Biosynthesis of Medium-to Long-Chain α , ω-Diols from Free Fatty Acids Using CYP 153 A Monooxygenase , Carboxylic Acid Reductase , and E . coli Endogenous Aldehyde Reductases

α,ω-Diols are important monomers widely used for the production of polyesters and polyurethanes. Here, biosynthesis of α,ω-diols (C8–C16) from renewable free fatty acids using CYP153A monooxygenase, carboxylic acid reductase, and E. coli endogenous aldehyde reductases is reported. The highest yield of α,ω-diol was achieved for the production of 1,12-dodecanediol. In the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor regeneration system, 5 g/L of 1,12-dodecanediol was synthesized in 24 h reaction from the commercialω-hydroxy dodecanoic acid. Finally, 1.4 g/L 1,12-dodecanediol was produced in a consecutive approach from dodecanoic acids. The results of this study demonstrated the scope of the potential development of bioprocesses to substitute the petroleum-based products in the polymer industry.

Vegetable oil derivatives (e.g., FFAs) are important renewable resources [1,2].Unfortunately, limited attention has been paid to synthesize diols from FFAs that can serve as good starting materials for green processes, including the production of α,ω-diols [13,15].We describe herein the production of medium-to long-chain α,ω-diols from FFAs using CYP153A monooxygenase, carboxylic acid reductase (CAR) and E. coli endogenous aldehyde reductases (ALRs) (Figure 1).First, hydroxylation of FFAs was carried out using CYP153A33 from Marinobacter aquaeolei (MaqCYP153A33), a promising biocatalyst due to its exceptional regioselectivity (>95%) for the ω-position [16].Next, a catalytically efficient carboxylic acid reductase (CAR) from Mycobacterium marinum (MmCAR) [17] recently reported was used for selective reduction of carboxylic acid functional group of ω-hydroxy FFAs to corresponding aldehydes.It exhibited high catalytic efficiency (kcat/Km) against benzoic acid and the C 6 -C 16 fatty acids [17].However, microbial hosts such as E. coli genome encoded for 44 endogenous aldehyde reductases (ALRs), and among them 13 are highly active that converted aldehydes to alcohols ranging from C 2 to C 18 [18,19].To efficiently convert ω-hydroxy fatty aldehydes to α,ω-diols, E. coli cells as a source of endogenous ALRs were used.
for the production of α,ω3-diols in E. coli by employing engineered P450BM3 [9].Microbial synthesis of medium-to long-chain α,ω-diols suffers major challenges such as low product yields, low productivities, and narrow substrate range of enzymes used [9,13,15].Although some preparative-scale reactions have been reported [13], novel approaches are highly desirable to overcome bottlenecks in microbial synthesis of α,ω-diols.In this light, here we report highly efficient and sequential biocatalytic conversion of free fatty acids (FFAs) to medium-to long-chain α,ω-diols.

Establishment of MmCAR Reaction System
CARs need to undergo posttranslational activation via phosphopantetheinylation using phosphopantetheinyl transferase (PPTase) [17,[20][21][22].Therefore, MmCAR was co-expressed (Figure S1) with surfactin phosphopantetheinyl transferase (Sfp), a PPTase from Bacillus subtilis.SDS-PAGE analysis confirmed the production of MmCAR and Sfp recombinant proteins in soluble form (Figure S2).Whole-cell (0.3 gCDW/mL) reaction was carried out at 30 °C and 200 rpm using MmCAR with or without co-expressing Sfp cells in potassium phosphate buffer (100 mM, pH 7.5) in the presence of 1% (w/v) glucose and 10 mM MgCl2.Co-expressed cells produced 9.2 mM of benzyl alcohol.However, cells only expressing MmCAR synthesized 0.5 mM of benzyl alcohol (Figure S3).In both cases, only negligible amount of benzylaldehyde was detected (data not shown).This result clearly suggested that Sfp had a pivotal role in the activation of MmCAR and that activities of endogenous ALRs were sufficiently high enough to produce alcohols (Figure S4).MmCAR-Sfp co-expressed cells

