Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid
© Sun et al; licensee BioMed Central Ltd. 2011
Received: 11 July 2011
Accepted: 13 September 2011
Published: 13 September 2011
Mandelic acid (MA), an important component in pharmaceutical syntheses, is currently produced exclusively via petrochemical processes. Growing concerns over the environment and fossil energy costs have inspired a quest to develop alternative routes to MA using renewable resources. Herein we report the first direct route to optically pure MA from glucose via genetic modification of the L-phenylalanine pathway in E. coli.
The introduction of hydroxymandelate synthase (HmaS) from Amycolatopsis orientalis into E. coli led to a yield of 0.092 g/L S-MA. By combined deletion of competing pathways, further optimization of S-MA production was achieved, and the yield reached 0.74 g/L within 24 h. To produce R-MA, hydroxymandelate oxidase (Hmo) from Streptomyces coelicolor and D-mandelate dehydrogenase (DMD) from Rhodotorula graminis were co-expressed in an S-MA-producing strain, and the resulting strain was capable of producing 0.68 g/L R-MA. Finally, phenylpyruvate feeding experiments suggest that HmaS is a potential bottleneck to further improvement in yields.
We have constructed E. coli strains that successfully accomplished the production of S- and R-MA directly from glucose. Our work provides the first example of the completely fermentative production of S- and R-MA from renewable feedstock.
Mandelic acid (MA), an important fine chemical, has been widely used in the synthesis of cephalosporin antibiotics , the preparation of various other pharmaceuticals [2–4], as well as the resolution of racemic alcohols and amines [5, 6]. Its stereoisomers generally are prepared using a chemical approach [7–9]. However, it has also been reported that its stereoisomers can be prepared via enzymatic routes; for example, stereospecific nitrilases are used to produce S- or R-MA [10–13] and enantioselective microbes are employed to degrade S-MA or R-MA to yield R- or S-enantiomers [14–16], respectively. In addition, a system combining the two microbes Pseudomonas polycolor and Micrococcus freudenreichii has also been explored for the production of R-MA from the racemate . However, these commodity chemicals are manufactured entirely from petroleum-based feedstock such as benzaldehyde and mandelonitrile. Growing concerns over the environment and fossil resources costs have inspired a quest to develop more sustainable processes that afford these products from renewable feedstock at lower cost. Thus, biological processes based on renewable resources such as glucose that would provide direct one-step production of chiral MA in a microbial fermentation process would be of commercial interest.
Thus far, no direct biosynthetic pathway to free chiral MA has been identified in nature, although some organisms possess an MA catabolic pathway . Engineering a microbe for the production of a heterologous compound requires the establishment of a new biochemical pathway. To construct synthetic pathways for producing S-MA and R-MA in E. coli, two problems need to be resolved. Firstly, suitable starting intermediates must be available. Secondly, the introduction of enzymes catalyzing reactions to produce the desired products must be feasible.
In this study, we have modified E. coli strains that successfully achieved the production of S- and R-MA directly from glucose. To our knowledge, this is the first report describing the engineering of the L-phenylalanine pathway in E. coli to produce enantiomerically pure S-MA and R-MA directly from a renewable resource such as glucose.
Fermentation of glucose via expression of HmaS in E. coli to produce S-MA directly
In order to construct a pathway to produce S-MA via a modified L-phenylalanine pathway in E. coli, an enzyme with S-mandelate synthesis activity is required to convert phenylpyruvate to S-MA. Thus, HmaS from A. orientalis was cloned and over-expressed in E. coli; the specific activity of HmaS was determined to be 10.18 mU/mg total protein. Production of MA in the reaction broth was identified by HPLC equipped with a chiral column, and the results illustrated that HmaS was able to convert phenylpyruvate to S-MA (Additional file 1), but not for the control (Additional file 1). Surprisingly, trace amounts of R-MA were detected as well, the results revealing that HmaS is not a rigorous stereo-inverting enzyme for catalyzing phenylpyruvate to S-MA. The enantiomeric excess (ee), > 98%, was calculated according to a previous report . Therefore, this enzyme could be used further for the preparation of S-MA in E. coli.
