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De novo biosynthesis of (S)- and (R)-phenylethanediol in yeast via artificial enzyme cascades Jifeng Yuan, Benedict Ryan Lukito, and Zhi Li ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.9b00123 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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ACS Synthetic Biology

De novo biosynthesis of (S)- and (R)-phenylethanediol in yeast via artificial enzyme cascades Jifeng Yuan, Benedict Ryan Lukito, and Zhi Li* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore

* Corresponding author Email address: [email protected] (A/P Zhi Li)

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ABSTRACT Due to the oil depletion and global climate change, sustainable manufacturing of fine chemicals from renewable feedstocks has gained an increasing attention in the scientific community. In the present study, we attempted to engineer Saccharomyces cerevisiae towards de novo synthesis of (S)- or (R)-phenylethanediol, an important pharmaceutical intermediate. More specifically, the biocatalytic cascades contain: L-phenylalanine undergoes deamination/decarboxylation to styrene by using phenylalanine ammonia lyase (PAL) and ferulic acid decarboxylase (FDC), followed by S-selective epoxidation of styrene to give (S)-styrene oxide with styrene monooxygenase (SMO); regioselective hydrolysis of (S)-styrene oxide with epoxide hydrolase from Sphingomonas HXN-200 (SpEH) or from potato (StEH) gives rise to (S)- or (R)-phenylethanediol. In this work, we found the artificial enzyme cascades could be functionally expressed in the heterologous host of S. cerevisiae. Small-scale shake flask studies revealed that the engineered yeast cell factories produced approximately 100~120 mg/L of (S)- or (R)-phenylethanediol after 96 h cultivation. To the best of our knowledge, this is the first attempt to explore an artificial route with styrene as an intermediate for producing phenylethanediol in S. cerevisiae. We envision that our engineering strategy will open a new research field for synthesizing other vicinal diol derived chemicals in yeast. Key words: Artificial enzyme cascades; (S)- and (R)-phenylethanediol; styrene monooxygenase; epoxide hydrolase; de novo synthesis; Saccharomyces cerevisiae


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Sustainable manufacturing of chemicals from renewable feedstocks has gained an increasing attention because of the oil depletion and global climate change. Recently, there is a rising effort in utilizing microbial system for the synthesis of chemicals as an alternative to petroleum-based chemical synthesis. By using genetically tractable host organisms such as Escherichia coli and Saccharomyces cerevisiae, different value-added chemicals have been synthesized from inexpensive and renewable resources by large-scale industrial processes1-3. The advances of metabolic engineering and synthetic biology have enabled the bioconversion of renewable resources to produce a variety of biobased bulk chemicals. However, the bioproduction of non-natural high-value fine chemicals still faces many challenges, including the lack of efficient pathways towards the non-natural chemicals. Enantiopure phenylethanediol is an important pharmaceutical intermediate, which has been reported for the synthesis of chiral non-natural amino acids, and hydroxy acids4-6. Conventional approach for vicinal diol synthesis was achieved by Sharpless dihydroxylation and two-step reaction involving epoxidation of olefins followed by subsequent hydrolysis of epoxides7-10. However, these approaches were mostly associated with the involvement of heavy metal oxide catalyst as well as separation of unstable and toxic epoxide intermediates. Previously, we have demonstrated that a highly-active cascade biotransformation system enabled efficient conversion of biobased L-phenylalanine to one of the enantiomerically vicinal diols, (S)- or (R)-phenylethanediol via the whole-cell biocatalysis from E. coli co-expressing phenylalanine ammonia lyase (PAL), phenylacrylic acid decarboxylase (PAD), styrene monooxygenase (SMO), and epoxide hydrolase from Sphingomonas HXN-200 (SpEH) or from Solanum tuberosum (StEH)11-13.



