin (Pirn) M7 for 2-methyl branch installation. Ethyl branch modification was accomplished by AT exchange to ethylmalonyl-CoA specific ATs from Rim Ml 3, salinomycin (Sin) pathway Ml, M9, M14, FK-520 pathway M4, or fluvirucin (Flu) pathway Ml or M5. We also introduced reductive loop (RL) exchange, replaced Rim Ml KR with coelimycin (Cpk) pathway M2 dehydratase-ketoreductase (DH-KR), and showcased removal of 3 -OH for C2- C3 double bond generation.

[0027] (3) Terminal domains: TRs were not applied in PKS engineering before. For PKS termination, we selected eight TRs from natural sources for polyketide aldehyde production. These are Cpk PKS TR (TRI), B24891 TR (TR2), cyclizidine TR (TR3), iminimycin TR (TR4), streptazone TR (TR6), TR7 and TR8 from unknown pathways, and TR9 from venediol pathway. For carboxylic acid production, three thioesterases (TEs) were also selected for our build, which is erythromycin (DEBS) TE (TE1), pikromycin TE (TE2), and Rim TE (TE3).

[0028] (4) Post-PKS enzymes: To modify the polyketide aldehyde for alcohol or amine production, different post-PKS enzymes were incorporated into our engineering pipelines. Specific alcohol dehydrogenases (ADHs) were used for aldehyde to alcohol conversion, and specific transaminases (TAs) were required for producing amino alcohols with a terminal amine group. If needed, specific carboxylic acid reductases (CARs) were used for reducing carboxylic acid to form aldehyde, enabling the subsequent aldehyde to alcohol production. All protein sequence information are listed in Table 2.

[0029] Table 2. Enzyme information.

[0030] Medium-chain alcohols are valuable and biosynthetically challenging target molecules in metabolic engineering. Polyketide synthases (PKSs) have long been proposed as promising megasynthase platforms for alcohol bioproduction, via reductive termination of natural PKS terminal reductase (TR) domains. Rimocidin PKS and TRs (rimPKS-TRs) have been engineered for successful bioproduction of 1,3 -butanediol, 1,3 -pentanediol, and 1,3- hexanediol in Streptomyces albus JI 074. It is shown that the PKS product profile can be adjusted by regulating intracellular coenzyme A substrate pools, as both feeding of L-valine, the butyryl-CoA precursor, and overexpression of a crotonyl-CoA reductase, FkbS, resulted in 1,3 -hexanediol titer increase from 33 mg / L to 239 mg / L in shake flasks. 1,3 -hexanediol can serve as the precursor molecule of many industrially relevant alcohols, such as trans-2- hexenol and hexanol. Furthermore, rimPKS-TRs produce polyketide aldehyde products highly submissive to subsequent pathway design, as demonstrated by production of 4- aminobutan-2-ol, 1 -aminopentan-3 -ol, and l-aminohexan-3-ol via PKS transaminases. Herein presented is a terminal reductase-based PKS biosynthetic platform, which lays the foundation for expanding current PKS design space and biosynthesizing previously inaccessible alcohols and other aldehyde-derived molecules.

[0031] The present invention works by the rational design and integration of engineered PKS megasynthase genes into host microbes for fermentative bioproduction test and improvement. In some embodiments, the host cell is Streptomyces albus JI 074, based on its established genetic integration techniques and slow consumption rates of target molecules, such as 1,3- diols. It is predicted rimocidin PKS as a natural butyryl starter unit acceptor and annotated its gene cluster. Candidate PKS TR genes are identified and analyzed based on literature search and phylogeny analysis. With these bioinformatic knowledge, rimPKS-TRs was designed and demonstrated robust phiC31 / VWB / phiBTl / pSAM2 integrase-based plasmid integration intoStreptomyces albus JI 074. Escherichia coli DHIO-beta was the donor cell, and Escherichia coli ET12567 / pUB307 was the helper cell for triparental conjugation. In some embodiments, spectinomycin, apramycin, kanamycin, and / or hygromycin are used as the selection marker in different rounds of conjugation experiments. Successful integrations are validated by genome PCRs, and the engineered host strains were fermented for bioproduction of target molecules.

[0032] Despite successful engineering of modular type I PKSs for three decades, PKS termination was always practiced via incorporation of terminal thioesterase (TE) domains. Natural PKSs containing terminal TRs were reported and TRs were suggested to bear great potential in PKS engineering, but no previous study validated the use of TRs in an engineered PKS system. This invention is novel because, for the first time, it is confirmed that TRs can be incorporated in PKS engineering for production of alcohols and other aldehyde-derived products, amines for example. Furthermore, the rimocidin PKS is the first confirmed PKS that naturally loads butyryl starter units. It is demonstrated that PKS-TR can be a great biosynthetic platform for amine production, as expression of TR-cognate transaminases successfully produced corresponding amino alcohols in Streptomyces .

[0033] In some embodiments, the present invention is useful in the production of aldehyde derivatives that can be produced from PKS retro-biosynthesis, such as alcohols, alkane / alkene biofuels, carboxylic acids, amines, and the like. One example is trans-2- hexenol, a plant-based fragrance molecule without any known biosynthetic pathway.

[0034] The present invention comprises one or more of the following: (1) use of TRs or cognate transaminases in PKS engineering, (2) a natural butyryl starter unit-containing PKS engineering, and / or (3) alcohol or amine production by an engineered modular PKS. No previous literature has reported any of the foregoing features.

[0035] In some embodiments, the growing or culturing step of the method uses a media comprising a renewable carbon source, such as lignocellulosic biomass. In some embodiments, the alcohol or derivative thereof compound is produced using a one-pot pretreatment saccharification and fermentation process. In some embodiments, the nucleic acid encoding each enzyme described herein is codon optimized to the genetically modified host cell.

[0036] In some embodiments, the culturing or growing step (b) comprises the host cell growing by respiratory cell growth. In some embodiments, the culturing or growing step (b)takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process. In some embodiments, the culture comprises a biomass, such as a lignocellulosic biomass, or hydrolysate thereof. In some embodiments, the biomass is obtained from softwood feedstock (such as poplar), hardwood feedstock, grass feedstock, and / or agricultural feedstock, or mixture thereof.

[0037] In some embodiments, the culture or medium comprises a rich medium, such as LB (Lysogeny -Broth) or comprising one or more ingredients of LB, such as tryptone and / or yeast extract. In some embodiments, the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass. In some embodiments, the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose or galactose, or glycerol, or a mixture thereof. In some embodiments, the carbon source is fermentable. In some embodiments, the carbon source is non-fermentable. In some embodiments, the culture or medium comprises urea as a nitrogen source. In some embodiments, the culture or medium comprises an ionic liquid (IL).

[0038] In some embodiments, the invention comprises the use of a heterologous codon- optimized version of each nucleic acid encoding the described enzyme, which are optimized to the genetically modified host cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0040] Figure 1. Schematic representation of the rimocidin PKS-TR platform in Streptomyces . a, Using glucose (and L-valine) as the carbon source, engineered rimocidin PKS can load three CoA starter units and three extender units. Incorporation of a PKS TR led to production of diverse products via programmed post-PKS modification by alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) or transaminases (TAs). Dashed lines indicate multiple steps in the precursor pathways. TD: terminal domain; CoL: CoA ligase; ACP: acyl carrier protein; KS: ketosynthase; AT: acyltransferase; KR: ketoreductase; TR: thioreductase; ADH: alcohol dehydrogenase; ALDH: aldehyde dehydrogenase; CAR: carboxylic acid reductase; TA: transaminase, b, Summary of all products in this study. Molecules highlighted in red are industrially valuable, and underlinedproducts were not microbially synthesized before.

[0041] Figure 2. PKS TR phylogeny, catalysis, and crystal structures, a, Phylogenetic analysis of PKS TRs in purple, PKS KRs in grey, mammalian fatty acid synthases (mFASs) in green, NRPS R domains in cyan, and alcohol dehydrogenases (ADHs) in primary metabolism in orange, b, TR9-catalysed reaction scheme of a natural substrate mimic, octanoyl-ACP9. c-d, Characterization of TR9 substrate scope and cofactor preference, with NADPH in c and NADH in d. NAD(P)H consumption was assessed by monitoring UV absorbance at 340 nm. Black: TR9 + NAD(P)H + octanoyl-ACP9; Blue: TR9 + NAD(P)H + octanoyl-CoA; Brown: TR9 + NAD(P)H + octanal; Green: NAD(P)H + octanoyl-CoA; Orange: NAD(P)H + octanal. For TR catalysis, the preferred substrates are ACPs or CoAs, and the preferred cofactor is NADPH. e, CpkC TR (TRI) crystal structure bound with NADP+, with protein colored in white and cofactor colored in blue, f, Active site view of TRI, R1824 and R1834 coordinate NADP+ ribose 2’ -phosphate, Y1956 is the catalytic proton donor. The NADP+ omit map (mFo-DFc, 3.0 o level) is colored in green.

[0042] Figure 3. Rimocidin PKS- and TR-based 1 ,3-diol bioproduction, a, Scheme of RimPKS-TR for production of 1,3-BDO, 1,3-PDO, and 1,3-HDO. b, LC-MS extracted ion chromatograms (EICs) of 1 ,3 -diols produced by S. albus RimMOMl-TRl (QD27). Black: S. albus RimMO (QD1) control; Blue: QD27; Orange: QD27 spiked with standards; Brown: 1,3- diol standards, c, 1,3-diol [M + Na]+ m / z detection, see Supplementary Figure 12 (all “Supplementary Figures” are found in the APPENDIX “SUPPLEMENTARY INFORMATION’’ in the fding of U.S. Provisional Patent Application Ser. No. 63 / 645,806, filed May 10, 2024, which is incorporated by reference in its entirety.) for detailed information, d-f, Characterization of 1,3-HDO enzymatic oxidation. NADPH (e) or NADH (f) accumulation was assessed by monitoring UV absorbance at 340 nm. Black: 1,3-HDO + NAD(P)+; Blue: TRI + 1,3-HDO + NAD(P)+; Brown: TR2 + 1,3-HDO + NAD(P)+; Green: TR7 + 1,3-HDO + NAD(P)+; Orange: TR9 + 1,3-HDO + NAD(P)+; Purple: TADH2 + 1,3- HDO + NAD(P)+. No TR catalyzed such oxidation reactions. TADH2 serves as a positive control.

[0043] Figure 4. Improving 1,3-diol production in Streptomyces albus J1074 RimPKS-TR, with 1,3-BDO colored in purple, 1,3-PDO in green, and 1,3-HDO in red. a, RimMOMl-TR 3 d cultivation results for screening TRs in R5 medium, b, Addition of butyryl-CoA precursor, L-valine, increased 1,3-HDO production titer and ratio in RimM0Ml-TR2 (QD28). c,Overexpression of FkbS or Rim J crotonyl-CoA reductase (CCR) in QD28 and increasing glucose concentration in R5 led to 765 mg / L total 1 ,3 -diol production in shake flasks, d, CCR-catalyzed reactions to convert crotonyl-CoA to butyryl- / ethylmalonyl-CoA. e, Overexpression of an exogenous alcohol dehydrogenase (ADH) YahK in engineered S. albus RimM0Ml-TR2 + FkbS (QD76) led to QD80 with increased 1 ,3 -diol total titers of 1008 mg / L after 7 d cultivation in R5 + 2% glucose + 15 mM L-valine. All cultivation data are represented as mean value and error bars indicate standard deviation (s.d.) of at least three biological replicates.

