FERMENTATIVE PRODUCTION OF N-BUTYLACRYLATE USING ALCOHOL ACYL TRANSFERASE ENZYMES

Abstract

Provided herein is a recombinant nucleic acid molecule, a recombinant microorganism, and a method for fermentative production of n-butylacrylate and other esters from alcohols and acyl-CoA units using alcohol acyl transferase enzymes.

Claims

1. A method for fermentative production of n-butylacrylate (n-BA) comprising the steps of i) providing a recombinant microorganism comprising a butanol producing pathway and an acryloyl-CoA producing pathway and expressing an AAT gene encoding an AAT enzyme having an n-BA forming activity; and ii) culturing said microorganism under conditions that allow for the production of n-BA; and iii) recovering n-BA from a fermentation broth.

2. The method of claim 1 wherein the AAT gene encoding an AAT enzyme having an n-BA forming activity is selected from the group consisting of: (I) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (II) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (III) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 under stringent conditions; and (IV) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof; and (V) a nucleic acid molecule encoding a polypeptide having at least 60% identity to a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof.

3. A recombinant microorganism comprising an introduced, increased, or enhanced activity and/or expression of a nucleic acid molecule encoding an AAT gene encoding an AAT enzyme having an n-butylacrylate (n-BA) forming activity.

4. The recombinant microorganism of claim 3 further comprising a butanol producing pathway and an acryloyl-CoA producing pathway.

5. The recombinant microorganism of claim 3 wherein the nucleic acid molecule encoding an AAT enzyme is selected from the group consisting of: (I) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (II) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (III) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 under stringent conditions; and (IV) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof; and (V) a nucleic acid molecule encoding a polypeptide having at least 60% identity to a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof.

6. The recombinant microorganism of claim 3, wherein the microorganism is selected from a genus of the group consisting of Saccharomyces, Yarrowia, Arxula, Kluyveromyces, Lactobacillus, Clostridium, Pseudomonas, Corynebacterium, Bacillus, Erwinia, Escherichia, Pantoea, Streptomyces, Zymomonas and Rhodococcus.

7. A composition comprising one or more recombinant microorganisms according to claim 3.

8. The composition of claim 7 further comprising a medium and a carbon source.

9. A method for producing a recombinant microorganism producing n-BA comprising the steps of: (I) introducing, increasing, or enhancing the activity and/or expression of an AAT gene encoding an AAT enzyme having an n-BA forming activity in a microorganism; and (II) further introducing in the microorganism a butanol producing pathway and an acryloyl-CoA producing pathway.

10. The method of claim 9, wherein the AAT gene is selected from the group consisting of: (I) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (II) a nucleic acid molecule having at least 80% identity to a nucleic acid molecule of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (III) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 under stringent conditions; and (IV) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof; and (V) a nucleic acid molecule encoding a polypeptide having at least 60% identity to a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof.

11. The method of claim 9, wherein the microorganism is selected from a genus of the group consisting of Saccharomyces, Lactobacillus, Clostridium, Pseudomonas, Corynebacterium, Bacillus, Erwinia, Escherichia, Pantoea, Streptomyces, Zymomonas and Rhodococcus.

12. A recombinant expression construct comprising a promoter functional in a microorganism functionally linked to a nucleic acid molecule having a sequence selected from the group consisting of: (I) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (II) a nucleic acid molecule comprising a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13; and (III) a nucleic acid molecule hybridizing to the complement of a nucleic acid molecule having SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13 under stringent conditions; and (IV) a nucleic acid molecule encoding a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof; and (V) a nucleic acid molecule encoding a polypeptide having at least 60% identity to a polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14, or a functional fragment thereof, wherein the promoter is heterologous to the nucleic acid molecule.

13. A recombinant vector comprising the recombinant expression construct of claim 12.

14. A method of culturing or growing a recombinant microorganism comprising inoculating a culture medium with one or more recombinant microorganism according to claim 3 and culturing or growing the one or more recombinant microorganism in the culture medium.

15. A use of a recombinant microorganism according to claim 3 or a composition for the fermentative production of n-BA.

16. A process for fermentative production of n-BA comprising the steps of: I) growing the recombinant microorganism according to claim 3 in a fermenter; and II) recovering n-BA from a fermentation broth obtained in step I).