Unlike E. coli, which is not generally recognized as safe, S. cerevisiae has been welldemonstrated for industrial-scale production of chemicals and drug precursors due to its tolerance and robustness towards industrial conditions. For example, S. cerevisiae has recently been used for high-level production of artemisinic acid14 and fatty acids-derived oleochemicals15. In addition, yeast-generated products are much more likely to be accepted to consumers. In the present study, we sought to develop S. cerevisiae cell factories towards de novo synthesis of (S)- or (R)-phenylethanediol. The novel biocatalytic cascades to synthesize enantiopure phenylethanediol from L-phenylalanine were designed as shown in Fig. 1. More specifically, the cascade reactions contain: L-phenylalanine undergoes deamination/decarboxylation to styrene via phenylalanine ammonia lyase (PAL2) from Arabidopsis thaliana16 and ferulic acid decarboxylase (FDC1) from S. cerevisiae17, followed by S-selective epoxidation of styrene to give (S)-styrene oxide using SMO from Pseudomonas putida S1218; regioselective hydrolysis of (S)-styrene oxide with epoxide hydrolase from Sphingomonas HXN-200 or from S. tuberosum, respectively.

Figure 1 Schematic diagram of artificial metabolic route towards (S)- and (R)phenylethanediol synthesis in yeast. The pathway intermediates G6P, E4P, PEP, DAHP, CHOR and PREPH are defined as glucose-6-phosphate, erythrose 4-phosphate, phosphoenolpyruvate, 3-deoxy-D-arabino-heptulosonate-7-phosphate, chorismate and prepherate, respectively. 2-Phenylethanol (2-PE) represents the byproduct from the Ehrlich pathway. The red “X” indicates the gene deletion to abolish 2-PE and L-tryptophan byproduct formation. PAL2, phenylalanine ammonia lyase from A. thaliana; FDC1, ferulic acid decarboxylase from S. cerevisiae; SMO, styrene monooxygenase from P. putida S12;


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SpEH and StEH, epoxide hydrolase from Sphingomonas HXN-200 and Solanum tuberosum. Results and Discussion CRISPR/Cas9-mediated genome editing for integrating feedback-resistant ARO4 and ARO7. In budding yeast, L-phenylalanine is synthesized from precursors of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) via the shikimate pathway, and the entire biosynthetic pathway contains ten enzymatic steps: ARO3/ARO4, ARO1, ARO2, ARO7, PHA2, and ARO8 (Fig. 1a). According to literature, ARO3 is subjected to feedbackregulation to high level of L-phenylalanine, whereas ARO4 and ARO7 are subjected to feedback-regulation to L-tyrosine19-21. Previously, it was reported that introducing feedback resistant enzymes involved in the shikimate pathway in E. coli could significantly improve L-tyrosine and L-phenylalanine22-24. Similar strategy has also been demonstrated to be effective for an enhanced production of aromatic amino acid derived flavonoids and alkaloids in budding yeast25-28. In the present study, in order to relieve feedback inhibition caused by the accumulation of L-phenylalanine, we sought to introduce feedback resistant versions of ARO4*(K229L)29, 30

and ARO7*(G141S)30 into yeast chromosomes under strong galactose-inducible

promoter. The yeast S. cerevisiae was reported to naturally produce small amounts of fusel alcohols such as 2-phenylethanol via the Ehrlich pathway31-33. To minimize the byproduct formation of 2-phenylethanol, it is desirable to further delete ARO10 to increase the L-



phenylalanine flux towards products-of-interest. Therefore, we sought to delete ARO10 by integrating ARO4*-ARO8 cassette (Fig. 2a). Based on the previous findings which showed that overexpression of ARO8 would allow more L-phenylalanine productions34, we introduced another copy of ARO8 for overexpression via abolishing TRP1 with ARO7*ARO8 cassette (Fig. 2a). In the present study, all genome modifications were achieved via CRISPR/Cas9-mediated genome editing, which was previously reported to be an efficient method in budding yeast35, 36. As can be seen from Fig. 2b, diagnostic PCR verification confirmed all mutant yeast strains with the genotype of aro10::TADH1-ARO4*-PGAL1/10ARO8-TCYC1 and trp1::TADH1-ARO7*-PGAL1/10-ARO8-TCYC1, and the resulting strains were designated as strain BY2M herein. During the HPLC analysis, we also found that deleting ARO10 gene in the engineered yeast cells nearly abolished the 2-phenylethanol biosynthesis via the Ehrlich pathway (data not shown), which would allow potential accumulation of L-phenylalanine in our engineered yeast strains.