Establishment of MmCAR Reaction System
CARs need to undergo posttranslational activation via phosphopantetheinylation using phosphopantetheinyl transferase (PPTase) [17,[20][21][22].Therefore, MmCAR was co-expressed (Figure S1) with surfactin phosphopantetheinyl transferase (Sfp), a PPTase from Bacillus subtilis.SDS-PAGE analysis confirmed the production of MmCAR and Sfp recombinant proteins in soluble form (Figure S2).Whole-cell (0.3 g CDW /mL) reaction was carried out at 30 • C and 200 rpm using MmCAR with or without co-expressing Sfp cells in potassium phosphate buffer (100 mM, pH 7.5) in the presence of 1% (w/v) glucose and 10 mM MgCl 2 .Co-expressed cells produced 9.2 mM of benzyl alcohol.However, cells only expressing MmCAR synthesized 0.5 mM of benzyl alcohol (Figure S3).In both cases, only negligible amount of benzylaldehyde was detected (data not shown).This result clearly suggested that Sfp had a pivotal role in the activation of MmCAR and that activities of endogenous ALRs were sufficiently high enough to produce alcohols (Figure S4).MmCAR-Sfp co-expressed cells were then studied further using various FFAs.Whole-cell (0.3 g CDW /mL) reactions were performed using 10 mM octanoic acid (C 8 ), decanoic acid (C 10 ), dodecanoic acid (C 12 ), tetradecanoic acid (C 14 ), and hexadecanoic acid (C 16 ).The amount of produced 1-octanol, 1-decanol, 1-dodecanol, 1-tetradecanol, and 1-hexadecanol was 1.8, 9.8, 9.9, 1.6, and 0.6 mM, respectively (Figure S5).GC analysis revealed that there was no accumulation of aldehydes during the reaction process, implying that endogenous ALRs were sufficient enough to reduce aldehydes to alcohols.This can be explained by the reduction of carboxylic acid functional groups of FFAs to their aldehyde counterparts and concomitant reduction of these aldehydes to alcohols by ALRs (Figure S4).To confirm ALR-mediated reduction of aldehydes, a separate reaction was carried out using E. coli BW25113 (∆fadD, DE3) host cells.Complete conversion of 10 mM dodecanal and benzaldehyde to 1-dodecanol and benzyl alcohol, respectively, was achieved in 6 h, implying that endogenous ALRs from E. coli BW25113 (∆fadD, DE3) host cells had catalytic efficiency (Figure S6).These results were consistent with those of Akhtar et al. (2013) showing that alcohols (C 8 -C 18 ) could be produced over a short time period (5 h) using MmCAR when natural oils were used as sources of FFAs [17].

pH Optimization
To examine the effect of pH on bioconversion of ω-OHDDA to 1,12-dodecanediol using MmCAR, whole-cell reaction was performed at three different pH (6.5, 7.5 and 8.5).Substrate concentration was increased to 30 mM.Optimum activity was measured at pH 7.5, with 19.5 mM product formed in 24 h, whereas only 7.4 and 9.7 mM of products were synthesized at pH 6.5 and 8.5, respectively (Figure S7).Higher concentration of ω-OHDDA (beyond 30 mM) resulted in decreased productivity.This might be due to the toxic effects of higher concentrations of ω-OHDDA to living cells by [23].

Cofactor Regeneration
Regenerations of nicotinamide cofactors (NADPH) and adenosine triphosphate (ATP) are very important in CAR reactions to restore consumed cofactors.For regeneration of ATP, several enzymatic reactions have been reported including PPK2 which mainly catalyzes polyphosphate-dependent phosphorylation [21].Whole-cell reaction was performed in the above mentioned conditions employing Arthrobacter aurescens PPK2 co-expressing MmCAR and Sfp with an additional use of 2 mM polyP [21].In 24 h of reaction, produced amount of 1,12-dodecanediol was 22.5 mM (4.6 g/L) (Figure 3).To examine the effect of pH on bioconversion of ω-OHDDA to 1,12-dodecanediol using MmCAR, whole-cell reaction was performed at three different pH (6.5, 7.5 and 8.5).Substrate concentration was increased to 30 mM.Optimum activity was measured at pH 7.5, with 19.5 mM product formed in 24 h, whereas only 7.4 and 9.7 mM of products were synthesized at pH 6.5 and 8.5, respectively (Figure S7).Higher concentration of ω-OHDDA (beyond 30 mM) resulted in decreased productivity.This might be due to the toxic effects of higher concentrations of ω-OHDDA to living cells by [23].