Description

FIGURES

[0274] FIG. 1 pYP137 construction vector for overexpression of Aco in S. cerevisiae

[0275] FIG. 2 pYP096, pYP106 construction vector for overexpression of pct in S. cerevisiae

[0276] FIG. 3 pYP083 construction vector for overexpression of AAT in S. cerevisiae

[0277] FIG. 4 nBA (a) and nBP (b) production in S. cerevisiae by feeding propionate and butanol. TYC-166 is the test strain with ACO, Pct-Me, and Cm-AAT2 expressed. TYC-181 is the negative control strain with Pct-Me and Cm-AAT2 but without ACO expressed.

EXAMPLES

[0278] The following examples serve to illustrate the invention without limiting the scope thereof.

Chemicals and Common Methods

[0279] Unless indicated otherwise, cloning procedures carried out for the purposes of the present invention including restriction digest, agarose gel electrophoresis, purification of nucleic acids, ligation of nucleic acids, transformation, selection and cultivation of bacterial cells are performed as described (Sambrook et al., 1989). Sequence analyses of recombinant DNA are performed with a laser fluorescence DNA sequencer (Applied Biosystems, Foster City, Calif., USA) using the Sanger technology (Sanger et al., 1977). Unless described otherwise, chemicals and reagents are obtained from Sigma Aldrich (Sigma Aldrich, St. Louis, USA), from Promega (Madison, Wis., USA), Duchefa (Haarlem, The Netherlands) or Invitrogen (Carlsbad, Calif., USA). Restriction endonucleases are from New England Biolabs (Ipswich, Mass., USA) or Roche Diagnostics GmbH (Penzberg, Germany). Oligonucleotides are synthesized by IDT (Coralville, USA).

1. In Vivo Production of Acryloyl-CoA in S. cerevisiae

[0280] 1.1 Heterologous Expression of Short-Chain acyl-CoA Oxidase (Aco) in S. cerevisiae

[0281] Short-chain acyl-CoA (coenzyme A) oxidase catalyses an oxidation reaction with saturated acyl-CoAs (e.g. propionyl-CoA) to enoyl-CoAs (e.g. acryloyl-CoA). Nucleotide sequence of the Aco gene (GB: AB017643.1) from Arabidopsis thaliana was obtained from the NCBI (http://www.ncbi.nlm.nih.gov/). The nucleotide sequence was codon optimized for expression in yeast with an N-terminal 6?-His tag based on the standard codon usage table in IDT Gene synthesis service (Seq ID No. 57). The 1337 bp of ACO gene was synthesized by IDT (Coralville, USA). The ACO gene fragment flanked by BamHI and HindIII restriction sites was inserted in a vector with 2 micron and pBR322 origin of replicon, ura3 and bla gene as markers to yield pYP137 (high-copy E. coli/S. cerevisiae shuttle vector; complements Ura-auxotrophy in S. cerevisiae: pBR322; CEN4-origin; AmpR; URA3, ACO under control of truncated HXT71-392 promoter and CYC1 terminator, Seq ID No. 67). The construct is subjected to be introduced in S. cerevisiae with various combinations of other genes in the pathway (FIG. 1).

1.2 Heterologous Expression of Propionyl-CoA Transferase in S. cerevisiae

[0282] Enzymatic activity of pct is to transfer CoA to short carbon length acids by forming thioester bond. This yield various acyl-CoAs as intermediates of various biosynthetic pathways. Propionyl-CoA transferases use acetyl-CoA as a CoA donor to create propionyl-CoA from propionate. Pct genes used for this experiment from Megasphaera elsdenii, (CCC72964) and from Cupriavidus necator (CAJ93797) were codon-optimized for expression in S. cerervisiae and synthesized by GeneScript (Piscataway, USA) and IDT (Coralville, USA), yielding pct-Me (Seq ID No. 59) and pct-CN (Seq ID No. 61) (Prabhu et. al 2012). These genes were cloned into gene overexpression plasmids by homologous recombination methods described in prior publications (Gietz et. al., 2007). The pct genes were inserted seamlessly into the E. coli/S. cerevisiae shuttle plasmid to overexpress the protein from the strong constitutive promoter ADH1 and TRPL3 terminator with tryptophan auxotrophic marker in 2micron base high-copy expression vector. The resulting plasmids pYP096 (Seq ID No. 65), and pYP106 (Seq ID No. 66) are listed in the Table 1 (FIG. 2).