Figure 2 Metabolically engineered budding yeast towards styrene biosynthesis. (a) Schematic illustration of CRISPR/Cas9-mediated genome editing. The ARO4*(K229L)ARO8 and ARO7*(G141S)-ARO8 cassettes are integrated into ARO10 and TRP1 locus by CRISPR/Cas9-mediated genome editing. (b) Diagnostic PCR analysis of genome integration of ARO4*(K229L) and ARO7*(G141S) cassettes. Label 1-2, mutant strains; control, wild type strain. Lane 1 and 2 represent the two colonies which were randomly picked and subjected to PCR analysis. (c) Enhanced styrene levels in the engineered yeast strains. Plasmid P425-PAL2/FDC1 was transformed into strain BY4741 (Control) and


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BY2M (Engineered), respectively. Cells were cultivated in 14 mL shake-tubes supplemented with 2 mL of culture medium containing 1.8% (w/v) galactose + 0.2% (w/v) glucose. Data represent the average and standard deviation of three independent experiments. Styrene production in the engineered yeast cells. Due to the L-phenylalanine could not freely diffuse outside cell, it is a relatively tedious procedure to accurately measure the intracellular L-phenylalanine level. To further confirm whether our engineering strategy could yield an elevated pathway activity towards Lphenylalanine biosynthesis, we next sought to design a styrene-producing strain to investigate styrene productions in the engineered yeast. In the present study, a two-gene module that comprises phenylalanine ammonia lyase (PAL2) from A. thaliana and the native ferulic acid decarboxylase (FDC1) from S. cerevisiae were chosen37-40, which would convert L-phenylalanine into trans-cinnamic acid and further into diffusible styrene molecule. For a comparison, the reference strain BY4741 was also transformed with the same plasmid containing PAL2 and FDC1. As shown in Fig. 2c, there is a clear improvement of styrene level in the engineered strain BY2M, which corresponds to approximately 200% increase over the control strain BY4741. For unknown reasons, there was a reduced growth rate for the engineered yeast strain BY2M during the fermentation process. Nevertheless, these results clearly indicated that our engineering strategy of simultaneously deleting side pathways via the replacement of feedback-resistant shikimate enzymes was effective in improving the L-phenylalanine biosynthetic pathway activity. Engineering the budding yeast towards (S)-phenylethanediol biosynthesis.



Upon successful construction of strain BY2M with an enhanced pathway activity towards L-phenylalanine biosynthesis, we next attempted to further engineer strain BY2M for (S)phenylethanediol production. Previously, our group has demonstrated that styrene monooxygenase (SMO) encoded by the two-component system of StyA and StyB from P. putida S12 could be functionally expressed in E. coli, which enabled cascade biotransformation of styrene into various high-value chemicals41. In addition, epoxide hydrolase from Sphingomonas HXN-200 (SpEH) was found to selectively convert (S)styrene oxide into (S)-phenylethanediol with ee value above 98%41-43. To date, none of these enzymes has been reported for the heterologous expression in S. cerevisiae to evaluate their enzymatic activities in yeast. Considering the budding yeast is the workhorse for industrial applications, it prompted us to investigate the phenylethanediol biosynthesis in budding yeast (Fig. 1). More specifically, two-component system of styrene monooxygenase (StyA and StyB) was fused together using P2A sequence, to favor the expression under a single promoter. The GAL1/10 dual expression plasmid was used to express SMO-SpEH, and the resulting plasmid was designated as P423-SMO/SpEH. To further increase the metabolic flux towards phenylethanediol biosynthesis, we also constructed another plasmid with the native prephenate dehydratase (encoded by PHA2) to divert more flux towards Lphenylalanine and additional copy of PAL2 (P426-PAL2/PHA2) to further pull Lphenylalanine to provide more trans-cinnamic acid intermediate. As our group is also working on L-tyrosine derived chemicals, we choose to use 2-micron plasmid-based expression of PHA2 instead of chromosome integration so that plasmid-based expression of TYR1 in the future would divert the metabolic flux towards L-tyrosine. The current