[0044] Figure 5. Expanding RimPKS-TR biosynthetic platform for medium- and branched- chain diol and carboxylic acid production, a, Scheme of PKS-TR engineering via AT exchange and post-PKS modification, b, Bioproduction of 2-methyl-l,3-diols in S. albus RimM0Ml(Rim / PimM7 AT)-TRs, with 2M-1,3-BDO colored in yellow, 2M-1,3-PDO colored in blue, and 2M-1,3-HDO colored in green, c, Bioproduction of 2-ethyl-3- hydroxyhexanoic acid (orange) in engineered S. albus RimM0Ml(RimM13 AT)-TR2 / TEs. TE 1-3: DEBS TE, Pikromycin TE, and Rimocidin TE. d, Bioproduction of insect repellent 2E-1,3-HDO (cyan) in 5. albus RimM0Ml(RimM13 AT)-TR2 + FkbS + ADH + CAR. All data were collected after 7 d cultivation in R5 + 2% glucose + 15 mM L-valine, as mean value and error bars indicate standard 361 deviation (s.d.) of at least three biological replicates.

[0045] Figure 6. Utilization of PKS TR-cognate transaminases (TAs) for amino alcohol production, a, Scheme of PKS-TR engineering via PKS TR-cognate transamination. AA: amino acid, b, Bioproduction of l-amino-3 -alcohols in S. albus RimMOMl-TRs + TAs after 7 d cultivation in R5 + 2% glucose. 15 mM L-valine was also added in the QD98 and QD99 groups. In QD99, RimM7 AT exchange was applied for methyl-amino-alcohol production, c, Protein abundance of TRI (black) and TR2 (white), d, Protein abundance of TAI (black) and TA2 (white). All cultivation data are represented as mean value and error bars indicate standard deviation (s.d.) of at least three biological replicates.

[0049] Figure 10. Detection of 2-ethyl-3 -hydroxyhexanoic acid produced in S. albus. A, three PKS pathway designs for 2-ethyl-3 -hydroxyhexanoic acid production were tested in S. albus. AT: acyltransferase; DD: docking domain; KS: ketosynthase; B, EIC profiles of 2-ethyl-3- hydroxyhexanoic acid in QD28 control (black), QD40 (blue), QD42 (brown), QD51 (pink) after 3 d cultivation in R5 + 2% Glucose + 5 mM L-valine, and 2-ethyl-3 -hydroxyhexanoic acid standard (cyan).DETAILED DESCRIPTION OF THE INVENTION

[0050] Before the invention is described in detail, it is to be understood that, unless otherwise indicated, this invention is not limited to particular sequences, expression vectors, enzymes, host microorganisms, or processes, as such may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

[0051] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0052] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0053] As used in the specification and the appended claims, the singular forms "a," "an,"and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "cell" includes a single cell as well as a plurality of cells; and the like.

[0054] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

[0055] The terms "optional" or "optionally" as used herein mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instances where a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0056] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0057] The term “about” refers to a value including 10% more than the stated value and 10% less than the stated value.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0059] The terms “host cell” and "host microorganism" are used interchangeably herein to refer to a living biological cell, such as a microbe, that can be transformed via insertion of an expression vector. Thus, a host organism or cell as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell. As will be appreciated by one of ordinary skill in the art, a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus.

[0060] The term "heterologous" as used herein refers to a material, or nucleotide or amino acid sequence, that is found in or is linked to another material, or nucleotide or amino acid sequence, wherein the materials, or nucleotide or amino acid sequences, are foreign to each other (i.e., not found or linked together in nature).

[0061] The terms "expression vector" or "vector" refer to a compound and / or composition that transduces, transforms, or infects a host cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell, or in a manner not native to the cell. An "expression vector" contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the host cell, such as a virus, liposome, protein coating, or the like. The expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements. Further, the expression vector must be one that can be transferred into a host cell and replicated therein. Particular expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence. Such plasmids, as well as other expression vectors, are well known to those of ordinary skill in the art.

[0062] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non- ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may alsoinclude modified nucleotides that permit correct read-through by a polymerase. "Polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0063] The term "promoter," as used herein, refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans- acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and / or rate of transcription of a gene. For example, a promoter can be a cis- acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5' and 3' untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on / off, regulate, modulate, etc.} gene transcription. Promoters are located 5' to the transcribed gene, and as used herein, include the sequence 5' from the translation start codon (i.e., including the 5' untranslated region of the mRNA, typically comprising 100-200 bp). Most often the core promoter sequences lie within 1-2 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls. In the context of this application, a promoter is typically referred to by the name of the gene for which it naturally regulates expression. A promoter used in an expression construct of the invention is referred to by the name of the gene. Reference to a promoter by name includes a wildtype, native promoter as well as variants of the promoter that retain the ability to induce expression. Reference to a promoter by name is not restricted to a particular species, but also encompasses a promoter from a corresponding gene in other species.

[0064] A polynucleotide is "heterologous" to a host cell or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide coding sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).

[0065] The term "operatively linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0066] The nucleic acid constructs of the present invention comprise nucleic acid sequences encoding one or more of the subject enzymes. The nucleic acid of the subject enzymes are operably linked to promoters and optionally control sequences such that the subject enzymes are expressed in a host cell cultured under suitable conditions. The promoters and control sequences are specific for each host cell species. In some embodiments, expression vectors comprise the nucleic acid constructs. Methods for designing and making nucleic acid constructs and expression vectors are well known to those skilled in the art.

[0067] Sequences of nucleic acids encoding the subject enzymes are prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis or cloning. For direct chemical synthesis, formation of a polymer of nucleic acids typically involves sequential addition of 3'-blocked and 5'-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain on the3'-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like. Such methodology is known to those of ordinary skill in the art and is described in the pertinent texts and literature (e.g., in Matteuci et al. (1980) Let. Let. 521 :719; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637). In addition, the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired nucleic acid sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; e.g., U.S. Pat. No. 4,683,195).

[0068] Each nucleic acid sequence encoding the desired subject enzyme can be incorporated into an expression vector. Incorporation of the individual nucleic acid sequences may be accomplished through known methods that include, for example, the use of restriction enzymes (such as BamHI, EcoRI, Hhal, Xhol, Xmal, and so forth) to cleave specific sites in the expression vector, e.g., plasmid. The restriction enzyme produces single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. Annealing is performed using an appropriate enzyme, e.g., DNA ligase. As will be appreciated by those of ordinary skill in the art, both the expression vector and the desired nucleic acid sequence are often cleaved with the same restriction enzyme, thereby assuring that the ends of the expression vector and the ends of the nucleic acid sequence are complementary to each other. In addition, DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.

[0069] A series of individual nucleic acid sequences can also be combined by utilizing methods that are known to those having ordinary skill in the art (e.g., U.S. Pat. No. 4,683,195).

[0070] For example, each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences. When the PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and can act as primers for each other Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are "spliced" together. In this way, a series of individual nucleic acid sequences may be "spliced" together and subsequently transduced intoa host microorganism simultaneously. Thus, expression of each of the plurality of nucleic acid sequences is effected.

[0071] Individual nucleic acid sequences, or "spliced" nucleic acid sequences, are then incorporated into an expression vector. The invention is not limited with respect to the process by which the nucleic acid sequence is incorporated into the expression vector. Those of ordinary skill in the art are familiar with the necessary steps for incorporating a nucleic acid sequence into an expression vector. A typical expression vector contains the desired nucleic acid sequence preceded by one or more regulatory regions, along with a ribosome binding site, e.g., a nucleotide sequence that is 3-9 nucleotides in length and located 3-11 nucleotides upstream of the initiation codon in E. coli. See Shine et al. (1975) Nature 254:34 and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349, 1979, Plenum Publishing, N.Y.

[0072] Regulatory regions include, for example, those regions that contain a promoter and an operator. A promoter is operably linked to the desired nucleic acid sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase enzyme. An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein- binding domain where a repressor protein can bind. In the absence of a repressor protein, transcription initiates through the promoter. When present, the repressor protein specific to the protein-binding domain of the operator binds to the operator, thereby inhibiting transcription. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding repressor protein. An example includes lactose promoters (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the LacI repressor protein from binding to the operator). Another example is the tac promoter. (See deBoer et al. (1983) Proc. Natl. Acad. Sci. USA, 80:21-25.) As will be appreciated by those of ordinary skill in the art, these and other expression vectors may be used in the present invention, and the invention is not limited in this respect.

[0073] Although any suitable expression vector may be used to incorporate the desired sequences, readily available expression vectors include, without limitation: plasmids, such as pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRIOO, pCR4, pBAD24, pUC19; bacteriophages, such as Ml 3 phage and X phage. Of course, such expression vectors may only be suitable for particular host cells. One of ordinary skill in the art, however, can readilydetermine through routine experimentation whether any particular expression vector is suited for any given host cell. For example, the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector. In addition, reference may be made to the relevant texts and literature, which describe expression vectors and their suitability to any particular host cell.

[0074] The expression vectors of the invention must be introduced or transferred into the host cell. Such methods for transferring the expression vectors into host cells are well known to those of ordinary skill in the art. For example, one method for transforming E. coli with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate. Other salts, e.g., calcium phosphate, may also be used following a similar procedure. In addition, electroporation (i.e., the application of current to increase the permeability of cells to nucleic acid sequences) may be used to transfect the host microorganism. Also, microinjection of the nucleic acid sequencers) provides the ability to transfect host microorganisms. Other means, such as lipid complexes, liposomes, and dendrimers, may also be employed. Those of ordinary skill in the art can transfect a host cell with a desired sequence using these or other methods.

[0075] For identifying a transfected host cell, a variety of methods are available. For example, a culture of potentially transfected host cells may be separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of the desired nucleic acid sequence. In addition, when plasmids are used, an often-used practice involves the selection of cells based upon antimicrobial resistance that has been conferred by genes intentionally contained within the expression vector, such as the amp, gpt, neo, and hyg genes.

[0076] When the host cell is transformed with at least one expression vector. When only a single expression vector is used (without the addition of an intermediate), the vector will contain all of the nucleic acid sequences necessary.

[0077] Once the host cell has been transformed with the expression vector, the host cell is allowed to grow. For microbial hosts, this process entails culturing the cells in a suitable medium. It is important that the culture medium contain an excess carbon source, such as a sugar (e.g., glucose) when an intermediate is not introduced. In this way, cellular production of the alcohol or derivative thereof ensured. When added, any intermediate is present in anexcess amount in the culture medium.

[0078] Any means for extracting or separating the alcohol or derivative thereof from the host cell may be used. For example, the host cell may be harvested and subjected to hypotonic conditions, thereby lysing the cells. The lysate may then be centrifuged and the supernatant subjected to high performance liquid chromatography (HPLC) or gas chromatography (GC).Host cells

[0079] In some embodiments, the host cells are genetically modified in that heterologous nucleic acid have been introduced into the host cells, and as such the genetically modified host cells do not occur in nature. The suitable host cell is one capable of expressing a nucleic acid construct encoding one or more enzymes described herein. The gene(s) encoding the enzyme(s) may be heterologous to the host cell or the gene may be native to the host cell but is operatively linked to a heterologous promoter and one or more control regions which result in a higher expression of the gene in the host cell.

[0080] Each introduced enzyme can be native or heterologous to the host cell. Where the enzyme is native to the host cell, the host cell is genetically modified to modulate expression of the enzyme. This modification can involve the modification of the chromosomal gene encoding the enzyme in the host cell or a nucleic acid construct encoding the gene of the enzyme is introduced into the host cell. One of the effects of the modification is the expression of the enzyme is modulated in the host cell, such as the increased expression of the enzyme in the host cell as compared to the expression of the enzyme in an unmodified host cell.