TABLE-US-00001 TABLE 1 List of plasmids encoding for different propionate CoA-transferase Enzyme Accession #, Plasmid Plasmid description Key Species/Protein Seq ID No. pYP096 high-copy E. coli/S. cerevisiae Pct-Me Megasphaera CCC72964, shuttle vector; complements elsdenii Seq ID No. 65 Trp-auxotrophy in S. cerevisiae: pBR322; 2 ?m-ori; AmpR; TRP1; ADH1 promoter and RPL3 terminator, contains codon-optimized pct of M. elsdenii pYP106 high-copy E. coli/S. cerevisiae Pct-Cn Cupriavidus CAJ93797, shuttle vector; complements necator Seq ID No. 66 Trp-auxotrophy in S. cerevisiae: pBR322; 2 ?m-ori; AmpR; TRP1; ADH1 promoter and RPL3 terminator, contains codon-optimized pct of Cupriavidus necator

2. In Vivo Production of lactoyl-CoA in S. cerevisiae

[0283] In order to produce lactoyl-CoA in the cytosol of S. cerevisiae, conversion of pyruvate to lactate and lactate to lactoyl-CoA has to occur by heterologous enzymes. Lactate dehydrogenase is responsible to convert pyruvate to lactate with NADH, and lactate dehydrogenase (IdhA) from E. coli was expresses in yeast after codon optimization, yielding IdhA-sc (Seq ID No. 69). To overexpress IdhA-sc in S. cerevisiae, a yeast shuttle expression vector was constructed. The IdhA-sc was inserted seamlessly by homologues recombination between the HXT7 promoter and CYC1 terminator on a plasmid (high-copy E. coli/S. cerevisiae shuttle vector; complements His-auxotrophy in S. cerevisiae: pBR322; 2 ?-ori; AmpR; HIS3). The resulting plasmid was named pYP024 (Seq ID No. 71). This plasmid was transformed into S. cerevisiae W303-1A strain to yield strain TYC-006. TYC-006 was cultured in the synthetic media and the cultured broth contained lactate which was converted from lactate. This lactate was converted further to lactoyl-CoA by expression of propionyl-CoA transferase, pct-Me (Seq ID No. 59), and pct-Cn (Seq ID No. 61) in the S. cerevisiae strain with IdhA-sc.

3. Heterologous Expression of Alcohol Acyl Transferase (AAT) in P. pastoris and S. cerevisiae.

[0284] AAT is responsible for forming an ester bond between alcohols and acyl-CoAs. Various putative AATs were chosen for analysis (see Table 2). Selected AAT genes were subjected to go through activity screening tests in vitro. The methylotrophic yeast P. pastoris was chosen as expression host for the evaluation of expression of candidate genes encoding putative AATs. Expression constructs were set up in the plasmid pD902 (DNA 2.0) which provides the strong methanol-inducible AOX promoter. The plasmid was modified by inserting the PARS1 element, which allowed the episomal replication of the plasmid (Cregg, J. M., et al., 1985). Selected candidate AATs were constructed into pD902e plasmids (Seq ID No. 68) and transformed in P. pastoris GapChap. In order to overexpress for example Cm-AAT2 in S. cerevisiae, the gene was cloned in a high copy overexpression vector with a strong promoter and a terminator, yielding pYP083 (Seq ID No. 64, high-copy E. coli/S. cerevisiae shuttle vector; confers geneticin resistance: pBR322; 2 ?m-ori; AmpR; lac Z; kanMX; truncated HXT71-392 promoter and CYC1 terminator CDS: N-terminal His tagged CmAAT2_Cucumis melo) (FIG. 3).