To produce S-MA in E. coli, wild-type strains of E. coli W3110 harboring pSUFAAQ were cultured in fermentation medium in shake flasks. This resulted in the production of 0.092 g/L S-MA within 24 h, and a large amount of L-phenylalanine was detected as well. This proved that the artificial operon was functionally expressed in E. coli. Therefore, this strain could be used as a basis strain for further improvement to the production of S-MA directly from glucose. The biosynthetic pathway for S-MA is shown schematically in Figure 1.
Combined deletion of competing pathways to optimize the production of S-MA
To further increase the carbon flux to phenylpyruvate, the branch pathways of L-tyrosine and L-tryptophan were disrupted based on tyrB and aspC double mutants (BC) to reduce divergence from chorismate. Then we deleted the chorismate mutase/prephenate dehydrogenase encoded by tyrA and the anthranilate synthase encoded by trpE. As expected, for tyrA or/and trpE deletion based on double mutants, the production of S-MA increased to 0.30 g/gDCW (BCAE) with a 2-fold increase compared to BC and a 5-fold increase compared to wild-type.
We still detected amounts of phenylpyruvate (approximately 0.02 g/gDCW) that accumulated from BC to BCAE (Figure 3), which implied that the activity of HmaS was insufficient to convert all precursors of phenylpyruvate to S-MA. We postulate that combined deletion of the competing pathways led to increased phenylpyruvate accumulation and resulted in driving HmaS to synthesize more S-MA. Therefore, HmaS might be a potential bottleneck for further optimization of S-MA production and its derivatives.
Characterization of the fermentation of recombinant strain BCAE/pSUFAAQ
In addition, 1.95 g/L of the byproduct acetic acid was detected in 24 h, and the titer significantly increased to 6.87 g/L within 84 h (Figure 4B). Acetic acid accumulation could cause many obstacles to the industrial production of interesting products. To minimize acetate formation, many strategies have been developed for this purpose during E. coli aerobic fermentations .
Use of Hmo together with DMD to convert S-MA via phenylglyoxylate to R-MA in vitro
Thus far, no enzyme with R-mandelate synthase activity has been identified that could catalyze the precursor of the amino acid to R-MA. Therefore, to promote fermentation production of R-MA in vivo, two other enzymes are required to convert S-MA via phenylglyoxylate to R-MA. In this study, Hmo from S. coelicolor and DMD from R. graminis were cloned and expressed in E. coli. Then, the specific activities of Hmo and DMD were determined to be 0.257 mU/mg and 87.44 mU/mg, respectively. These enzymes were used for further studies.
To test whether Hmo together with DMD could transform S-MA to R-MA in vitro, these two genes were cloned into a vector of pTrc99a under IPTG inducible promoter Ptrc resulting in pTrc99aSD. Then an assay for the simultaneous catalysis of S-MA to R-MA via the enzymes Hmo and DMD was performed in vitro. As shown in Additional file 1, R-MA was detected, but not for the control (Additional file 1). Thus, the conversion of S-MA via phenylglyoxylate to R-MA proved the functional expression of these two enzymes in E. coli, and paved the way for further synthesis of R-MA using their activities in vivo.
Co-expression of hmaS, hmo, and dmd in engineered E. coli to produce R-MA directly from glucose fermentation
We have demonstrated that expression of HmaS in E. coli can ferment glucose to produce S-MA and proved that the simultaneous expression of Hmo and DMD can convert S-MA via phenylglyoxylate to R-MA in vitro. Therefore, we presumed that co-expression of HmaS, Hmo, and DMD in E. coli could produce R-MA from glucose directly.