[0081] The genetically modified host cell can be any bacterial cell capable of production of the alcohol or derivative thereof of the present invention in accordance with the methods of the invention.

[0082] In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the host cell is a bacterial cell selected from the Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella, Ralstonia, Rhizobia, or Vitreoscilla taxonomical class. Bacterial host cells suitable for the invention include, but are not limited to, Escherichia, Corynebacterium, Pseudomonas, Streptomyces, and Bacillus. In some embodiments, the Escherichia cell is anE. coli, E. albertii, E. fergusonii, E. hermanii, E. marmotae, or E. vulneris. In some embodiments, the Corynebacterium cell is Corynebacterium glutamicum, Corynebacterium kroppenstedtii, Corynebacterium alimapuense, Corynebacterium amycolatum, Corynebacterium diphtheriae, Corynebacterium efficiens, Corynebacterium jeikeium, Corynebacterium macginleyi, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium renale, Corynebacterium striatum, Corynebacterium ulcerans, Corynebacterium urealyticum, or Corynebacterium uropygiale. In some embodiments, the Pseudomonas cell is a P. putida, P. aeruginosa, P. chlororaphis, P. fluor escens, P. pertucinogena, P. stutzeri, P. syringae, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafluva, or P. plecoglossicida. In some embodiments, the Streptomyces cell is a S. coelicolor, S. lividans, S. venezuelae, S. ambofaciens, S. avermitilis, S. albus, or S. scabies. In some embodiments, the Bacillus cell is a B. subtilis, B. megaterium, B. licheniformis, B. anthracis, B. amyloliquefaciens, B. pumilus, B. brevis, B. aminovorans, or B. fusiformis. In some embodiments the bacterial cell is a Gram-positive bacterium, such as a Streptomyces species, such as any Streptomyces species or strain taught herein.

[0083] The genetically modified host cell can be any yeast capable of production of the alcohol or derivative thereof in accordance with the methods of the invention.

[0084] In some embodiments, the host cell is a yeast. Yeast host cells suitable for the invention include, but are not limited to, Yarrowia, Candida, Bebaromyces, Saccharomyces, Schizosaccharomyces and Pichia cells. In one embodiment, Saccharomyces cerevisae is the host cell. In one embodiment, the yeast host cell is a species of Candida, including but not limited to C. tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. panapsilosis and C. zeylenoides. In one embodiment, Candida tropicalis is the host cell.

[0085] In some embodiments, the yeast host cell is a non-oleaginous yeast. In some embodiments, the yeast host cell is a basidiomycete. In some embodiments, the yeast host cell is an oleaginous yeast. In some embodiments, the oleaginous yeast is a Rhodosporidium species. In some embodiments, the Rhodosporidium species is Rhodosporidium toruloides. In some embodiments, the Rhodosporidium toruloides is strain IFO 0880.

[0086] In some embodiments, the host cell comprises a nucleic acid encoding the one ormore enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell. In some embodiments, the encoding of the one or more enzymes to the nucleic acid is codon optimized to the host cell. In some embodiments, the nucleic acid is vector or replicon that can stably reside in the host cell. In some embodiments, the nucleic acid is stably integrated into one or more chromosomes of the host cell.

[0087] In some embodiments, the providing step (a) comprises introducing a nucleic acid encoding the one or more enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell into the host cell.

[0088] In some embodiments, the culturing or growing step (b) comprises the host cell growing by respiratory cell growth. In some embodiments, the culturing or growing step (b) takes place in a batch process or a fed-batch process, such as a high-gravity fed-batch process. In some embodiments, the culture or medium comprises hydrolysates derived or obtained from a biomass, such as a lignocellulosic biomass. In some embodiments, the culture or medium comprises one or more carbon sources, such as a sugar, such as glucose or galactose, or glycerol, or a mixture thereof. In some embodiments, the carbon source is fermentable. In some embodiments, the carbon source is non-fermentable.

[0089] The present invention provides for a method for constructing a genetically modified host cell of the present invention, comprising (a) introducing a nucleic acid encoding the one or more enzymes operatively linked to a promoter capable of expressing the one or more enzymes in the host cell into the host cell.

[0090] One can modify the expression of a gene encoding any of the enzymes taught herein by a variety of methods in accordance with the methods of the invention. Those skilled in the art would recognize that increasing gene copy number, ribosome binding site strength, promoter strength, and various transcriptional regulators can be employed to alter an enzyme expression level.Suitable biomass

[0091] The biomass can be obtained from one or more feedstock, such as softwood feedstock, hardwood feedstock, grass feedstock, and / or agricultural feedstock, or a mixture thereof.

[0092] Softwood feedstocks include, but are not limited to, Araucaria (e.g. A. cunninghamii, A. angustifolia, A. araucana); softwood Cedar (e.g. Juniperus virginiana, Thuja plicata, Thuja occidentalis, Chamaecyparis thyoides Callitropsis nootkatensis); Cypress (e.g. Chamaecyparis, Cupressus Taxodium, Cupressus arizonica, Taxodium distichum, Chamaecyparis obtusa, Chamaecyparis lawsoniana, Cupressus semperviren); Rocky Mountain Douglas fir; European Yew; Fir (e.g. Abies balsamea, Abies alba, Abies procera, Abies amabilis); Hemlock (e.g. Tsuga canadensis, Tsuga mertensiana, Tsuga heterophylla); Kauri; Kaya; Larch (e.g. Larix decidua, Larix kaempferi, Larix laricina, Larix occidentalis); Pine (e.g. Pinus nigra, Pinus banksiana, Pinus contorta, Pinus radiata, Pinus ponderosa, Pinus resinosa, Pinus sylvestris, Pinus strobus, Pinus monticola, Pinus lambertiana, Pinus taeda, Pinus palustris, Pinus rigida, Pinus echinata); Redwood; Rimu; Spruce (e.g. Picea abies, Picea mariana, Picea rubens, Picea sitchensis, Picea glauca); Sugi; and combinations / hybrids thereof.

[0093] For example, softwood feedstocks which may be used herein include cedar; fir; pine; spruce; and combinations thereof. The softwood feedstocks for the present invention may be selected from loblolly pine (Pinus taeda), radiata pine, jack pine, spruce (e.g., white, interior, black), Douglas fir, Pinus silvestris, Picea abies, and combinations / hybrids thereof. The softwood feedstocks for the present invention may be selected from pine (e.g. Pinus radiata, Pinus taeda); spruce; and combinations / hybrids thereof.

[0094] Hardwood feedstocks include, but are not limited to, Acacia; Afzelia; Synsepalum duloificum; Albizia ; Alder (e.g. Alnus glutinosa, Alnus rubra ); Applewood; Arbutus ; Ash (e.g. F. nigra, F. quadrangulata, F. excelsior, F. pennsylvanica lanceolata, F. latifolia, F. profunda, F. americana ); Aspen (e.g. P. grandidentata, P. tremula, P. tremuloides ); Australian Red Cedar ( Toona ciliata ); Ayna ( Distemonanthus benthamianus ); Balsa ( Ochroma pyramidale ); Basswood (e.g. T. americana, T. heterophylla ); Beech (e.g. F. sylvatica, F. grandifolia ); Birch; (e.g. Betula populifolia, B. nigra, B. papyrifera, B. lenta, B. alleghaniensis / B. lutea, B. pendula, B. pubescens ); Blackbean; Blackwood; Bocote; Boxelder; Boxwood; Brazilwood; Bubing a; Buckeye (e.g. Aesculus hippocastanum, Aesculus glabra, Aesculus flava / Aesculus octandra ); Butternut; Catalpa; Cherny (e.g. Prunus serotina, Prunus pennsylvanica, Prunus avium ); Crabwood; Chestnut; Coachwood;Cocobolo; Corkwood; Cottonwood (e.g. Populus balsamifera, Populus deltoides, Populus sargentii, Populus heterophylla ); Cucumbertree; Dogwood (e.g. Cornus florida, Cornusnuttallii ); Ebony (e.g. Diospyros kurzii, Diospyros melanida, Diospyros crassiflora ); Elm (e.g. Ulmus americana, Ulmus procera, Ulmus thomasii, Ulmus rubra, Ulmus glabra ); Eucalyptus ; Greenheart; Grenadilla; Gum (e.g. Nyssa sylvatica, Eucalyptus globulus, Liquidambar styraciflua, Nyssa aquatica ); Hickory (e.g. Carya alba, Carya glabra, Carya ovata, Carya laciniosa ); Hornbeam; Hophornbeam; Ipe; Iroko; Ironwood (e.g. Bangkirai, Carpinus caroliniana, Casuarina equisetifolia, Choricbangarpia subargentea, Copaifera spp., Eusideroxylon zwageri, Guajacum officinale, Guajacum sanctum, Hopea odorata, Ipe, Krugiodendronferreum, Lyonothamnus lyonii ( L. floribundus ), Mesua ferrea, Olea spp., Olneya tesota, Ostrya virginiana, Parrotia persica, Tabebuia serratifolia ); Jacaranda; Jotoba; Lacewood; Laurel; Limba; Lignum vitae; Locust (e.g. Robinia pseudacacia, Gleditsia triacanthos ); Mahogany; Maple (e.g. Acer saccharum, Acer nigrum, Acer negundo, Acer rubrum, Acer saccharinum, Acer pseudoplatanus ); Meranti; Mpingo; Oak (e.g. Quercus macrocarpa, Quercus alba, Quercus stellata, Quercus bicolor, Quercus virginiana, Quercus michauxii, Quercus prinus, Quercus muhlenbergii, Quercus chrysolepis, Quercus lyrata, Quercus robur, Quercus petraea, Quercus rubra, Quercus velutina, Quercus laurifolia, Quercus falcata, Quercus nigra, Quercus phellos, Quercus texana ); Obeche; Okoume; Oregon Myrtle; California Bay Laurel; Pear; Poplar (e.g. P. balsamifera, P. nigra , Hybrid Poplar ( Populus x canadensis )); Ramin; Red cedar; Rosewood; Sal; Sandalwood; Sassafras; Satinwood; Silky Oak; Silver Wattle; Snakewood; Sourwood; Spanish cedar; American sycamore; Teak; Walnut (e.g. Juglans nigra, Juglans regia); Willow (e.g. Salix nigra, Salix alba ); Yellow poplar ( Liriodendron tulipifera ); Bamboo; Palmwood; and combinations / hybrids thereof.

[0095] For example, hardwood feedstocks for the present invention may be selected from Acacia, Aspen, Beech, Eucalyptus , Maple, Birch, Gum, Oak, Poplar, and combinations / hybrids thereof. The hardwood feedstocks for the present invention may be selected from Populus spp. (e.g. Populus tremuloides ), Eucalyptus spp. (e.g. Eucalyptus globulus ), Acacia spp. (e.g. Acacia dealbata ), and combinations thereof.

[0096] Grass feedstocks include, but are not limited to, C4 or C3 grasses, e.g. Switchgrass, Indiangrass, Big Bluestem, Little Bluestem, Canada Wildrye, Virginia Wildrye, and Goldenrod wildflowers, etc, amongst other species known in the art.

[0097] Agricultural feedstocks include, but are not limited to, agricultural byproducts such as husks, stovers, foliage, and the like. Such agricultural byproducts can be derived from cropsfor human consumption, animal consumption, or other non-consumption purposes. Such crops can be corps such as com, wheat, rice, soybeans, hay, potatoes, cotton, or sugarcane.

[0098] The feedstock can arise from the harvesting of crops from the following practices: intercropping, mixed intercropping, row cropping, relay cropping, and the like.