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design of plasmid-based expression of gene at the branching point will allow more flexibility for different applications. The cell growth of the yeast-producing (S)-phenylethanediol is shown in Fig. S5 in the SI. As can be seen from Fig. 3a, HPLC analysis of strain BY2M_S-PED revealed additional peak around 4 min which had the same retention time as the authentic standard phenylethanediol from Sigma-Aldrich, suggesting that SMO and SpEH could be functionally expressed in the heterologous host of budding yeast. As can be seen from Fig. 3b, further quantitation analysis indicated that (S)-phenylethanediol in strain BY2M_SPED reached around 100 mg/L after 96 h cultivation. In addition, we also measured the product level at 120 h. However, there was no further improvement on (S)phenylethanediol after the prolonged cultivation. Further GC-MS analysis revealed there was an additional peak around retention time 7.6 min for the engineered yeast cells, and the mass spectrum confirmed the identity of the peak to be phenylethanediol (Fig. 3c). In addition, GC-MS analysis also suggested that there was no accumulation of styrene and (S)-styrene oxide intermediates in the cell broth. Further chiral HPLC revealed that (S)phenylethanediol produced from yeast gave the ee value above 97% (Fig. 3d). To the best of our knowledge, there is no de novo synthesis of phenylethanediol from sugar in yeast. Our work represented the first report to investigate the styrene degradation pathway in budding yeast to facilitate the synthesis of (S)-phenylethanediol from sugar41-43.

Figure 3 Biosynthesis of (S)-phenylethanediol in budding yeast via styrene monooxygenase from P. putida S12 and epoxide hydrolase from Sphingomonas HXN-200.



(a) HPLC analysis of (S)-phenylethanediol production in the engineered yeast strain BY2M_S-PED. Strain BY2M_Control is used as the reference strain. (b) (S)phenylethanediol production profile by engineered yeast strains. Strain BY2M_S-PED was generated by transforming BY2M with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/SpEH. All strains were cultivated in shake-flasks supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose. Data represent the average and standard deviation of three independent experiments. (c) GC-MS analysis: GC chromatogram of (S)phenylethanediol produced from the engineered yeast strains (Left); MS analysis of (S)phenylethanediol peak at 7.6 min produced from the engineered yeast strains. (d) Chiral HPLC analysis: racemic phenylethanediol (standard) (Left); (S)-phenylethanediol produced from the engineered yeast strains (Right). Engineering the budding yeast towards (R)-phenylethanediol biosynthesis. Previously, it was reported that epoxide hydrolase from Solanum tuberosum (StEH) could convert (S)-styrene oxide into (R)-phenylethanediol with ee value above 95% in E. coli4143.

In the present work, we decided to further investigate (R)-phenylethanediol biosynthesis

in our engineered yeast platform. The cell growth of the yeast-producing (R)phenylethanediol is shown in Fig. S5 in the SI. As can be seen from Fig. 4a, (R)phenylethanediol could also be produced upon introducing the enzyme cascades (PAL2FDC1-SMO-StEH) in strain BY2M, and the retention time of (R)-phenylethanediol appeared around 4 min. Notably, strain BY_R-PED showed a relatively low production rate in the early 48 h and could eventually reach around 110 mg/L (R)-phenylethanediol after 96 h cultivation (Fig. 4b), indicating that the maximum amount of carbon source to be diverted to phenylethanediol synthesis in strain BY_R-PED was similar to that of strain


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BY_S-PED. GC analysis showed no accumulation of styrene and (S)-styrene oxide in the end of the reaction. In addition, chiral HPLC revealed that (R)-phenylethanediol produced from yeast had the enantiopurity above 99% (Fig. 4c).