To produce R-MA ultimately from glucose, a new artificial biosynthetic pathway was assembled in E. coli. The scheme of this new pathway is shown in Figure 1. Based on the findings described above, we constructed an artificial biosynthetic pathway containing HmaS, Hmo, and DMD, with the corresponding genes obtained from A. orientalis, S. coelicolor, and R. graminis, respectively. Then, an engineered E. coli strain BCAE with the recombinant plasmid pSUFAAQSD (Figure 2B) was created.
Batch cultivations of BCAE/pSUFAAQSD were performed in shake flasks. During the fermentation process, cell density, R-MA production, glucose assimilation, and accumulation of the byproduct acetic acid were monitored (Figure 4C and 4D). The results illustrated that 0.68 g/L of R-MA with a yield of 7.8% (g/g) from glucose was achieved within 24 h and reached 37% of theoretical maximum, and the titer of R-MA reached 0.88 g/L in 84 h. The fermentation broth was analyzed by HPLC equipped with a chiral column (Additional file 2), and the products were further identified by GC-MS (Additional file 4). Combining these data showed that the compound in the broth was confirmed as R-MA.
Therefore, in this study we demonstrated that combined expression of the three enzymes in engineered E. coli strains resulted in the first completely fermentative synthesis of R-MA directly from glucose.
HmaS might be a potential bottleneck for further improvement of chiral MA production
Furthermore, the kinetics parameters of HmaS were measured: the Km of HmaS was 0.45 ± 0.04 mM with phenylpyruvate as substrate, which was approximately 70-fold greater than the Km with its native substrate 4-hydroxyphenylpyruvate (6.5 ± 0.8 μM) . This indicates that more phenylpyruvate is needed, compared to its native substrate, to achieve the same effect for HmaS synthesis of the corresponding products. Therefore, the Km of HmaS for phenylpyruvate needs to be modified to improve the catalytic efficiency at lower phenylpyruvate concentrations. This would be useful not only in terms of HmaS competing with other enzymes for catalyzing phenylpyruvate to S-MA, but would also be beneficial in alleviating phenylpyruvate toxicity towards the host at lower concentrations.
Therefore, in combining these data we infer that HmaS will be an important target for further strain improvement to enhance S-MA and R-MA production.
Chiral mandelic acid, an important medical intermediate, is mainly prepared by chemical or enzymatic approaches. In this study, a new fermentative route was created using an engineered E. coli to produce optical isomers based on renewable resources. The L-phenylalanine pathway was modified and an artificial pathway using the enzymes HmaS, Hmo, and DMD from A. orientalis, S. coelicolor, and R. graminis, respectively, was constructed.
Many enzymatic methods have been reported for the preparation of stereoisomers [10, 13]. However, none of them were able previously to produce mandelate enantiomers from glucose directly in a single microbial fermentation step. Thus far, substrates of enzymatic routes such as benzaldehyde and mandelonitrile have not been identified among the microbial primary metabolic pathways. If in the future some enzymes are discovered for catalyzing the primary metabolites to benzaldehyde or mandelonitrile, such multistep enzyme-catalyzed processes might be developed in the microbe for the production of mandelate isomers from glucose. However, the conversion rate would not be superior to that in this report due to the requirement for more heterogeneous enzymes and the extra reaction steps required.
Due to a lack of R-mandelate synthase, which could catalyze phenylpyruvate to R-MA directly, a relatively complex artificial pathway was constructed in this work for the production of R-MA. If R-mandelate synthase were available, the method for preparation of R-MA described here would be optimized using R-mandelate synthase only. We tried to find this kind of enzyme in some microorganisms using bioinformatics means. Unfortunately, thus far we have not been able to find it due to limited resources; however, it should be possible to construct enzymes with R-mandelate synthase activity through directed evolution.