[0099] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

[0100] It is to be understood that, while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0101] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0102] The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.EXAMPLE 1A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxyacids

[0103] Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here, we present a novel approach utilizing a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and NADPH-dependent terminal thioreductases (TRs), previously untapped in engineered PKSs. Reduction of the terminal aldehyde with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specifictransaminases enables production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A (CoA)-specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl-CoA-specific ATs enables production of branched-chain diols and amino alcohols. In total, we demonstrated production of nine 1 ,3 -diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl- 1,3 -hexanediol), six amino alcohols, and two carboxylic acids using our PKS platform in Streptomyces albus. Finally, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g / L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform.

[0104] We aimed to design a biosynthetic platform suitable for production of 1,3- BDO, 2-E-l,3-HDO, or nearly any medium- or branched-chain alcohol and derivative, with a key feature of programmable access to precisely altered chemistry.

[0105] Modular type I polyketide synthases (PKSs) are megasynthases that utilize coenzyme A (CoA) substrates to produce complex natural products, many of which are used as pharmaceuticals 11. The chemical structure of PKS products is strictly determined by the order of each enzymatic domain and module. Because the structure of the molecule it produces is encoded in the DNA sequence, scientists have long dreamed of designing PKSs to produce nearly any organic molecule 12,13. And recently, PKSs have been redesigned to incorporate unnatural functionalities, such as fluorination 14,15 or to produce new-to-nature molecules, such as short-chain ketones 16 and triketide lactones 17,18. Thus, we hoped to investigate if PKSs can be used as a platform to biosynthesize many different diols and related molecules that have not been biosynthesized or molecules that require distinct biosynthetic pathways. For PKS production of 1,3-BDO and 2-E-l,3-HDO, two prerequisites are an initiation module that selects for acetyl- and butyryl- starter units and an extension module with ketosynthase-acyltransferase-ketoreductase-acyl carrier protein (KS-AT-KR- ACP) architecture to install an OH on the third carbon (Fig. 1, panel a). Finally, we must terminate polyketide biosynthesis with an alcohol if we want to produce a diol with an alcohol on the first carbon (or with an amine if we want to produce an amino alcohol with an amine on the first carbon); unfortunately, most natural PKSs terminate polyketide biosynthesis with thioesterases (TEs), which either directly hydrolyze the ACP intermediates to produce carboxylic acids or catalyze macrocyclization to give lactones or lactams 19. Indeed, the monotony of TE-mediated termination is a major hurdle for expanding currentPKS design space.

[0106] One appealing alternative to overcome this challenge is the use of terminal thioreductases (TRs)13. PKS chain release catalyzed by TRs was revealed in the recent decade, with only a handful of examples 20-26, including the coelimycin and cyclizidine PKSs. They are often coupled with cognate transaminases (TAs) in the biosynthetic gene clusters (BGCs): the TR catalyzes reductive cleavage of the acyl substrate to produce an aldehyde, which is subsequently converted to amine by the TA 20. Apart from the few studies to understand natural PKS TRs, they have not been used in engineered PKSs. Here, we biochemically and structurally characterized PKS TRs and confirmed that TRs catalyze two electron reductive cleavage and produce aldehydes. Based on the fact that aldehydes are highly reactive intracellularly, we designed an engineered PKS-TR-based biosynthetic platform for bioproduction of a wide range of aldehyde derivatives, illustrated by production of nine 1 ,3-diols including 1,3-BDO and 2-E-l,3-HDO, six amino alcohols, and at least two 3-hydroxy acids, fourteen of which have not been previously biosynthesized (Fig. 1, panel b). This platform utilizes PKS substrate promiscuity and assembly-line architecture to provide designable carbon skeletons, and TR catalysis and programmed post-PKS modification to diversify Cl chemistry, with potential to produce a wide range of bioproducts including diols, alcohols, amines, and hydrocarbon biofuels.RESULTSPKS TRs terminate polyketides with an aldehyde group.

[0107] Comprehensive PKS TR engineering was not previously reported, thus we first built a PKS TR library based on phylogeny analysis (Fig. 2, panel a, and Supplementary Figure 1) and selected nine TRs to study (TR1-9). The selected TRs uniformly covered the phylogenetic tree, and seven of them had validated pathway products. Among them, the TR from the coelimycin PKS BGC (TRI or CpkC TR) was reported to be an NADH-dependent reductase 20, capable of reducing octanoyl-CoA, a natural substrate mimic (Figure 8), to octanol in two steps (Fig. 2, panel b). Our phylogenetic analysis suggested a close evolutionary relationship between PKS TRs and NADPH-dependent PKS ketoreductases (KRs) 27 and a distant relationship with NADH-dependent alcohol dehydrogenases 28 (ADHs), disagreeing with this NADH-dependent literature report (Fig. 2, panel a). To gain further insight into TR catalysis, we overexpressed TRI, TR2, TR7, and TR9 in A. coliBL21(DE3) and purified these TRs to > 95% homogeneity (Supplementary Figure 3a-3d). We also purified the TRI -cognate ACPI with a maltose binding protein (MBP) tag fused at the N-terminus of ACPI to improve protein solubility (Supplementary Figure 3e). We then loaded octanoyl-CoA onto MBP-ACP1 in vitro with Sfp, a promiscuous phosphopantetheinyl transferase 29. After removal of excess octanoyl-CoA via dialysis, octanoyl-ACPl was tested as a TRI substrate together with octanoy 1 -Co A and octanal. TRI did not reduce octanoy 1- CoA or octanal when NADH was used as cofactor (Supplementary Figure 4b) but it did reduce octanoyl-ACPl. With NADPH, TRI reduced octanoyl-ACPl and octanoyl-CoA at a much faster rate than octanal (Supplementary Figure 4a). These results indicate that TRI is an NADPH-preferred reductase. To conclusively address substrate scope and cofactor preference of PKS TRs, we next purified ACP9 without the MBP tag and tested reactivity with TR9 (Supplementary Figure 3f). TR9 is from the venediol BGC, with 63% sequence identity to TR125. TR9 only used NADPH as cofactor, and readily reacted with the natural substrate analog octanoyl-ACP9 or octanoyl-CoA but would not react with NADH or octanal (Fig. 2 panels c-d and Supplementary Figure 4c). From these data, we concluded that TRs are NADPH-dependent termination enzymes that produce aldehydes and are incapable of further reducing aldehydes to alcohols.

[0108] To understand the structural basis for TR cofactor recognition, we determined the crystal structure of CpkC TR (TRI) bound with NADP+ to 1.8 A resolution (Fig. 2, panel e). The TRI structure consists of the N-terminal nucleotide binding domain and the C- terminal substrate binding domain, featuring an invariant “TGXXGXXG” (SEQ ID NO:45) motif, and two conserved arginines at 1824 and 1834 coordinating the ribose 2’-phosphate in NADP+, providing structural explanation for NADP+ preference 28,30 (Fig. 2, panel f, and Figure 7). Overall, these structural features align well with published tyrosine-dependent “extended” short-chain reductase / dehydrogenase (SDR) structures 31,32. We performed structural homology search with Dali server 33 and found the closest structural homologs to be nonribosomal peptide synthetase MxaA terminal reductase (NRPS R) domain31 with root mean squared deviation (r.m.s.d.) of 1.14 A, and carboxylic acid reductase (SrCAR) R domain34 with r.m.s.d. of 1.97 A. The major structural differences between PKS TR, NRPS R and CAR R lie in the post- / >5 loop and al 0-al 1 “helix-turn-helix” (HTH) motif (Supplementary Figure 6), which are the putative ACP or peptidyl carrier protein (PCP) binding sites. The HTH motif is absent in other common types of SDRs, supporting the importance of HTH for ACP / PCP recognition 35. Furthermore, the limited structuralsimilarity between PKS TR, NRPS R and CAR R HTH regions suggest distinct substrate binding patterns for these three types of reductases in secondary metabolism.Engineering rimocidin PKS and TRs for 1 ,3 -diol production

[0109] With a functional TR in hand and an understanding of its catalytic potential, we next sought to use it to produce aldehyde-derived alcohols, including 2-E-l,3-HDO. We used retrobiosynthesis software, ClusterCAD RetroTide, which we developed previously to design PKSs for specific products36. We designed PKS domain architectures for 2-E-l,3- HDO production, which contains a butyryl-CoA-specific loading module, an ethylmalonyl- CoA-specific extension module with KS-AT-KR-ACP architecture, and a TR termination domain. Based on that information, we searched for suitable PKS candidates and narrowed our candidates to the rimocidin (Rim) PKS. Rimocidin-like natural products have been isolated from several Streptomyces species 37,38. Depending on which substrates are loaded onto the PKS, the products are CE-108 and rimocidin in Streptomyces diastaticus var. 108 and BUI 6 and rimocidin in Streptomyces mauvecolor strain BUI 6. Only partial BGC information was available, thus we analyzed the Rim BGCs and validated their PKS architecture with antiSMASH39 (Supplementary Figure 7). Since the Rim PKS loading module RimMO has a novel CoA ligase loading domain architecture, (CoL)-ACPl-KS-AT- ACP2, we investigated its loading mechanism as the first step. However, we failed to obtain soluble RimMO when we expressed it in E. coli. Instead, we turned our attention to a RimMO homolog protein in the natamycin / pimaricin (Pirn) PKS pathway, PimSO, which has the same domain architecture 40 (Supplementary Figure 8a). We expressed the gene encoding PimSO in E. coli, purified it, and confirmed this novel PKS initiation mode with an acetyl- starter unit (Supplementary Figures 8b-8c and 9). Despite the same initiation module architecture as Rim PKS, Pirn PKS is unable to load the butyryl- start unit required for 2-E-l,3-HDO production. Thus, we tested whether the Rim PKS can be fused with TRs and produce 1,3- diols in a microbial host (Fig. 3, panel a).

[0110] Using bacteriophage integrases 41, we integrated the native rimMO gene driven by a constitutive PrpsL(RO) promoter into Streptomyces albus J 1074 and Streptomyces coelicolor Ml 152, two common heterologous hosts for PKS engineering. RimMO protein abundance in these Streptomyces hosts was assessed by proteomics analysis, which showed that RimMO was present at 1.2 ± 0.3% relative protein abundance in S. albus and absent in S. coelicolor (Supplementary Figure 10a). We also screened rimMO expressionunder two other constitutive promoters, Pgapdh(EL) and kasOP*42,43. PgapdhfEL) -driven rimMO expression peaked the screening test, with 8.3 ± 0.9% protein abundance, while kasOP* -driven rimMO expression was not detected. With S. albus RimMO (QD1) in hand, we examined potential target molecule consumption by our Streptomyces hosts, a phenomenon that we have observed when engineering other hosts to produce new-to-nature molecules 44,45. Neither of the two target molecules, 1,3-pentanediol (1,3-PDO) and 1,3 -hexanediol (1,3-HDO), were catabolized by our Streptomyces hosts when added to cultures (Supplementary Figure 11). This drove us to build S. albus RimMOMl-TRl (QD27) and look for 1 ,3 -diol production. For the chimeric RimMl-TRl design, we identified the N-terminal boundary of TRI based on the crystal structure, fused the N-terminus of TRI with the C- terminus of RimMl ACP, and integrated the chimeric Pgapdh(EL)-rimMl-TRl into QD1, making QD27. LC-MS analysis was used for detection of 1 ,3 -diol products. We successfully detected production of 6.7 mg / L 1,3-BDO, 12.3 mg / L 1,3-PDO, and 0.76 mg / L 1,3-HDO in TSB medium after 72 hours, demonstrating the feasibility of PKS-TR engineering for diol bioproduction for the first time (Fig. 3, panels b-c, Supplementary Figure 12). In comparison, we also constructed S. albus RimMOMl-DEBS TE (QD18) and looked for 3-hydroxy acid production, but with no success (Supplementary Figure 13a). 3 -Hydroxypentanoic acid and 3-hydroxyhexanoic acid, two putative QD18 products, were quickly consumed by S. albus J1074 after direct feeding, providing a possible explanation (Supplementary Figure 13b-13c). This presents a vivid example of PKS-TR advantage over traditional PKS-TE engineering, as polyketide-based carboxylic acids may be prone to P-oxidation, resulting in loss of desired product and ultimately design failure. Apart from providing alternative Cl chemistry as more reactive aldehyde, PKS-TR may also protect the designed carbon skeleton from host consumption and degradation.