Cofactor Regeneration
Regenerations of nicotinamide cofactors (NADPH) and adenosine triphosphate (ATP) are very important in CAR reactions to restore consumed cofactors.For regeneration of ATP, several enzymatic reactions have been reported including PPK2 which mainly catalyzes polyphosphate-dependent phosphorylation [21].Whole-cell reaction was performed in the above mentioned conditions employing Arthrobacter aurescens PPK2 co-expressing MmCAR and Sfp with an additional use of 2 mM polyP [21].In 24 h of reaction, produced amount of 1,12-dodecanediol was 22.5 mM (4.6 g/L) (Figure 3).For regeneration of NADPH in MmCAR-catalyzed reactions, a Bacillus subtilis glucose dehydrogenase (GDH) was co-expressed to ensure constant supply of cofactors the whole-cell biotransformation processes [24].After 24 h of reaction, 24.7 mM (5.0 g/L) of 1,12-dodecanediol was produced from 30 mM substrate, higher than the yield of MmCAR-PPK2 system (Figure 3).The production of 1,12-dodecanediol showed a substantial improvement (66-fold) compared to that reported by Fujii et al. (2006) who demonstrated 79 mg/L product from 1-dodecanol using E. coli expressing CYP153A from Acinetobacter sp.OC4 [10].Results of these experiments clearly demonstrated that the carboxylic acid functional group of ω-OHFAs was reduced to the aldehyde functional group by whole-cell catalysts expressing CAR, Sfp and GDH where the aldehyde functional group was further reduced to the alcoholic functional group by E. coli endogenous ALRs.For regeneration of NADPH in MmCAR-catalyzed reactions, a Bacillus subtilis glucose dehydrogenase (GDH) was co-expressed to ensure constant supply of cofactors the whole-cell biotransformation processes [24].After 24 h of reaction, 24.7 mM (5.0 g/L) of 1,12-dodecanediol was produced from 30 mM substrate, higher than the yield of MmCAR-PPK2 system (Figure 3).The production of 1,12-dodecanediol showed a substantial improvement (66-fold) compared to that reported by Fujii et al. (2006) who demonstrated 79 mg/L product from 1-dodecanol using E. coli expressing CYP153A from Acinetobacter sp.OC4 [10].Results of these experiments clearly demonstrated that the carboxylic acid functional group of ω-OHFAs was reduced to the aldehyde functional group by whole-cell catalysts expressing CAR, Sfp and GDH where the aldehyde functional group was further reduced to the alcoholic functional group by E. coli endogenous ALRs.