Although we have successfully achieved the preparation of S-MA and R-MA in engineered E. coli, the byproduct of L-phenylalanine was still formed by the double mutant. It could be that the branched-chain amino acid aminotransferase encoded by ilvE is able to catalyze phenylpyruvate to L-phenylalanine , so the deletion of ilvE should eliminate L-phenylalanine formation to optimize the production of S-MA and R-MA in the future.
We observed the accumulation of large quantities of acetic acid during the fermentation process (Figure 4B and 4D). Acetic acid excretion by E. coli during aerobic growth on glucose is a major obstacle to recombinant protein production and chemical products, and gene disruptions have been successfully employed to minimize acetic acid formation and increase the production level of desired chemicals [27–30]. In this work, to improve MA production, we have deleted acetate-forming genes including poxB, pta, ackA, and acs to reduce acetic acid formation. Results showed that single gene deletions did not reduce acetate accumulation, suggesting that alternative pathways were also involved in acetate production (Additional file 5). As expected, simultaneous deletion of poxB, ackA and acs greatly minimized acetate accumulation (Additional file 6). However, the acetate reduction led to no increase in the carbon flux to MA production, which might be caused by significantly reduced cell growth rates.
In order to improve the production of S-MA, two genes (ppsA and tktA)  were co-expressed in the engineered S-MA-producing strains to enhance the supply of precursors of PEP and E4P. In the resulting strains the productivity of S-MA improved whereas the titer did not improve (data not shown). This result indicated that some other bottleneck exists downstream of the S-MA synthesis pathway.
HmaS is not only a key enzyme for S-MA synthesis but is also the first step in R-MA synthesis. We have previously investigated the effects of different mutants on S-MA production. The results implied that the activity of HmaS was insufficient to convert all phenylpyruvate to S-MA, leading to the accumulation of phenylpyruvate in the culture. To further explore the activity of HmaS, we also conducted phenylpyruvate feeding experiments. Although higher concentrations of phenylpyruvate slightly enhanced the yield of S-MA, the conversion rate of HmaS was significantly reduced with increasing phenylpyruvate. Furthermore, we measured the kinetic parameters of HmaS and found that its affinity towards the non-natural substrate phenylpyruvate was much lower than that towards its natural substrate . This suggests that, in order to synthetize S-MA, HmaS needs a higher concentration of the substrate phenylpyruvate as the driving force. Therefore, combining these data we infer that HmaS will potentially be the bottleneck for further improvement of S-MA and R-MA synthesis. Thus, the catalytic activity of HmaS for converting phenylpyruvate to S-MA should be improved first through protein engineering to further improve the titers and yields of S-MA and R-MA production.
We have established a functional artificial pathway in E. coli and developed the first completely fermentative route for producing optically pure S-MA and R-MA from glucose, though further strain improvement and fermentative process optimization, including metabolic flux analysis, will be needed to improve the production of S-MA and R-MA. The successful construction of S-MA- and R-MA-producing E. coli in this study provides a solid basis for the development of other engineered bacteria, which could yield other commercially attractive bio-products or their derivatives from renewable resources.