Figure 4 Biosynthesis of (R)-phenylethanediol in budding yeast via styrene monooxygenase from P. putida S12 and epoxide hydrolase from S. tuberosum. (a) HPLC analysis of (R)-phenylethanediol production in the engineered yeast strain BY2M_R-PED. Strain BY2M_Control is used as the reference strain. (b) (R)-phenylethanediol production profile by engineered yeast strains. Strain BY2M_R-PED was generated by transforming BY2M with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/StEH. All strains were cultivated in shake-flasks supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose. Data represent the average and standard deviation of three independent experiments. (c) Chiral HPLC analysis of (R)-phenylethanediol produced from the engineered yeast strains. Conclusion Enantiopure phenylethanediol are very useful building blocks for the synthesis of biologically active molecules and pharmaceuticals4-6. In the present study, we demonstrated an artificial metabolic route for (S)- and (R)-phenylethanediol biosynthesis in S. cerevisiae. Phenylalanine ammonia lyase (PAL2) from A. thaliana, styrene monooxygenase (SMO) from P. putida S12 and epoxide hydrolase from Sphingomonas HXN-200 or from S. tuberosum were all functionally expressed in the heterologous host of S. cerevisiae. By engineering the endogenous L-phenylalanine biosynthetic pathway



coupled with the artificial enzyme cascades, the engineered yeast cell factories could yield approximately 100~120 mg/L of (S)- or (R)-phenylethanediol after 96 h cultivation. (R)-Phenylethanediol has been produced from E. coli with a titer of 1.23 g/L from glucose44. However, our yield from yeast (~100 mg/L) is relatively low compared to that of E..coli. Considering more than 1 g/L of shikimate pathway derived chemicals such as 4hydroxymandelic acid could be achieved in S. cerevisiae45, future work will be focused on elucidating of the metabolic bottleneck towards phenylethanediol biosynthesis and further improving the product titers. In addition, we will also attempt to expand the synthesis of other vicinal diol derived fine chemicals such as non-natural amino acids and hydroxy acids, as well as other biologically active molecules and pharmaceuticals. Methods Genetic modification in budding yeast. Genome modification of budding yeast was performed via CRISPR/Cas9-mediated editing tool as described previously35. The guide RNA targeting at ARO10 and TRP1 locus was designed using the online tool CHOPCHOP (https://chopchop.rc.fas.harvard.edu). Oligonucleotides used for genome editing are listed in Table S1. The feedback resistant ARO4 and ARO7 were generated using overlapping extension PCR, and assembled together with ARO8, to yield plasmid P426-ARO4*/ARO8 and P426-ARO7*/ARO8 (Table 1). These intermediate plasmids were used as template for PCR amplification of the entire genome integration cassettes. Briefly, 50 μL of yeast cells together with approximately 2-4 μg mixture of PCR amplified genome integration cassettes and 500 ng


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of gRNA expressing plasmid was electroporated in a 0.2 cm cuvette at 1.6 kV. After electroporation, cells were immediately mixed with 6 mL 1:1 mix of 1 M sorbital:YPD medium and recovered a rotary shaker for 1-2 h. Following that, cells were collected by centrifugation at 3000 rpm for 5 min on a centrifuge, washed and re-suspended in 200 μL ddH2O. Subsequently, 100 μL cells were plated on SC-Ura-Leu agar plates. Colonies were randomly picked from the plate and subjected to the diagnostic PCR analysis of genome integration events. For the verification of genome integration events, colonies were first picked from the plate and streaked on fresh SC-Ura-Leu agar plates to eliminate false positives. Cells were lysed by 20 mM NaOH for 30 min at 98°C on the thermocycler. The diagnostic PCR program was set as follows: 1 cycle of 95°C for 2 min; amplification, 30 cycles of 95°C for 10 s, 50°C for 15 s and 72°C for 90 s; 1 cycle of 68°C for 2 min. Universal primer pair of F_GAL10_Scr and R_ADH1_Scr was used to verify the genome integration event of ARO4*-ARO8 and ARO7*-ARO8. Only colonies with the two-band pattern with the correct size corresponding to ARO4* and ARO7* gene were considered to have the ARO4*-ARO8 and ARO7*-ARO8 assembled into the yeast chromosomes. Table 1 List of plasmids used in the present study Name pML107 P426-gARO10 P426-gTRP1 P426ARO4*/ARO8 P426ARO7*/ARO8 P426-PAL2/PHA2