[0111] Because TR is incapable of aldehyde reduction, the aldehyde to 1 ,3 -diol conversion that occurred in our engineered S. albus QD27 was likely catalyzed by unknown ADHs in the host. To confirm this hypothesis, we tested whether four purified TRs can oxidize the 1 ,3 -diols that QD27 produced to 3-hydroxy aldehydes, as ADH-catalyzed reactions between alcohols and aldehydes are usually reversible (Fig. 3, panel d). For example, promiscuous tomato alcohol dehydrogenase 2 (TADH2) readily catalyzed NAD+- dependent oxidation on 1,3-BDO, 1,3-PDO, or 1,3-HDO46 (Fig. 3, panels e-f, and Supplementary Figures 14-15). In comparison, none of the four TRs accepted these 1 ,3-diols as the substrate, suggesting that native S. albus ADHs catalyze the final reduction step in 1,3-diol biosynthesis without the need to incorporate exogenous ADHs (Fig. 3, panels e-f).Exploring engineering strategies of a tunable PKS-TR platform

[0112] Our next goal was to increase the titers of all 1 ,3 -diols and also ratio of 1,3- HDO to other diols. We first screened for a series of common Streptomyces growth media including TSB, R5, M042, ISP2, and ISP4 (Figure 9). When grown in R5 medium, QD27 produced the highest titers of 1 ,3-diols in 72 hours (38.6 mg / L). Based on this result, for the following experiments, we chose R5 as the standard medium, which includes 1% glucose. Next, we tested eight RimMl-TR2 / 3 / 4 / 6 / 7 / 8 / 9 chimaeras and confirmed 1 ,3 -diol production in all designs except for RimMl-TR4 and RimMl-TR7. From the six successful designs, RimM0Ml-TR2 (QD28) was the best 1 ,3 -diol producer (Fig. 4, panel a). Finally, prolonged 10-day cultivation of QD28 led to an increase of 1,3 -diol titers to 264 mg / L, with 135.4 mg / L1.3-BDO, 99.7 mg / L 1,3-PDO, and 29.1 mg / L 1,3-HDO produced in shake flasks (Fig. 4, panel b). Interestingly, although Pgapdh(EL)- wQn rimMO expression was higher than PrpsL(RO)-rimMO expression in engineered S. albus, 1 ,3 -diol production in the latter strain was optimal (Supplementary Figure 10b).

[0113] Because 1,3-HDO was still a minor product in QD28, we hoped to further improve its titer and product ratio. Substrate-promiscuous PKSs have been proposed as biosynthetic platforms elegantly tuned by their CoA substrate regulation, with a few successful engineering reports 16. In S. albus, addition of L-valine increased intracellular butyryl-CoA and ethylmalonyl-CoA levels47, both of which are putative building blocks for1.3-HDO and 2-E-l,3-HDO biosynthesis. To validate the hypothesis that L-valine was converted to 1,3-HDO involving engineered RimPKS-TR catalysis, we fed in [13C]labelled L-valine and observed [13C]labelled 1,3-HDO as the final product (Supplementary Figure 17). We then added 15 mM L-valine in R5 medium and detected a slight increase of overall1.3-diol titers from 264.2 mg / L to 287.2 mg / L, while featuring significant improvement of both 1,3-HDO titer (from 29.1 mg / L to 94.5 mg / L) and ratio (from 11% to 33%, Fig. 4, panel b).

[0114] Next, we tried to further increase butyryl-CoA and ethylmalonyl-CoA substrate pools by enzymatically modifying their biosynthetic pathways. Ethylmalonyl-CoA is the third most used extender unit in PKS biosynthesis48, and several BGCs that extend with ethylmalonyl-CoA have annotated crotonyl-CoA carboxylase / reductase (CCR) genes inthem, presumably for enhancing the CoA substrate supply 49,50 (Fig. 4, panel d). We selected two candidates, rzmJCCR in the rimocidin BGC37 and JkbS CCR in the FK520 BGC51. Both CCR genes were integrated into S. albus QD28, and the resulting strains showed improved 1,3-HDO titer of 238.9 mg / L (QD76 with JkbS integration) and 210.4 mg / L (QD77 with rimJ integration) after 7-day cultivation. Remarkably, QD76 completely reversed the product ratio and produced 309.2 mg / L 1 ,3-diols with 77% being 1,3-HDO, demonstrating robustness of tuning PKS product profiles by adjusting CoA substrate pools. Moreover, increasing glucose concentration in R5 from 1% to 2% led to total production of 517.3 mg / L 1,3-diols, and R5 + 4% glucose produced 765.1 mg / L 1,3-diols in shake flasks (Fig. 4, panel c). Finally, we integrated five exogenous ADH genes (yqhD, chnD, yahK, yjgB and ScADH 2)4, 52, 53 into QD76, creating strains QD78-82, and tested whether overexpression of these ADHs can improve 1 ,3-diol production. Among them, theyahK- integrated strain QD80 and the jyg / / -integrated strain QD81 improved production, with QD80 being the best producer and achieving 1008.5 mg / L 1 ,3 -diol titer after 7-day cultivation in R5 + 2% glucose + 15 mM L-valine, of which 77% was 1,3-HDO (Fig. 4, panel e). These results convincingly demonstrate the efficiency and tunability of our PKS-TR platform.Bioproduction of medium-chain and branched-chain diols

[0115] Branched-chain diol biosynthesis remains a major challenge to this date due to the relatively narrow scope of available pathways for biosynthesis. Amino acids with branched side-chains such as L-valine and L-isoleucine can serve as precursors for branched- chain diol biosynthesis through CoA-independent biosynthesis routes4, but the lack of available enzymes to modify amino acid side-chain length and chemical diversity hinders their further application. We aimed to use our PKS-TR platform via AT domain exchange to produce a series of branched-chain diols (Fig. 5, panel a). Among our targets, 2-methyl-l,3- butanediol (2-M-l,3-BDO) has been produced in E. coll at low titer (12.1 mg / L), whereas 2- methyl-l,3-pentanediol (2-M-l,3-PDO), 2-methyl- 1,3 -hexanediol (2-M-l,3-HDO), and three 2-ethyl-l,3-diols were previously biosynthetically inaccessible4. For methyl-branched diol production, we replaced the malonyl-CoA-specific AT in the PKS extension module with a methylmalonyl-CoA-specific AT from module 7 of the rimocidin BGC (RimM7 AT) constructing S. albus RimM0Ml(RimM7 AT)-TR2 (QD66) and S. albus RimM0Ml(RimM7 AT)-TR2 + FkbS (QD83). We selected 7 days in R5 + 2% glucose + 15 mM L-valine as our standard cultivation method, under which QD66 produced 425.3 mg / L 2-M-l,3-BDO, 56.2mg / L 2-M-l,3-PDO, and 40.0 mg / L 2-M-l,3-HDO (Fig. 5, panel b). Furthermore, no unbranched-chain diol was detected, highlighting a precise PKS engineering strategy (Supplementary Figures 18-20). Next, TR screens showed that TR7 was the best TR accepting methyl-branched substrates, resulting in production of 743.6 mg / L 2-methyl-l,3- diols (Fig. 5, panel b). We also tested a RimM7 AT homolog in Pirn PKS, PimM7 AT, and achieved a similar 2-methyl-l,3-diol titer of 572.8 mg / L in RimM0Ml(PimM7 AT)-TR7 (QD69). In QD69, FkbS CCR overexpression created QD85 strain with increased both 2-M- 1,3-HDO titer (from 18.6 mg / L to 79.2 mg / L) and ratio (from 3% to 31%). The relatively low ratio of 2-M-l,3-HDO in the product profile is likely due to accumulation of its carboxylic acid derivative, 2-methyl-3 -hydroxy hexanoic acid, as FkbS overexpression also led to 8.6- fold increase of 2-methyl-3 -hydroxyhexanoic acid production (Supplementary Figure 21).

[0116] We next moved on to construct a microbe for 2-E-l,3-HDO production by replacing the malonyl-CoA-specific AT with an ethylmalonyl-CoA-specific AT. RimM13, the last module of Rim PKS, has the same domain composition as RimMl, except RimM13 AT loads ethylmalonyl-CoA based on pathway analysis. This drove us to test a series of RimPKS-TR chimeric designs, including exchanging only RimMl AT with RimM13 AT (QD40), RimMl AT-KR-ACP with RimM13 AT-KR-ACP (QD51), or the entire RimMl with RimM13 (QD42) (Figure 10). Among these designs, none produced 2-E-l,3-HDO; only S. albus RimM0Ml(RimM13 AT)-TR2 (QD40) produced 2.6 mg / L 2-ethyl-3- hydroxyhexanoic acid (Fig. 5, panel c). To validate whether 2-ethyl-3 -hydroxyhexanoic acid was produced via enzymatic condensation of the butyryl starter unit and ethylmalonyl extender unit, we performed [13C]labelled L-valine feeding, and observed both half- and fully-[ 13C]labelled 2-ethyl-3 -hydroxyhexanoic acid, confirming its biosynthetic route (Supplementary Figure 23). We also tested three other TEs as the termination domains; compared to DEBS / Pik / Rim TE, TR2-containing QD40 remained the best 3 -hydroxy acid producer. FkbS CCR overexpression in QD40 led to QD86 with an increased 2-ethyl-3- hydroxyhexanoic acid titer at 3.9 mg / L, albeit still with no 2-E-l,3-HDO production.