Bacterial strains and culture media
Strains and plasmids used in this study
Wild type SCP1- SCP2-
Rhodotorula graminis di Menna, anamorph, mutant derived from KGX39
lacZ ΔM15 endA1 recA1 relA1 gyrA96 deoR nupG λ-
endA1 glnV44 sbcB15 rpsL thi-1 Δ(lac-proAB) [F' traD36 proAB + lacI q lacZ ΔM15] hsdR 4 (rK-mK+)
F- omp gal dcm lon hsdS B (rB-mB-) λ(DE3)
F- λ- ΔlacZ4787 (::rrnB-3), ΔtrpE:: FRT-kan- FRT, rph-1 hsdR514
Wild type E. coli W3110 F- λ- rph-1 INV (rrnD rrnE)
W3110 ΔtyrB::FRT, ΔaspC::FRT
W3110 ΔtyrB::FRT, ΔaspC::FRT, tyrA16::Tn10
W3110 ΔtyrB::FRT, ΔaspC::FRT, ΔtrpE::FRT
W3110 ΔtyrB::FRT, ΔaspC::FRT, tyrA16::Tn10, ΔtrpE::FRT
kan PT7 expression vector
pET24a derivative, hmaS Ao from A. orientalis
pET24a derivative, dmd from R. graminis
bla P trc lacI q pBR322 ori expression vector
pTrc99a derivative, hmo
pTrc99a derivative, hmo, dmd
bla P tac pBR322 ori expression vector
pKK223-3 derivative, pheA fbr from E. coli
Hae II fragment of pUC18 containing lacZα and MCS, p15A replicon with chloramphenicol acetyl transferase gene (cat)
pSU2718 derivative, aroF fbr from E. coli
pSU2718 derivative, aroF fbr , pheA fbr
pSU2718 derivative, aroF fbr , pheA fbr , lacI q
pSU2718 derivative, aroF fbr , pheA fbr , lacI q , hmaS Ao
pSU2718 derivative, aroF fbr , pheA fbr , lacI q , hmaS Ao , hmo, dmd
araC ParaBAD gam bet exo repA 101ts oriR 101 cat
t s bla cat
LB medium was used for culturing the general strains for cloning. The engineered E. coli strains were cultivated in shake flasks in fermentation medium to test S- or R-MA production in vivo.
The fermentation medium contained the following components: D-glucose (20 g/L), KH2PO4 (1.0 g/L), (NH4)2SO4 (16 g/L), MgSO4 7H2O (1.0 g/L), L-tyrosine (0.2 g/L), L-tryptophan (0.1 g/L), L-aspartic acid (3 g/L), FeSO4 7H2O (0.01 g/L), MnSO4 H2O (0.008 g/L), CaCO3 (20 g/L). The stock solution of D-glucose, CaCO3, and MgSO4 was autoclaved separately whereas L-tryptophan, FeSO4, and MnSO4 were sterilized using a 0.22-μm filter.
The gene encoding feedback resistant mutant aroF fbr P148L  including the native promoter was obtained by overlap PCR, and the genomic DNA of E. coli W3110 was used as the PCR template. Two overlapping DNA fragments were amplified using primer pairs P148L-F/aroFSacI-R and P148L-R/aroFSacI-F, respectively. Then, using the two overlapping DNA fragments as the templates, another PCR reaction was performed with primer pair aroFSacI-F/aroFSacI-R and the whole length mutant aroF fbr gene was obtained. The resulting product was digested with Sac I and cloned into pSU2718 cut with the same enzyme, creating pSUaroFfbr.
The gene encoding feedback resistant mutant pheA fbr G309C  was constructed with the primers pheA-M-F/pheA-M-R and pheA-MN-FM/pheA-M-RM using overlap PCR (as described above). The purified PCR fragments of mutant pheA fbr were double digested with Eco R I and Hin d III and then cloned into pKK223-3, creating pKKpheAfbr. Then the gene pheA fbr with the promoter Ptac and RBS (ribosome binding site) was amplified from pKKpheAfbr with primers PTacpheA*-F and PTacpheA*-R. The resulting PCR product was ligated into pSUaroFfbr vector, creating pSUFA.
The gene lacI q was amplified from pTrc99a with primers lacIq-F and lacIq-R. Then, it was cut with Nhe I and ligated into the same site of pSUFA, creating pSUFAQ.
The gene hmaS was amplified from the genomic DNA of A. orientalis HCCB10007 (GenBank: HQ679900) using the primers hmaSAo-F and hmaSAo-R. The PCR fragments were digested with Nde I and Hin d III, and cloned into pET24a vector, generating pEThmaSAo. hmaS with the RBS of pET24a was amplified by PCR using primers pETrbs+hmaSAo-F and pETrbs+hmaSAo-R. The corresponding PCR fragments were purified and cut with Sal I and Bam H I, then cloned into pSUFAQ, generating the recombinant plasmid pSUFAAQ for the production of S-MA (the scheme of plasmid pSUFAAQ can be seen in Figure 2A).