Production of α,ω-Diols from FFAs
2.4.1.Production of α,ω-Diols by One-Pot, One-Step Reaction 'One-pot, one step' reaction is desirable for cascade reaction.To start the one-pot, one-step reaction, E. coli cells expressing MaqCYP153A33/CamAB for ω-hydroxylation of DDA and E. coli cells expressing MmCAR-GDH/Sfp were grown separately.After the expression, both types of cells were harvested, and the cell pellets were washed, and re-suspended in 100 mM potassium phosphate buffer with 1% (w/v) glucose (pH: 7.5).A total of 0.3 gDCW/mL of whole-cell was used in the reaction with 1:1 ratio.The reaction was initiated by adding 10 mM DDA (stock in DMSO, 5% (v/v) final concentration), and 10 mM MgCl 2 .The reactants were incubated at 30 • C and 200 rpm.After 15 h of whole-cell reaction, 6.8 mM 1-dodecanol and 1.0 mM 1,12-dodecanediol was produced, respectively (Figure 4).These findings suggest that one-pot, one-step reaction of FFAs predominantly produced fatty alcohols rather than α,ω-diols.This is because, FFAs acted as common active substrates for both MmCAR and MaqCYP153A33 enzymes where MmCAR was more active than MaqCYP153A33.Consequently, one-pot reaction was not feasible for the production of α,ω-diols from FFAs.Therefore, next we tried sequential reaction.In first step, ω-OHFAs were produced from FFAs by MaqCYP153A33/CamAB and in second step α,ω-diols were synthesized from biotransformed ω-OHFAs by MmCAR/Sfp.E. coli cells expressing MmCAR-GDH/Sfp were grown separately.After the expression, both types of cells were harvested, and the cell pellets were washed, and re-suspended in 100 mM potassium phosphate buffer with 1% (w/v) glucose (pH: 7.5).A total of 0.3 gDCW/mL of whole-cell was used in the reaction with 1:1 ratio.The reaction was initiated by adding 10 mM DDA (stock in DMSO, 5% (v/v) final concentration), and 10 mM MgCl2.The reactants were incubated at 30 °C and 200 rpm.After 15 h of whole-cell reaction, 6.8 mM 1-dodecanol and 1.0 mM 1,12-dodecanediol was produced, respectively (Figure 4).These findings suggest that one-pot, one-step reaction of FFAs predominantly produced fatty alcohols rather than α,ω-diols.This is because, FFAs acted as common active substrates for both MmCAR and MaqCYP153A33 enzymes where MmCAR was more active than MaqCYP153A33.Consequently, one-pot reaction was not feasible for the production of α,ω-diols from FFAs.Therefore, next we tried sequential reaction.In first step, ω-OHFAs were produced from FFAs by MaqCYP153A33/CamAB and in second step α,ω-diols were synthesized from biotransformed ω-OHFAs by MmCAR/Sfp.
Finally, to check the utility of these sequential biocatalytic process, the production of 1,12-dodecanediol was studied using biotransformed ω-OHDDA as a substrate in all three systems (Figure 7).However, owing to lower productivity of ω-OHDDA (8.2 mM) from MaqCYP153A33 reactions, only 7 mM of biotransformed ω-OHDDA was used for subsequent whole-cell reactions involving MmCARs.Nevertheless, the highest rate (96%) was achieved in MmCAR-GDH system after 15 h of reaction, with production yield of 1.4 g/L (Figure 7).

Plasmid Construction and Gene Manipulation
All bacterial strains, plasmid vectors and custom designed oligonucleotides used in this study are listed in Table 1.Genomic DNA of Mycobacterium marinum ATCC ® BAA535™ was obtained from American Type Culture Collection (ATCC).Gene encoding M. marinum carboxylic acid reductase (CAR, UniProt: B2HN69) was amplified from genomic DNA using a PCR thermocycler (Veriti ® 96-Well Thermal Cycler; AB Applied Biosystems, Foster City, CA, USA) and cloned into pETDuet-1 first multiple cloning site (MCS1).A phosphopantetheinyl transferase, Sfp (GI: P39135) from Bacillus subtilis and polyphosphate kinase 2, PPK2 (GI: ABM08865.1)from Arthrobacter aurescens genes were chemically synthesized and codon-optimized for E. coli.Synthesis and codon optimization were performed by Cosmo Genetech (Cosmo Genetech, Seoul, Korea).PPK2 and glucose dehydrogenase (GDH) gene of Bacillus subtilis were cloned into the second multiple cloning site (MCS2) of pETDuet-1 vector separately with CAR.Sfp was cloned into pET24ma vector.All DNA manipulations for cloning were performed following standard protocols [26].E. coli DH5α was used for cloning purposes.All primers used in this study were purchased from Cosmo Genetech.They are listed in Table 1.