[0117] We reasoned that endogenous ADHs in S. albus were not capable of reducing ethyl-branched aldehydes to diols, in agreement with our observation that overexpression of MaCAR carboxylic acid reductase in QD40 did not produce 2-E-l,3-HDO. To enzymatically convert 2-ethyl-3 -hydroxyhexanoic acid to 2-E-l,3-HDO, we then tested three CARs (MaCAR / SrCAR / MmCAR) and two ADHs (YahK / YjgB) in QD86, and all the resultingQD88-QD93 microbes produced 2-E-l,3-HDO, with SrCAR + YahK combination (QD89) producing the highest 2-E-l,3-HDO titer (3.0 mg / L, Fig. 5, panel d, and Supplementary Figure 24). Moreover, LC-MS peaks matching 2-E-l,3-BDO and 2-E-l,3-PDO theoretical [M + Na]+ m / z were observed (Supplementary Figure 25), together with 2-E-l,3-HDO production demonstrating successful engineering of 2-ethyl- extender units into our diol biosynthetic pipelines.Bioproduction of amino alcohols via post-PKS transamination

[0118] Amino alcohols are also important specialty chemicals in the polymer and pharmaceutical industries, and they can be synthesized using the PKS platform we constructed to synthesize diols. Moreover, most PKS pathways in nature have dedicated post- PKS decoration enzymes with extraordinary diversity and peculiarity, including TR-cognate transaminases, yet their application in PKS engineering has not been reported20,54. To explore this possibility (Fig. 6, panel a), we synthesized TAI and TA2 genes from Cpk and B24891 BGCs, and integrated them into S. albus RimMOMl-TRl (QD27) or RimMOMl- TR2 (QD28). The four resulting strains (QD94-QD97) successfully produced 4-aminobutan- 2-ol, 1 -aminopentan-3 -ol, and l-aminohexan-3-ol, confirming the activity of TAs (Fig. 6, panel b, and Supplementary Figures 26-27). Despite the fact that post-PKS TAs are often TR- cognate in natural PKSs, our choice of TAs was not strictly restricted by the TRs selected for PKS termination, because QD96 with TR2 + TAI combination was the best amino alcohol producer in R5 + 2% glucose (425.1 mg / L 4-aminobutan-2-ol, 112.1 mg / L 1 -aminopentan-3 - ol, and 17.1 mg / L l-aminohexan-3-ol, 554.2 mg / L in total). Moreover, we successfully tuned the amino alcohol product profile by the same CoA substrate regulation strategies: 15 mM L- valine supplemented to QD98 (FkbS overexpression in QD96) cultures led to dramatically increased l-aminohexan-3-ol titer (from 17.1 mg / L to 364.4 mg / L) and product ratio (from 3% to 70%). The AT exchange was also compatible with PKS-TR-TA-based branched-chain amino alcohol production, as S. albus RimM0Ml(RimM7 AT)-TR2 + TAI + FkbS (QD99) produced 21.6 mg / L 4-amino-3-methyl-2-butanol, as well as l-amino-2-methyl-3 -pentanol and l-amino-2-methyl-3 -hexanol (Fig. 6, panel b, and Supplementary Figures 28-29).

[0119] Next, we evaluated the impact of protein abundance on amino alcohol titers. Proteomics analysis confirmed expression of all incorporated genes in S. albus and showed higher RimMl-TR2 level than RimMl-TRl chimaera (Fig. 6, panel c), explaining higher product titers when using RimMl-TR2 to produce either 1 ,3 -diols or amino alcohols.Furthermore, the TAI protein level was 5 times higher than that of TA2 (Fig. 6, panel d), also in agreement with the amino alcohol production data. All together, these data demonstrate that optimal protein expression level is key to high product titers by these engineered PKS systems in Streptomyces hosts.

[0120] Another possible key factor that may play a critical role in PKS-TR-based bioproduction was intracellular NAD(P) levels, as both PKS KRs and TRs are NADPH- dependent, and many ADHs are NADH-dependent. We measured the NAD(P) levels in several of our engineered S. albus strains, and concluded that they were similar in all tested strains (Supplementary Figure 30), ruling out the possibility of cofactor influence on observed product titers.DISCUSSION

[0121] Nature designs and produces a wide range of structurally complex molecules with modular type I PKSs, which provide parts to engineer megasynthases for a variety of unnatural molecules that would be difficult or impossible to biosynthesize in any other way. In this study, we developed PKSs with terminal reductases (TRs) to enable biosynthesis of medium- and branched-chain aldehydes. Compared to previously established pathways that can be used to form C-C bonds, such as reverse P-oxidation 55, PKSs have three major advantages. First, because PKSs liberate carbon dioxide during Claisen condensation, they have a thermodynamic advantage over thiolase-catalyzed non-decarboxylative condensation reactions. Taking the 2-ethyl-3 -hydroxyhexanoic skeleton as an example, the PKS biosynthesis route has a 3 kcal / mol advantage in Gibbs standard free energy over the thiolase pathway (Supplementary Table 5). Second, the PKS biosynthesis route is a platform to many different molecules because of the great structural diversity in the starter choice as well as the extender choice due to AT-gated substrate loading (e.g., desmethyl-, methyl-, ethyl-, allyl-, hydroxy-, methoxy-, and amino-, to name a few48). This is particularly advantageous over other routes for adding branches at designated even positions. Furthermore, because of the stepwise catalytic feature and ACP protection of the intermediates, PKS product profiles are more amenable to fine tuning. Rimocidin PKS naturally initiates with acetyl-, propionyl-, and butyryl- starter units, based on which we successfully engineered RimPKS-TRs for bioproduction of a series of unbranched 1 ,3-diols, including the humectant 1,3-BDO. We also validated the plausibility of the canonical PKS AT exchange engineering strategy when terminating with TRs, presenting a platform suitable for medium-chain methyl diol and ethyldiol biosynthesis, featuring the insect repellent 2-E-l,3-HDO. Finally, natural PKS pathways provide lavish enzymatic toolkits including many novel post-PKS decoration enzymes. In this study we showcased the TR-cognate TAs, incorporation of which led to bioproducts with terminal -NH2 in our engineered RimPKS-TR + TA hosts.

[0122] Moreover, we achieved tunable product profiles of RimPKS-TRs through substrate CoA pool engineering. PKS pathways are often tightly regulated in nature, as shown by wide presence of PKS pathway specific transcription regulators within the BGCs56, and more recent discovery of pyrroloquinoline quinone (PQQ) gene clusters that co-evolved with PKS BGCs and enhanced natural product production 57. Another common PKS-related regulation approach prioritizes regulating CoA substrate biosynthesis, investigation of which benefited identification of key steps in engineering such PKSs. For example, 2(5)-ethylmalonyl CoA biosynthesis in Streptomyces has been heavily investigated 58: the major route is a CCR-catalyzed reaction to convert crotonyl-CoA to ethylmalonyl- CoA, and a minor pathway is propionyl-CoA carboxylase-catalyzed butyryl-CoA carboxylation. Butyryl-CoA is a major product of L-valine catabolism, and multi omics analysis revealed a strong suppression of CCR gene expression upon L-valine supplementation 47. This observation is indicative of a CCR shortage in our engineered system and may dampen butyryl-based 1,3-HDO production, thus we overexpressed a secondary FkbS or RimJ CCR and reversed the diol production ratio with a single step of engineering, leading to 1,3-HDO product ratio increase from 33% to 77%.

[0123] Lastly, terminal TR domains in PKS BGCs were reported in multiple biosynthetic studies, yet their application in PKS engineering was largely uninvestigated; our work presents the first PKS TR engineering report to the best of our knowledge. A common theme of these natural TR-containing pathways are amine production through TR-catalyzed reductive cleavage of the thioester bond and subsequent TA-catalyzed transamination. TRs were reported to be NADH-dependent, which was unexpected given the fact that all PKSs are secondary metabolic enzymes, and similar PKS KRs are NADPH-dependent. In this study, we concluded that TRs are evolutionarily related to PKS KRs and also NADPH-dependent. Moreover, our observation that TRs do not catalyze aldehyde reduction agrees with the fact that PKS TRs always have cognate TAs in the BGCs reacting on TR-produced aldehydes. The incapability of aldehyde reduction by PKS TRs separates them from NRPS Rs, many of which can catalyze four electron reduction to alcohols 31. The CpkC TR structure has anopen and solvent-exposed substrate binding pocket, which may facilitate the aldehyde escaping the active site after reductive cleavage from the ACP, preventing further reduction to alcohols. Besides modular type-I PKS and NRPS pathways, TRs are also widely present in other secondary metabolism, including putative iterative type I PKSs in Dictyostelium discoideum59,6Q, but none of these Dictyostelium TRs (DD TRs) cooperated with Rim PKS, and the chimeric Rim PKS-DD TR proteins had no detected function when expressed in S. albus or E. coli.

[0124] Taken together, our study presents the first comprehensive PKS-TR engineering effort targeting diols, amino alcohols and carboxylic acids, which provides valuable toolkits for PKS retrobiosynthesis and lays the foundation for transforming PKS design schemes to produce previously inaccessible alcohols and amines. Our engineering efforts may expand the narrow scope of available biosynthetic pathways for medium- and branched-chain products; further chemical diversification on the Cl aldehyde group can also generate polyketide-based alkenes and alkanes, many of which are interesting biofuels and pharmaceutical intermediates.METHODSMaterials

[0125] Streptomyces albus J 1074, Streptomyces coelicolor Ml 152, E. coliET12567 / pUZ8002, E. coli ET12567 / pUB307, E. coli BAP1 were collected as previously described 61,62. Streptomyces rimosus subsp. rimosus ATCC 10970 and Streptomyces natalensis ATCC 27448 were purchased from ATCC. Streptomyces tsukubaensis NRRL 18488 was purchased from NRRL. E. coli BL21(DE3), DH5a, and DH10P were purchased from New England Biolabs.

[0126] The pSC and p41 Streptomyces integration vectors were collected as previously described 63, pOSV807 (Addgene plasmid 126600) and pOSV809 (Addgene plasmid 126602) Streptomyces integration vectors were purchased from Addgene. pHIS (Addgene plasmid 29653) and pMBP (Addgene plasmid 29656) E. coli expression vectors were purchased from Addgene as well. pG-KJE8 chaperone plasmid was purchased from Takara Bio.

[0127] 1,3-butanediol (309433), 2-methyl-butane-l,3-diol (S309524), 2-ethyl-hexane-1 ,3-diol (E29125), sodium acetate (S2889), sodium malonate (M4795), coenzyme A trilithium salt (C3019), acetyl coenzyme A lithium salt (A2181), malonyl coenzyme A lithium salt (M4263), [13C]labelled L-valine, ATP, NAD+, NADH, NADP+, and NADPH were purchased from Millipore Sigma. 1,3 -pentanediol (BDO 1000039) and 2-ethyl-3- hydroxyhexanoic acid (BD01079153) were purchased from BLDpharm. 1,3-hexanediol (EN300-142438), 2-methylpentane-l,3-diol (EN300-298644), 2-methylhexane-l,3-diol (EN300-7632942), 4-aminobutan-2-ol (EN300-76843), 1 -aminopentan-3 -ol (EN300-62209), l-aminohexan-3-ol (EN300-237616), and 4-amino-3-methylbutan-2-ol (EN300-683176) were purchased from Enamine. 3 -hydroxypentanoic acid (A282505) was purchased from AmBeed. 3-hydroxyhexanoic acid (H825320) was purchased from Toronto Research Chemicals.Cloning of integration plasmids and Streptomyces integration

[0128] All integration plasmids were constructed using the same Gibson assembly protocol, with triparental conjugation into S. albus JI 074, and biparental conjugation into S. coelicolor Ml 152. For A albus J1074 triparental conjugation, using p41_rimB27 (p41 plasmid carrying Pgapdh(EL)- rimMl-TRT) as an example, we PCR amplified the VWB integrase gene-containing p41 backbone, rimMl piece and TRI piece using PrimeSTAR GXL enzyme premix. The plasmid backbone and inserts were ligated through Gibson assembly, and the recombinant plasmid was then transformed into chemically competent E. coll DH5a. Plasmid-containing DH5a was inoculated into 5 mL Luria-Bertani (LB) broth overnight at 37 °C with apramycin (50 μg / mL) for plasmid mini-prep. The plasmid sequence was verified by whole-plasmid sequencing. The validated plasmid was subsequently transformed into chemically competent A. coll DH10P, which was selected for on LB agar containing apramycin (50 μg / mL). Helper cells ET12567 / pUB307 were also plated and selected for on LB agar containing kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL). Integration plasmid-containing DH10P and helper cells ET12567 / pUB307 were then inoculated in 10 mL LB broth at 37 °C with proper antibiotics until they reached an O.D. 600 nm value of 0.4. They were centrifuged at room temperature to remove the remaining media, washed with sterile water twice, and finally resuspended in 250μL - 1 mL LB. 500μL S. albus spores that were kept at -80 °C were heat-activated at 50 °C for 10 min and mixed with 250μL plasmid- containing DH10P and 250μL ET12567 / pUB307. They were then concentrated to 100 μL final volume, plated on mannitol soy agar (2% mannitol, 2% soy flour, and 10 mM MgC12),and incubated at 30 °C for 16 h. 1 mL nalidixic acid (50 μg / mL) and apramycin (50 μg / mL) were overlaid the next day to eliminate those S. albus that failed to integrate the recombinant plasmid.