The gene hmo was amplified from the genomic DNA of S. coelicolor A3(2) M145 (GenBank: AL939115) with the primers hmo-F and hmo-R. The PCR fragments were purified, then digested with Nco I and Kpn I, and cloned into pTrc99a vector, creating pTrc99ahmo.
The gene dmd was amplified from the total cDNA of R. graminis ATCC20804 (GenBank: AJ001428) with the primers dmd-F and dmd-R, and cloned into the Nde I and Eco R I sites of pET24a to create pET24admd. Then, the fragments including dmd gene and the RBS of pET24a were amplified with the primers pETrbs+dmd-F and pETrbs+dmd-R. The corresponding PCR fragments were cloned into the Kpn I and Pst I sites of pTrc99ahmo to generate pTrc99aSD. Then the DNA fragments containing genes hmo and dmd with Ptrc promoter were obtained by PCR with the primers pTrcSD-F and pTrcSD-R, and inserted into the Bam H I site of pSUFAAQ to create pSUFAAQSD for the production of R-MA (the scheme of plasmid pSUFAAQSD can be seen in Figure 2B).
Plasmids were isolated using the AxyPrep Plasmid Miniprep Kit (Axygen, Hangzhou, China). AxyPrep DNA Gel Extraction Kit was used to isolate DNA fragments from agarose gels (Axygen, Hangzhou, China). All PCR fragments were sequencing identified by BioSune (Shanghai, China). DNA polymerase KOD plus for PCR reactions was purchased from ToYoBo (Shanghai, China). All restriction enzymes and Taq DNA polymerase were purchased from Fermentas (Shanghai, China). Rapid DNA ligase and alkaline phosphatase were obtained from TaKaRa (Shanghai, China). Oligonucleotides were ordered from BioSune (Shanghai, China).
Primers used for gene cloning in this study
5'-GCTATTCAGATCTAACGCTTCCGTCGCCAGTGG - 3'
5'-AACGAGCTC ACCGGAAAGTCCTCGGGCATAAG - 3'
5'-AACGAGCTC CGACTTCATCAATTTGATCGCGTAA - 3'
5'-GGGAATTC TATGACATCGGAAAACCCGTTAC - 3'
Eco R I
5'- ATCCGGAAGCTT TTCATCAGG - 3'
Hin d III
5'- AACAAGCCTGTGCGCTGG - 3'
5'- TCAACCAGCGCACAGGCTTGTTGC - 3'
5'- GATCCGAAGCTT ATCGACTGCACG - 3'
Hin d III
5'- AGTCGAC GCTTTTCATCAGGTTGG - 3'
5'- CGCTAGC CCTGACGGGCTTGTCTG - 3'
5'- CGCTAGC TTCCGATGGCTGCCTG - 3'
5'- CGCAT ATG CAGAATTTCGAGATCGACTAC - 3'
5'- CAAGCTT AACGTACGTCATCGCCG - 3'
Hin d III
5'-GATATACC ATG G GCAGCAGCCATC-3'
5'- CCGTCGAC AAATAATTTTGTTTAACTTTAAG - 3'
5'- CGGCCGGATCC TTGAAGATCTC - 3'
Bam H I
5'-CCAT ATG CCTCGCCCTCGCGTC-3'
Eco R I
Bam H I
Bam H I
Construction of deletion mutants of E. coli
Primers used for gene deletion and verification in this study
5'-TTTAACCACCTGCCCGTAAACCTGGAGAACCATCGCGTG ATTCCGGGGATCCGTCGACC- 3'
5'-ACTGCAGGCTGGGTAGCTCCAGCCTGCTTTCCTGCATTA TGTAGGCTGGAGCTGCTTC- 3'
5'- CTGTTGCTAATTGCCGTTCG - 3'
5'- CACGTAGAACGATGGCATCA - 3'
5'-CGGACTTCCCTTCTGTAACCATAATGGAACCTCGTCATG ATTCCGGGGATCCGTCGACC- 3'
5'-AGCCCGCTTTTCAGCGGGCTTCATTGTTTTTAATGC TTA TGTAGGCTGGAGCTGCTTC- 3'
5' - CCTGCGTTTTCATCAGTAATAGTTGG - 3'
5' - CCTTATCCGGCCTACAAAATCG - 3'
5' - TATCCGTAACCGATGCCTGC - 3'
5' - GGGAAATCACCCGTTCAATG - 3'
5' - CGTACTGAAAGGTTGGTGGCG - 3'
5' - AGGAGAAAGCATCAGCACCG - 3'
Preparation of cell extract for enzymatic assays