TABLE-US-00002 TABLE 2 Overview of the AATs selected for expression and characterization SEQ ID gene NO name identifier Origin Origin reference 1 CM-AAT1 CAA94432 Cucumis melon Yahyaoui et melo al., 2002 El-Sharka- way et al., 2005 3 MpAAT1 AY707098 Malus apple Souleyre et al., 2005 pumila 5 VAAT CAC09062 Fragaria strawberry Beckwilder et vesca al., 2004 7 CM-AAT2 AAL77060 Cucumis melon El-Sharkaway et melo al., 2005 9 Md-AAT2 AAS79797 Malus apple Li et al., 2006 domestica 11 BEBT AF500200 Clarkia flower D'Auria et al., 2002 breweri 13 CbBEAT AAC18062 Clarkia flower Dudareva et breweri al., 1998 15 SAAT CAC09048 Fragaria ? strawberry Beekwilder et al., ananassa 2004 17 FaAAT2 JN089766) Fragaria ? strawberry Cumplido-Laso et ananassa al., 2012 19 AeAT9 HO772637 Actinidia kiwi G?nther et al., 2011 eriantha 21 Rh-AAT1 AAW31948 Rosa hybrid flower Guterman et cultivar al., 2006 23 CM-AAT4 AAW51126 Cucumis melon El-Sharkaway et melo al., 2005 25 ACT WP_001010387 (Staphylococcus bacterium Rodriguez et sciuri) al., 2014 27 BanAAT AX025506 Musa banana Beekwilder et sapientum al., 2004 29 Glossy2 CAA61258 Zea mays maize Tacke et al., 1995 31 CM-AAT3 AAW51125 Cucumis melon El-Sharkaway et melo al., 2005 33 BAHDFox EMT69722 Fusarium fungi oxysporum 35 VpAAT1 FJ548611 Vasconcellea papaya Balbotin et al., 2010 pubescens 37 AMAT AY705388 Vitis grape Wang & De labrusca Luca, 2005 39 Pun1 AAV66311 Capsicum pepper Stewart et al., 2005 annum 41 Dv3MaT AAO12206 Dahlia flower Suzuki et al., 2002 variabilis 43 NtHCT CAD47830 Nicotiana tobacco Hoffmann et tababcum al., 2003 45 DBATAca ACI47063 Aspergillus fungi candidus 47 TSga WP_006129805 Streptomyces bacterium gancidicus 49 TSvi YP_004810992 Streptomyces bacterium violaceusniger 51 CAT YP_007500975 Shigella bacterium Rodriguez et sonnei al., 2014 53 EHT NP_009736 Saccharomyces yeast Rodriguez et cerevisiae al., 2014 55 ATF NP_015022 Saccharomyces yeast Rodriguez et cerevisiae al., 2014

4. In Vivo Production of Butanol in S. cerevisiae

[0285] A butanol producing S. cerevisiae (TYC-185) strain was established as described in Schadeweg, V. and E. Boles, 2016.

5. In Vivo Production of nBA by Feeding Substrate

[0286] We have established a pathway within S. cerevisiae that is able to create n-butylacrylate from feeding of propionate and n-butanol. The first step uses propionyl-CoA transferase (M. elsdenii) to convert propionate to propionyl-CoA. Then, the Acyl-CoA dehydrogenase (ACO) enzyme from A. thaliana to convert the propionyl-CoA to acryloyl-CoA. Further down the pathway, acryloyl-CoA and n-butanol become key intermediates, which are esterified by the activity of an alcohol acyltransferase (AAT) to the desired end product n-butylacrylate. We used TYC-072 modified strain of S. cerevisiae to introduce nBA biosynthetic pathway plasmids. TYC-072 was transformed with a set of plasmids, pYP137 (Seq ID No. 67), pYP096 (Seq ID No. 65), and pYP083 (Seq ID No. 64) from which Aco, pct-Me, and cm-AAT2 are overexpressed, to yield S. cerevisiae strain TYC-166. TYC-166 was cultured in synthetic defined SD media (Bacto-Yeast nitrogen base without amino acids, 1.7g; Glucose, 20g; Dropout mix, 2 g/1 L) with G418 and without TRP and URA. As a negative control, an empty control vector instead of the Aco vector was introduced into TYC-072 with pct-Me and cm-AAT2 overexpression vectors to yield S. cerevisiae strain TYC-181. These strains were grown in SE-TRP-URA+G418 selective minimal media (glutamic acid, 1 g; Bacto-Yeast nitrogen base without amino acids and ammonium sulfate, 1.7 g; Dropoutmix, 2 g; glucose 20 g/1 L) at 30? C. (Table 3).