[0129] For S. coelicolor Ml 152 biparental conjugation, using pSC rimAO (pSC plasmid carrying PrpsL(RO)-rimMO') as an example, the validated pSC rimAO plasmid obtained from E. coli DH5a was transformed into E. coli ET12567 / pUZ8002 through electroporation. E. coli ET12567 / pUZ8002, serving as the conjugal donor, was then grown and selected for in 10 mL LB broth containing spectinomycin (100 μg / mL), kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) at 37 °C until it reached an O.D. 600 nm value of 0.4. The following conjugation procedure with heat-activated S. coelicolor Ml 152 spores and antibiotics overlay (1 mL 50 μg / mL nalidixic acid and 400 μg / mL spectinomycin) was identical to the triparental conjugation experiments.

[0130] Conjugated Streptomyces colonies were inoculated into 3 mL Tryptic Soy Broth (TSB) medium with 2-3 plating beads in a 24-well block at 30 °C for 2 d, and 100 pL culture aliquots were taken and boiled at 98 °C for 20 min to verify successful integration via genome PCR amplification.Streptomyces collection and cultivation

[0131] 100 μL of Streptomyces culture with successful integration was spread evenly on a mannitol soy agar containing nalidixic acid (50 μg / mL) and grown at 30 °C for 7 d to allow sporulation. Spores were then harvested from the plate with 5 mL of 2* YT medium and filtered by a sterilized cotton syringe. The spore mixture was subsequently mixed with 2 - 3 ml of 60% glycerol stock and stored at -80 °C. For Streptomyces cultivation, spores were 1 : 100 inoculated in 3 mL TSB medium with 2-3 plating beads and cultured at 30 °C for 2 d. 1 mL of Streptomyces seed culture was then added to 30 mL broth in a 250 mL baffled shake flask for cultivation at 30 °C and 200 r.p.m. TSB medium: 17 g tryptone, 3 g soytone, 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose, in 1 L dELO, pH = 7.3. Standard R5 medium: 103 g sucrose, 0.25 g K2SO4, 10.12 g MgCh 6H2O, 10 g glucose, 0.1 g Difco casamino acids, 5 g Difco yeast extract, 5.73 g TES buffer, and 2 ml trace element solution were added in dH2O (1 L final). 1 mL trace element solution was prepared by 40 mg ZnCh, 200 mg FeCh 6H2O, 10 mg CuCh 2H2O, lOmg MnC12'4H2O, lOmg Na2B4O?- IOH2O, and 10 mg (NH4)eMo7O24-4H2O. Before use, 100 mL R5 was mixed with 1 mL KH2PO4 (0.5%), 0.4 mLCaCh 2H2O (5M), 1.5 mL L-proline (20%), and 0.7 mL NaOH (IN). ISP2: 4 g yeast extract, 10 g malt extract, 4 g dextrose, in 1 L dlbO. ISP4: 10 g soluble starch, 1 g MgSCh 7H2O, 1 g NaCl, 2 g (NH4)2SO4, 2 g CaCCh, 1 mL trace salts solution (0.1 g FeSCh 7H2O, 0.1 g MnCh 4H2O, 0.1 g ZnSCh 7H2O in 100 mL dLLO). M042 was prepared as previously reported 16. 1 mL of Streptomyces culture was collected at different time points (maximum 10 d) for LC-MS analysis.LC-MS sample preparation and analysis

[0132] 1 mL of Streptomyces culture was centrifuged at 4,000g for 5 min to collect the soluble fraction, which was subsequently mixed with the same volume of LC-MS grade MeOH, vortexed for 10 s, and centrifuged in Millipore Amicon Ultra-0.5 centrifugal filters (3 kDa) at 14,000g for 15 min. The follow-through solution was diluted in 50% LC-MS grade MeOH by 25 folds if necessary, and analyzed by LC-MS using the Agilent LC / MSD iQ single quadrupole mass spectrometer. Accurate mass measurements were performed via an Agilent Technologies 6520 Q-TOF LC / MS system.

[0133] For 1,3 -diol or 3 -hydroxy acid detection and quantification, 5 samples μL were injected onto Phenomenex Kinetex XB-C18 (2.6 pm, 100 x 3 mm, 100 A) LC Column, and analyzed with the following HPLC protocol: buffer A: water with 0.1% v / v formic acid; buffer B: MeOH with 0.1% v / v formic acid; flow rate: 0.42 mL / min; 20 - 72.1% buffer B gradient for 4.5 min, 72.1 - 95% buffer B gradient for 1.3 min, 95% buffer B for 3 min, 95 - 20% buffer B for 0.2 min, 20% buffer B for 2.2 min; mass detection rage: m / z = 50 - 400.

[0134] For amino alcohol detection and quantification, 5 samplesμ wLere injected onto Agilent Technologies ZORBAX Eclipse Plus C18 (3.5 pm, 4.6 mm * 150 mm) LC column, and analyzed with the following HPLC protocol: buffer A: water with 0.1% v / v formic acid; buffer B: acetonitrile with 0.1% v / v formic acid; flow rate: 0.40 mL / min, 2% buffer B for 0.5 min, 2 - 13% buffer B gradient for 4.5 min, 13 - 80% buffer B gradient for 0.1 min; flow rate changed to 1.0 mL / min, 80% buffer B for 2 min, 80 - 2% buffer B for 3.1 min, 2% buffer B for 1.1 min; flow rate changed to 0.4 mL / min, 2% buffer B for 1.1 min; mass detection rage: m / z = 70 - 300. l,3-diol / 3-hydroxy acid / [13C]labelled L-valine feeding

[0135] For 1,3 -di ol / 3 -hydroxy acid feeding experiments, 50 mg / L 1 ,3-diol or 3-hydroxy acid was added to 30 mL R5 + 1% glucose, and 1 mL of Streptomyces seed culture was then inoculated into the broth in a baffled shake flask for cultivation at 30 °C and 200 r.p.m. Culture samples were collected every 24 h for LC-MS quantification as previously described. For [13C]labelled L-valine feeding, 5 mM or 10 mM [13C]labelled L-valine was added to 30 mL R5 + 1% glucose. After Streptomyces seed culture inoculation, LC-MS samples were collected after 3 d cultivation.Cloning of expression plasmids

[0136] RimM0,pimS0, TADH2, ACP9, TRI, TR2, TR7, and TR9 genes were cloned into the pHIS vector via Ligation-Independent Cloning (LIC). ACPI was cloned into the pMBP vector. The resulting plasmids (pHIS rimA, pHIS_pimS0, pHIS_TADH2, pMBP ACPl, pHIS_ACP9, pHIS TRl, pHIS_TR2, pHIS_TR7, pHIS_TR9) were transformed into DH5a and miniprepped using QIAprep Spin Miniprep Kit. All the plasmid sequences were validated by whole-plasmid sequencing from Primordium Labs.Protein expression and purification

[0137] For protein expression, pHIS or pMBP expression plasmid was co- transformed with pG-JKE8 plasmid into E. coli BL21(DE3). For pHIS rimA or pHIS_pimS0, E. coli BAP1 was used for Ao / o-protein expression. A single colony was inoculated in 10 mL LB broth with 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and grown overnight at 37 °C, 200 r.p.m. The overnight culture was inoculated into 500 mL Terrific Broth + 4% v / v glycerol with 50 μg / mL kanamycin and 25 μg / mL chloramphenicol in a 2 L baffled flask, and incubated at 37 °C, 200 r.p.m. until O.D. 600 nm reached 0.6. The flask was then transferred to 20 °C, incubated for 1 h, 200 r.p.m., and induced with 1 mg / mL L-arabinose, 10 ng / mL tetracycline, and 0.2 mM IPTG. After 20 h induction, the cells were centrifuged at 4000g for 30 min and the cell pellet was harvested and stored at -20 °C.

[0138] For protein purification, the cell pellet was resuspended in 50 mL lysis buffer (10% v / v glycerol, 300 mM NaCl, 20 mM imidazole pH 7.5, 25 mM HEPES buffer pH 7.5, 0.1 mg / mL lysozyme, 0.05 mg / mL DNase, and 1 mM MgC12) and vortexed for 30 min. To completely lyse the cells, sonication (amplitude 40%, 30 s on, 1 min off, repeat for 3 cycles) was applied at 4 °C. The cells were centrifuged at 20000g for 30 min, and the lysate supernatant was collected in a 50 mL falcon tube after 0.22 pm filtration. The supernatant was subsequently subjected to Ni-NTA affinity chromatography in 4 mL / min flow rate, andwashed with 10 column volumes of Ni-NTA buffer (10% v / v glycerol, 300 mM NaCl, 20 mM imidazole pH 7.5, 25 mM HEPES buffer pH 7.5). The His-tagged protein was eluted with an imidazole gradient from 20 mM to 500 mM in 15 min. Fractions containing the target His-tagged protein were analyzed by SDS-PAGE, pooled and incubated with 5 mM ATP pH 7.0 for 1 h to completely dissociate attached chaperones. After ATP incubation, the protein solution was concentrated using Amicon Ultra- 15 centrifugal filters to a final volume of 4 - 5 mL. To achieve higher purity and better understanding of the protein oligomeric distribution, the protein solution was further subjected to size-exclusion chromatography (SEC) with a GE Hiload 16 / 60 Superdex 200 prep grade column equilibrated with 10% v / v glycerol, 50 mM NaCl, 25 mM HEPES pH 7.5 in 1 mL / min flow rate. SEC fractions were assessed by SDS- PAGE, and ideal fractions were concentrated to 5 - 40 mg / mL, aliquoted and flash cooled in liquid N2, and stored at -80 °C. All proteins have > 95% homogeneity, except for PimSO with -80% purity.Crystallization, X-Ray data collection and structure determination of CpkC TR

[0139] The CpkC TR sample was concentrated at 10 mg / ml. The cofactor NADP+ was added prior to crystallization trials to a final concentration of 5 mM. The CpkC TR in complex with NADP+ was screened against the crystallization set of solutions: Berkeley Screen64, MCSG-1 (Anatrace), ShotGun (Molecular Dimensions), PEG / Ion, Index, Crystal Screen, and PEGRx (Hampton Research). Crystals of CpkC TR were found in Berkeley Screen condition B3 composed of 0.4 M sodium chloride, 0.1 M BIS-Tris pH 6.5 and 30 % PEG 3,350. The crystal of CpkC TR was placed in a reservoir solution containing 20% (v / v) glycerol, then flash-cooled in liquid nitrogen. The X-ray data set for CpkC TR was collected at the Berkeley Center for Structural Biology beamline 5.0.1 at the Advanced Light Source at Lawrence Berkeley National Laboratory. The diffraction data were processed using the program Xia265. The crystal structure of CpkC TR - NADP+ was solved by molecular replacement with the program PHASER66 using initial coordinates of the CpkC TR model generated by ALPHAFOLD 67. The atomic positions obtained from the molecular replacement were used to initiate refinement within the Phenix suite 68. Structure refinement was performed using the phenix. refine program. Manual rebuilding was done using COOT 69. Root-mean-square deviations from ideal geometries for bond lengths, bond angles and dihedral angles were calculated with Phenix. refine 70. The stereochemical quality of the final model of CpkC TR was assessed by the program MOLPROBITY 71. Summary of crystalparameters, data collection, and refinement statistics can be found in Supplementary Table 4.Proteomics analysis