E. coli BL21(DE3) strains harboring the plasmids pEThmaSAo, pET24admd, pTrc99ahmo or pTrc99aSD were cultivated in tubes containing 4 mL LB medium with appropriate antibiotics at 37°C at 220 rpm overnight. Then 0.5 mL of culture broth were added to 50 mL of LB medium in 250-mL shake flasks that contained appropriate antibiotics at 30°C at 220 rpm. When OD600 reached 0.4~0.6, the cells were induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After 4 h, cells were harvested and washed with 200 mM potassium phosphate buffer (pH 7.5). Then the cell pellet was resuspended in 5 mL 200 mM potassium phosphate buffer (pH 7.5), and the cells were disrupted by French Press (Constant cell disruption systems, United Kingdom) at 25 kpsi. The lysate was centrifuged at 12000 rpm for 20 min (Eppendorf Centrifage 5810R) and the supernatant was used to determine activity. As a control, E. coli BL21(DE3) containing plasmid pET24a or pTrc99a was treated in the same manner. The total protein concentration of the supernatant was determined by the Bradford method , using bovine serum albumin (BSA) as a standard.
The HmaS assay was performed as described previously , with the exception that the 5-mL reaction mixture contained 200 mM potassium phosphate buffer (pH 7.5), 5 mM phenylpyruvate, 44 mM ascorbate, 0.3 mM FeSO4, and a final concentration of 1 mg/mL of soluble protein. The reaction was started by the addition of the cell-free extract at 28°C. After addition of 100 μL 1 M HCl to 500-μL samples to stop the reaction, the solution was centrifuged at 12000 rpm for 5 min (Eppendorf Centrifage 5430), and the products were analyzed by HPLC.
Production of R-MA in vitro
In the assay for R-MA production in vitro, the 5-mL reaction mixture contained 200 mM potassium phosphate buffer (pH 7.5), 5 mM S-MA, 2 mM NADH, 40 mM NAD+, and a final concentration of 1 mg/mL of soluble protein. The reaction was started by the addition of the crude extract at 30°C. Following the addition of 100 μL 1 M HCl to 500-μL samples to stop the reaction, the solution was centrifuged at 12000 rpm for 5 min (Eppendorf Centrifage 5430), and the supernatants were analyzed by HPLC.
Production of S- or R-MA in vivo
The engineered strains BCAE containing the plasmid pSUFAAQ or pSUFAAQSD were inoculated into 4 mL LB medium with chloramphenicol (25 mg/L) and incubated at 37°C at 220 rpm overnight. 4 mL of this culture were subsequently added to 250-mL shake flasks containing 50 mL of the fermentation medium with chloramphenicol (25 mg/L) and incubated at 37°C, 220 rpm. After 4~5 h incubation when the OD600 had reached 0.4~0.6, the cells were induced by adding 0.1 mM IPTG, and then the temperature was set to 33°C. Samples were taken at different time points during cultivation and centrifuged at 4°C at 12000 rpm for 5 min (Eppendorf Centrifage 5430). Then the supernatant was frozen at -20°C and prepared for analysis by HPLC, GC, and GC-MS. Cell densities were determined by spectrophotometer OD600 (Beckman coulter DU730, USA). Dry cell weight (g/L) was calculated using a conversion coefficient of 0.375 g/L/OD600.