TABLE-US-00003 TABLE 3 Strains to produce n-butylacrylate in S. cerevisiae. Strain Selection Name Description of strain plasmids markers TYC-72 MATa; ura3-52; trp1-289; leu2-3_112; none Auxotrophic: his3 ?1; MAL2-8C; SUC2 adh1::loxP Trp, Ura, adh3::loxP; adh4?::loxP, adh5?::loxP Leu, His ?adh1,3,4,5 (all with loxP), Ethanol non- producer TYC-166 Prepared from TYC-072, Ethanol pYP083 (Cm-AAT2) Dominant: G418 non-producer, overexpress Aco, pct-Me, pYP096 (pct-ME) Auxotrophic: and Cm-AAT2 pYP137 (ACO) Trp, Ura TYC-181 Prepared from TYC-072, Ethanol non- pYP004 (empty) Dominant: producer, overexpress pct-Me, and Cm- pYP083 (Cm- G418 AAT2 AAT2) Auxotrophic: pYP096 (pct-ME) Trp, Ura TYC-185 n-Butanol producer. Ethanol non- none Auxotrophic: producer, MATa; ura3-52; trp1-289; Ura, Trp, His leu2 3_112; his3?1; MAL2-8C; SUC2; adh1::loxP; adh2?::LEU2; adh3::loxP; adh4?::loxP; adh5::loxP; adh6?::coaA, natNT2; sfa1?::adhE, A267T/E568K, hphNT1; gpd2::ERG10, hbd, crt, ter, adhE2, EutE, KanMX

[0287] Strains were grown aerobically in test tubes from glycerol stocks in 10 mL of SE-TRP-URA+G418 minimal media overnight at 30 ? C. and 250 rpm. These cultures were then transferred into a 250 mL baffled glass shake flask and normalized to an OD600 of 0.2 for a 25 mL culture. 3.0 g/L Sodium Propionate and 0.5% butanol were fed to the cultures every 24 hours. An additional 2% of glucose was also fed after the first 24 hours and every 24 hours thereafter. Samples were taken at 3, 6, 9,12, 24, 36 and 48 hour time points for HPLC and Solid Phase Micro Extraction (SPME) detection. The SPME method was used to detect esters, specifically Butylacrylate, and Butyl propionate.

6. In Vivo Production of nBA from Glucose

[0288] In order to demonstrate nBA production in microorganism from glucose as a carbon source, multiple pathways are introduced to generate substrates to the final esterification step, which is performed by AAT enzymes. The two major pathways to produce two key intermediates are heterologous biosynthetic pathways for butanol and acryloyl-CoA. Two S. cerevisiae production host as described in the examples 1 and 4 showed production of acryloyl-CoA and butanol in separate experiments. We use the S. cerevisiae strain (TYC-185), which can produce butanol by reverse beta-oxidation described in example 4 as a base strain to add an acryloyl-CoA pathway and alcohol acyl transferase (AAT). AAT and genes for the acryloyl-CoA pathway, short chain acyl-CoA oxidase (ACO), propionyl-CoA transferase (pct-Me), methylmalonyl-CoA mutase, methylmalonyl-CoA decarboxylase, are integrated into the chromosome of the base strain with functional promoters and terminators. In addition, other acryloyl-CoA production pathways are used separately and/or collectively. Some of example of other acryloyl-CoA pathways are the lactate route and the 3HP route. The lactate route is composed of a set of enzymes to convert pyruvate to lactate and from lactate to lactoyl-CoA and then to acryloyl-CoA. A 3HP route is composed of a set of enzymes to convert malonyl-CoA or beta-alanine to 3-oxopropanate, which is converted to 3-hydroxypropanoate (3HP) and further to 3-hydroxypropanoyl-CoA and then form acryloyl-CoA. Other routes from glucose to 3HP to acryloyl-CoA are tested. Optimization of the protein expression is achieved by testing various promoters, integration loci, copy-number of genes, episomal plasmid expression, and culture conditions. Once all the necessary genes are expressed in S. cerevisiae, glucose is converted to acryloyl-CoA and together with butanol then esterified by an AAT to form nBA and/or other ester compounds.

7. Production of Other Ester Compounds

7.1 In Vivo Production of Other Esters

[0289] Various ester compound were produced in the engineered S. cerevisiae strains with expression of heterologous pathways for nBA formation. TYC-166 and TYC-181 strains, in which an AAT and pct gene were overexpressed, showed production of n-butylpropionate (nBP) as a by-product along with nBA production. Propionate was fed to the culture broth and transformed by the cells to propionyl-CoA due to the enzyme activity of pct-ME. The propionyl-CoA together with fed butanol were esterified by the AAT activity resulting in production of n-butyl propionate (nBP). Detailed experimental methods are described in example 5. nBP in the culture broth was detected by the methods described in Example 8. Additionally, in vivo production of butyllactate was demonstrated by expression of lactate dehydrogenase and AAT in yeast.