[0140] Streptomyces cultures were collected after 3 d. Cells were harvested and stored at -80 °C until further processing. Protein was extracted from cell pellets and tryptic peptides were prepared by following established proteomic sample preparation protocol 72. Briefly, cell pellets were resuspended in Qiagen P2 Lysis Buffer (Qiagen, Germany) to promote cell lysis. Proteins were precipitated with addition of 1 mM NaCl and 4x vol acetone, followed by two additional washes with 80% acetone in water. The recovered protein pellet was homogenized by pipetting mixing with 100 mM ammonium bicarbonate in 20% methanol. Protein concentration was determined by the DC protein assay (BioRad, USA). Protein reduction was accomplished using 5 mM tris 2- (carboxyethyl)phosphine (TCEP) for 30 min at room temperature, and alkylation was performed with 10 mM iodoacetamide (IAM; final concentration) for 30 min at room temperature in the dark. Overnight digestion with trypsin was accomplished with a 1 :50 trypsimtotal protein ratio. The resulting peptide samples were analyzed on an Agilent 1290 UHPLC system coupled to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer for discovery proteomics73. Briefly, peptide samples were loaded onto an Ascentis® ES-C18 Column (Sigma-Aldrich, USA) and were eluted from the column by using a 10 minute gradient from 98% solvent A (0.1 % FA in H2O) and 2% solvent B (0.1% FA in ACN) to 65% solvent A and 35% solvent B. Eluting peptides were introduced to the mass spectrometer operating in positive-ion mode and were measured in data-independent acquisition (DIA) mode with a duty cycle of 3 survey scans from m / z 380 to m / z 985 and 45 MS2 scans with precursor isolation width of 13.5 m / z to cover the mass range. DIA raw data files were analyzed by an integrated software suite DIA-NN74. The databases used in the DIA-NN search (library -free mode) are S. albus and S. coelicolor latest Uniprot proteome FASTA sequences plus the protein sequences of the heterologous proteins and common proteomic contaminants. DIA-NN determines mass tolerances automatically based on first pass analysis of the samples with automated determination of optimal mass accuracies. The retention time extraction window was determined individually for all MS runs analyzed via the automated optimization procedure implemented in DIA-NN. Protein inference was enabled, and the quantification strategy was set to Robust LC = High Accuracy. Output main DIA-NN reports were filtered with a global FDR = 0.01 on both the precursor level and protein group level. The Top3 method, which is the average MS signalresponse of the three most intense tryptic peptides of each identified protein, was used to plot the quantity of the targeted proteins in the samples 75,76. The generated mass spectrometry proteomics data have been deposited to the ProteomeXchange 63 Consortium via the PRIDE partner repository with the dataset identifier PXD046595 77. DIA-NN is freely available for download from webpage for: github.com / vdemichev / DiaNN.Enzymatic assays: PimSO substrate loading

[0141] PimSO was added in 100 reaμcLtion mix (10% v / v glycerol, 50 mM NaCl, 25 mM HEPES pH 7.5, 5 mM ATP, 1 mM acetate + CoA / malonate + CoA / acetyl-CoA / malonyl- CoA) in 7.5 mg / mL final concentration to initiate the substrate loading reaction. At 1 h , 4 h, and 24 h, 20 μ rLeaction was quenched with 1% formic acid, flash cooled in liquid N2, and stored at -80 °C for subsequent treatment. PimSO with no tested substrate added in the reaction mix was taken as a negative control. Proteins in the reaction samples were precipitated by addition of 1 mM NaCl and 4 x vol acetone, followed by two additional washes with 80% acetone in water. Proteins were resuspended with 100 mM ammonium bicarbonate, reduced in 5 mM tris 2- (carboxy ethyl)phosphine (TCEP) and alkylated in 10 mM iodoacetamide (IAM) before subjecting to trypsin digestion. The resulting peptides were analyzed using an Agilent 1290 Infinity liquid chromatography system coupled to an Agilent 6460 QQQ mass spectrometer (Agilent Technologies, Santa Clara, CA). Peptides (~10 pg) were separated on an Ascentis Express Peptide ES-C18 column (2.7 pm particle size, 160 A pore size, 50 x 2.1mm) fitted with a guard column (5 mm x 2.1 mm, Sigma Aldrich). The column was heated to 60°C. The mobile phase consisted of 0.1% formic acid in H2O (A) and 0.1% formic acid in acetonitrile (B). Peptides were eluted from the column by using a 3.5 minute linear gradient from 95% solvent A and 2% solvent B to 60% solvent A and 40% solvent B. Peptides were ionized using an Agilent let Stream ESI source operating in positive-ion mode with the following source parameters: Gas Temperature = 250°C, Gas Flow = 13 L / min, Nebulizer Pressure = 35 psi, Sheath Gas Temperature = 250°C, Sheath Gas Flow = 11 L / min, and Capillary Voltage = 3,500 V. Dwell times were set to 18ms. Data was acquired using Agilent MassHunter Data Acquisition (Version B.08.02). The MRM method for quantifying phosphopantetheine bearing peptides was built using the Skyline (version 21.2). LC-MS raw data were imported and analyzed in Skyline. The MRM transitions and their integrated peak areas are available on the LC-MS data sharing platform Panorama Public 78.Enzymatic assays: TR-catalyzed reduction

[0142] Purified Sfp was purchased from NEB. To perform Sfp treatment for the formation of Ao / o-ACPs, purified qpo-MBP-ACPl and qpo-ACP9 were added separately in 500 μL reaction mixture (10% v / v glycerol, 50 mM NaCl, 50 mM HEPES pH 7.5, 2.5 mM octanoyl-CoA, 4 pM Sfp, and 20mM MgCh) and incubated at 30 °C for 3 h. The reaction mixture was then placed in the dialysis buffer (10% v / v glycerol, 50 mM NaCl, 50 mM HEPES pH 7.5) at 4 °C for 3 h to remove any remaining free octanoyl-CoA. To study the substrate scope and co-factor preference of TR-catalyzed reduction, TRI and TR9 storing in buffer (10% v / v glycerol, 50 mM NaCl, 25 mM HEPES pH 7.5) were tested respectively with 20 pM final concentration in a 200 reacμtiLon mixture containing any of the three substrates (0.5 mM octanoyl-ACP, 1 mM octanoyl-CoA, or 1 mM octanal), along with either of the two cofactors (200 pM NADH and 200 pM NADPH). These experiments were conducted in a Corning 96-well clear bottom black microplate, and 340 nm absorbance was monitored for the detection of NAD(P)H elimination for a maximum of 16 h. All assays were repeated in duplicate.Enzymatic assays: 1,3-diol oxidation

[0143] 300 μL reaction mixture (10% v / v glycerol, 50 mM NaCl, 25 mM HEPES pH7.5, 0.5 mM NAD(P)+, 20 mM alcohol substrate) was added in a Corning 96-well clear bottom black microplate. To initiate the reaction, TADH2 or TR1 / 2 / 7 / 9 enzyme was added at 0.4 pM final concentration, and 340 nm absorbance was monitored for the detection of NAD(P)H accumulation for 2 h. All assays were repeated in triplicate.Intracellular NADP and NAD quantification

[0144] The measurements were conducted by following the manufacture of NADP / NADPH Quantification Kit (MAK038) and NAD / NADH Quantification Kit (MAK037) from Sigma Aldrich. 10 O.D. (O.D.600) of 5’. albus cell tissue were harvested after culturing in R5 medium for 3 d at 30 °C, 200 r.p.m. Cells were collected by centrifuge at 15,000g, 4 °C, 5 mins, the supernatants were discarded, and the cells were washed with pre-chilled PBS at 15,000g, 4 °C, 5 mins. Discarding the PBS and adding 500 of NADP / NAμDLPH Extraction buffer or NAD / NADH Extraction buffer for NADP / NADPH measurement and NAD / NADH extraction respectively. 0.5 mm Glass beads (BioSpec Products, Cat. No. 11079105) were added for bead beating with the following process, 3,800 HZ for 30 s, on ice for 1 min, and 3 cyclestotal. The extracted samples were sitting on ice for 10 min, then centrifuge at 15,000 g, 4 °C, 10 min, the supernatant was filtering through a 10 kDa cut-off spin filter (Merck Millipore, UFC501096). The filtered samples were used for measurement, and the samples measurement is following the procedure in the Kits. All assays were repeated in at least duplicate.

[0145] APPENDIX “SUPPLEMENTARY INFORMATION” was taught in the filing of U.S. Provisional Patent Application Ser. No. 63 / 645,806, filed May 10, 2024, which is incorporated by reference in its entirety. Further Supplementary Information can be also found obtained online from the following webpage: nature. com / articles / s41929-025-01299-5, which is incorporated by reference in its entirety.

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[0147] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

What is claimed is:

1. A system comprising a hybrid polyketide synthase (PKS) capable of producing an alcohol and / or a derivative thereof.

2. The system of claim 1, wherein the hybrid PKS is capable of producing a P-hydroxy aldehyde (BHA).

3. The system of claim 2, wherein the BHA has the following chemical structure:; wherein Ri and R2, each independently, — H or — alkyl.

4. The system of claim 1, further comprising: (a) a transaminase (TA) capable of converting the BHA into an amino alcohol, (b) an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, and / or (c) aldehyde dehydrogenase (ALDH) capable of converting the BHA into a 3-hydroxy acid (3HA).

5. The system of claim 4, comprising the TA and the system produces an amino alcohol having the following chemical structure:wherein Ri and R2, each independently, — H or — alkyl.

6. The system of claim 4, comprising the ADH and the system produces a diol having the following chemical structure:wherein Ri and R2, each independently, — H or — alkyl.

7. The system of claim 4, comprising the ALDH and the system produces a 3-hydroxyacid (3HA) having the following chemical structure:wherein Ri andR2, each independently, — H or — alkyl.

8. A genetically modified host cell comprising a hybrid polyketide synthase (PKS) capable of producing a P-hydroxy aldehyde (BHA).

9. The genetically modified host cell of claim 8, comprising: (a) a transaminase (TA) capable of converting the BHA into an amino alcohol, (b) an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, and / or (c) aldehyde dehydrogenase (ALDH) capable of converting the BHA into a 3-hydroxy acid (3HA).

10. The genetically modified host cell of claim 9, the (a) transaminase (TA), (b) alcohol dehydrogenase (ADH), and / or (c) aldehyde dehydrogenase (ALDH) are each independently endogenous to the host cell, or stably introduced into the host cell.

11. The genetically modified host cell of claim 9, wherein the genetically modified host cell is capable of producing the diol or the amino alcohol.

12. The genetically modified host cell of claim 11, wherein the diol or the amino alcohol is a straight-chain or branched-chain diol or amino alcohol.

13. The genetically modified host cell of claim 11, wherein the diol or the amino alcohol is a medium- or branched-chain diol or amino alcohol.

14. A method of producing an alcohol and / or a derivative thereof, said method comprising: (a) providing the genetically modified host cell of claim 8 optionally comprising (a) a transaminase (TA) capable of converting the BHA into an amino alcohol, (b) an alcohol dehydrogenase (ADH) capable of converting the BHA into a diol, and / or (c) aldehyde dehydrogenase (ALDH) capable of converting the BHA into a 3-hydroxy acid (3HA); and (b) culturing or growing the genetically modified host cell such that the genetically modified host cell expresses the hybrid PKS, and optionally the TA, ADH, and / or ALDH, and produces the BHA and / or derivative thereof.

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