Phenylpyruvate feeding experiments
To test HmaS conversion of phenylpyruvate to S-MA in vivo, feeding experiments were performed. The concentration gradient was as follows: 0 mM, 1 mM, 2 mM, 4 mM, and 8 mM. The phenylpyruvate was fed when the OD600 reached 0.4~0.6, meanwhile 0.1 mM IPTG was added to the cultures inducing expression of HmaS, and the fermentation temperature was adjusted from 37°C to 33°C for 24 h. Culture samples were analyzed by HPLC.
L-phenylalanine, phenylpyruvate, and S- or R-MA were quantified using a high-performance liquid chromatography system (Agilent Technologies 1,100 series) equipped with a Zorbax Eclipse XDB-C8 column (Agilent, 4.6 mm × 150 mm, 5 μm) at 40°C. The mobile phase consisted of 50 mM phosphate sodium buffer (pH 6.5) and methanol at a volume ratio of 95:5, at a flow rate of 1 mL/min, with UV detection was 215 nm. The pure substances were used as standards.
The S- or R-MA was identified by HPLC with a CHIRALCEL OD-H column (4.6 mm × 250 mm, 5 μm; Daicel Co., Japan) at 30°C. The mobile phase consisted of hexane, isopropanol, and trifluoroacetic acid in a volume ratio of 94:6:0.2, at a flow rate of 1.2 mL/min, and the UV detection wavelength was 228 nm. The pH of samples was adjusted to 1~2 using HCl, then S- or R-MA in the broth was extracted using an equal volume of ethyl acetate.
Glucose concentrations in the broth were determined by HPLC (Model 1200, Agilent) using a Sugar-Pak TM I column (Waters Corp., MA, USA), and the refractive index detector was used .
Standard samples were racemic mandelic acid ordered from Fluka, L-phenylalanine from Tokyo Chemical Industry (TCI, Shanghai, China), and phenylpyruvate (sodium), S-MA, and R-MA from Sigma.
The concentration of acetic acid in the supernatant was determined using gas chromatography (GC) (7890A, Agilent, Wilmington, DE, USA).
S- or R-MA was further identified by gas chromatography-mass spectrometry (GC-MS). Lyophilized samples were oximated with 20 mg/mL methoxyamine hydrochloride in pyridine at 30°C for 60 min. Then 80 μL pyridine and 20 μL N-methyl-N-[tert-butyldimethylsilyl] trifluoroacetamide were added to the samples for derivatization at 70°C for 30 min. Then 3 μL of derivatized sample were injected for GC-MS analysis after filtration. The GC-MS system (Agilent 6890-5973) was equipped with an HP-5MS column (30 m, 0.25 mm, 0.25 μm), and the electron impact (EI) mode of the mass spectrometer was set to 70 eV. The GC oven temperature was initially held at 60°C and raised with a gradient of 5°C per minute until it reached 180°C. It was then raised with a gradient of 10°C per minute until it reached 260°C. The flow rate of the mobile phase was set to 1 mL/min. All reagents used in this assay were ordered from Sigma.
This work was supported by the National Natural Science Foundation of China (Funded No. 31170102) and the National Basic Research Program of China (973: 2011CBA00806).
The authors wish to express thanks to Professor Jianhe Xu, Dr. Jianping Wu and Dr. Bo Wang for their advices, and also thanks to Dr. Yinhua Lu for his improvement of the manuscripts, finally, thanks to Mr. Jun Chen, Mr. Zhijun Zhang, Mr. Liuyang Diao, Dr. Yuanheng Cai, and Dr. Dalong Zhang for their advices and assistances.
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