7.2 In Vitro Production of nBA and Other Esters by AATs

[0290] In addition to nBA, in vitro formation of other ester compounds, such as butyl propionate, butyl lactate, butyl acetate, and ethyl acetate, were confirmed by in vitro enzyme activity assays using the activity of the purified AAT enzymes, which form ester compounds from acyl-CoAs and alcohols. The methylotrophic yeast P. pastoris GapChap, which provides a chaperonin co-expression, was chosen as expression host for the evaluation of expression of candidate genes encoding putative AATs. Plasmid constructs with N-terminal 6?-his-tagged AATs were cloned with the strong methanol-inducible AOX promoter. The cultures of individual constructs were pooled, washed once with 100 mM sodium phosphate buffer pH 7.5 and re-suspended in 50 mM sodium phosphate buffer pH 8.0 containing complete plus EDTA free protease inhibitor (Roche), 300 mM NaCl, 10 mM imidazole. A Branson Sonifier 250 was used to generate a crude cell extract; 8?5 min pulses with 50% duty cycle and output level 7 were used to disrupt the cells in an appropriate vessel on ice. After centrifugation the supernatant was filtered before loading onto a HisTrap HF Ni-NTA column (1 ml, GE Healthcare) equilibrated with buffer A (300 mM NaCl, 50 mM sodium phosphate buffer pH 8.0, 10 mM imidazole). Buffer A was also used for loading and washing. A gradient was applied by switching from buffer A to buffer B (300 mM NaCl, 50 mM sodium phosphate buffer pH 8.0, 500 mM imidazole) within 10 column volumes (CV). Elution was prolonged by 5 additional CV of buffer B. Fractions of 1 ml were collected and separately analyzed by SDS-PAGE and activity measurements (GC/MS) for the identification of AAT protein. Up to 3 fractions were pooled and desalted by size exclusion chromatography (PD10, GE-Healthcare). Final preparations contained 100 mM sodium phosphate buffer pH 7.5 and were stored on ice. Purity was analyzed by SDS-PAGE and densitometric analysis of the corresponding protein band. Total protein amount was determined by Micro-BCA Assay (Thermo Fisher). The assay to determine the activity of AATs was set up as follows: 100 mM potassium phosphate buffer pH 7.5, 5 mM alcohol (e.g. butanol), 0.5 mM acyl-CoA, 1 mg/mL BSA, and 20 ?l enzyme sample in a total volume of 100 ?l in a 2 mL glass vial, which was sealed immediately after setup. Samples were set up in duplicate and incubated at room temperature (RT) for 0, 2, 4, 8 and 24 h respectively. Subsequently enzymes were inactivated by heat denaturation at 65? C. for 20 min. Afterward samples were analyzed by the methods described in Example 8. Various AATs showed esterase activities to various substrates to form butylacrylate, butyl propionate, butyl lactate, butyl acetate, and ethyl acetate. (Table. 4)

TABLE-US-00004 TABLE 4 Activities of AATs towards the formation of variable compounds. SEQ ID butyl butyl butyl ethyl NO Enzyme acrylate propionate lactate acetate 2 Cm-AAT1 ? ? ? x 4 Mp-AAT1 ? ? ? ? 6 VAAT ? ? ? ? 8 CM-AAT2 ? ? ? ? 10 Md-AAT2 ? ? ? ? 12 BEBT ? ? x ? 14 CbBEAT ? ? x ? 16 SAAT x ? ? ? 18 Fa-AAT2 x ? ? ? 20 AeAT9 x ? ? ? 22 Rh-AAT1 x ? x ? 24 CM-AAT4 x ? x ? 26 ACT x ? x ? 28 BanAAT x x x ? ?: detected, x: not detected

8. Detection of nBA from Culture Broth

[0291] Solid phase micro extraction (SPME)/GC/MS was used to detect nBA and other ester compounds from the culture broth. SPME samples were prepared by adding 500?L of filtered (0.22 ?m) cultured media into the head space analysis vial. SPME was done with carboxen/polydimethylsiloxane fiber. Extraction was done at 40? C. for 15 min after samples were conditioned at 40? C. for 10 min. Desorption was carried out at injection port at 250? C., followed by GC separation (column DB-624) and MS detection (full scan mode). nBA was detected from the broth of TYC-166 culture. No nBA was detected from TYC-181, which was negative control experiment. Both strains produced n-butylpropionate as a by-product formed by esterification of butanol and propionyl-CoA (FIG. 4).