Protein Expression and Biotransformation
Previously reported E. coli BW25113 (DE3) ∆fadD strains [25] were utilized for biotransformation studies wherein fatty acid degrading β-oxidation pathway was blocked.Recombinant strains were obtained by introducing plasmid DNA into host strains via standard heat shock method of transformation.
Transformants were selected based on their antibiotic resistance [26].Cultivations were carried out at 37 • C. Fresh colonies from agar plates of E. coli BL21 (DE3) transformed with each plasmid vector were cultured 10 mL of Luria-Bertani (LB) medium containing 50 µg/mL of kanamycin (for pCYP153A33, and pSFP) and/or 100 µg/mL ampicillin (for pCamAB, pMmCAR, pCAR-PK2, and pCAR-GDH) at 37 • C overnight.These overnight pre-cultured cells were then inoculated into larger volume flasks for expression.They were cultured at 37 • C until cell concentration reached an optical density at 600 nm (OD 600 ) of 0.6-0.8 for IPTG induction.MaqCYP153A33 and CamAB protein expression were carried out in 1 L of Terrific-Broth (TB) in a 3 L flask.The induction was performed by adding 0.01 mM IPTG, 0.5 mM 5-ALA as heme precursor, and 0.1 mM FeSO 4 at 30 • C for 16 h.MmCAR with or without PPK2/GDH and SFP protein expression were carried out in 1 L of LB medium in 3 L flasks.Induction was performed by adding 0.1 mM IPTG at 20 • C for 16 h.
After expression, cells were harvested by centrifugation (4000 rpm, 20 min, 4 • C), washed with PBS, and resuspended in 100 mM potassium phosphate buffer (pH 7.5) containing 1% (w/v) glucose.Whole-cell reaction (0.3 g CDW /mL) was initiated by adding respective substrates (stock in DMSO, 5% (v/v) final concentration or cell free reaction mixtures containing non-purified ω-OHFAs).These reactants were incubated at 30 • C and 200 rpm.For whole-cell reaction of MmCAR, 10 mM MgCl 2 was added during the addition of substrates.Every 6th h, the pH of the reaction mixture was measured with a pH meter and adjusted to pH 7.5 with 5 M KOH.Then, 50 µL of 80% (w/v) glucose was added.
To prepare the non-purified ω-OHFAs substrate for the sequential reaction of MmCAR, and ALRs, the whole-cell reactions of MaqCYP153A33 were stopped after 24 h, and acidified with 6 M HCl.The cells were removed by centrifugation at 16,000 rpm.The clear supernatant thus obtained was used for the further biotransformation without any purification step.The presented amount of produced ω-OHFAs in the reaction mixtures were determined by GC analysis (vide infra).The pH of the reaction mixture was readjusted to 7.5 for the next step bioconversion.Cells containing MmCAR reaction system were resuspended in the reaction mixture, and potassium phosphate buffer (100 mM, pH 7.5) was added again on the basis of ω-OHFAs concentration.To compensate the depleted glucose in earlier reaction, again 1% (w/v) glucose was added, and the whole-cell reaction was carried out as mentioned above.

Analysis of Compounds by Gas Chromatography
Quantitative analysis was performed using a gas chromatography instrument (GC 2010 plus Series) with a flame ionization detector (GC/FID) fitted with an AOC-20i series auto sampler injector (Shimadzu Scientific Instruments, Kyoto 604-8511, Japan).Two microliters of the sample were injected by split mode (split ratio 20:1) and analyzed using a nonpolar capillary column (5% phenyl methyl siloxane capillary 30 m × 320 µm i.d., 0.25-µm film thickness, HP-5).Oven temperature program for fatty acid analysis was follows: 50 • C for 1 min, increase to 250 • C at 10 • C/min, and hold for 10 min.The inlet temperature was 250 • C while the detector temperature was 280 • C. The flow rate of the carrier gas (He) was at 1 mL/min.Flow rates of H 2 , air, and He in FID were 45 mL/min, 400 mL/min, and 20 mL/min, respectively.The initial oven temperature was 90 • C. It was then increased to 250 • C by 15 • C/min and held at this temperature for 5 min.Each peak was identified by comparing GC chromatogram with an authentic reference.Internal standards were added to the sample before extraction of the substrate.

Conclusions
In summary, this work demonstrated the first gram-scale bioconversion of FFAs to mediumto long-chain α,ω-diols as high-value building blocks for synthesis of polyamides, polyesters, and polyurethanes.The applicability of in vivo cofactor regeneration systems for cost-effective biocatalytic production of α,ω-diols was also demonstrated.This study suggests that further bioconversion of α,ω-diols to corresponding diamines and diacids is possible through green process [10,11,13].Furthermore, optimization of the MaqCYP153A33 catalyzed reaction for ω-hydroxylation of FFAs could facilitate higher product titres from FFAs to α,ω-diols.

Table 1 .
Plasmids and strains used in this study *.