Description Plasmid harboring Cas9 expression cassette Plasmid harboring gRNA targeting at ARO10 locus Plasmid harboring gRNA targeting at TRP1 locus pESC-URA derivative with PGAL10-ARO4*-TADH1; PGAL1-ARO8-TCYC1 pESC-URA derivative with PGAL10-ARO7*-TADH1; PGAL1-ARO8-TCYC1 pESC-URA derivative with PGAL10-PAL2-TADH1; PGAL1-PHA2-TCYC1


Reference 36

This study This study This study This study This study


P425-PAL2/FDC1 P423-SMO/SpEH P423-SMO/StEH

pESC-LEU derivative with PGAL10-PAL2-TADH1; PGAL1-FDC1-TCYC1 pESC-HIS derivative with PGAL10-SMO-TADH1; PGAL-SpEH-TCYC1 pESC-HIS derivative with PGAL10-SMO-TADH1; PGAL1-StEH-TCYC1

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Plasmid-based expression system for cascade biotransformation. Oligonucleotides used for plasmid constructions are listed in Table S2. For constructing plasmid-based expression of heterologous pathways, StyA and StyB encoding styrene monooxygenase were codon-optimized and synthesized from IDT technologies. In the present study, StyA and StyB were fused together using P2A sequence to enable expression under a single promoter. The codon-optimized sequence of StyA and StyB is listed in Table S3. Both PHA2 and FDC1 gene was PCR amplified from the genomic DNA of S. cerevisiae. PAL2, StEH and SpEH were PCR amplified from our previous constructed plasmids41. All the gene fragments were assembled into the receiving vector via a restriction-free approach46. Since we are using the bidirectional promoter expression system (one plasmid for two gene expressions), instead of leaving the expression cassette empty, we decided to express two copies of PAL2 from different plasmids. The resulting plasmids were designated as P426-PAL2/PHA2, P425-PAL2/FDC1, P423-SMO/SpEH and P423-SMO/StEH (as listed in Table 1). Subsequently, these plasmids were transformed into the yeast strains using standard lithium acetate heat-shock approach.

Table 2 List of strains used in the present study


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Name BY4741 BY2M BY_Styrene BY2M_Styrene BY2M_Control BY2M_S-PED BY2M_R-PED

Description MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 BY4741 derivative with Δaro10::PGAL10-ARO4*TADH1; PGAL1-ARO8-TCYC1, Δtrp1::PGAL10-ARO7*TADH1; PGAL1-ARO8-TCYC1 BY4741 derivative with plasmid P425-PAL2/FDC1 BY2M derivative with plasmid P425-PAL2/FDC1 BY2M derivative with empty plasmid pESC-URA, pESC-LEU and pESC-HIS BY2M derivative with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/SpEH BY2M derivative with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/StEH

Reference Euroscarf This study This study This study This study This study This study

Styrene biosynthesis by engineered yeast cells. To enable styrene production in yeast cells, both the control BY4741 and the engineered yeast BY2M were transformed with plasmid P425-PAL2/FDC1. Strains used in the present study are listed in Table 2. Experiments were carried out in 14 mL shake-tubes. Tubes containing 1 mL SC-Leu medium supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose were inoculated to an initial OD600 of 0.1 with fresh overnight cultures. 20% of hexadecane was added to perform two-phase fermentation and to minimize the evaporation of styrene. Each time, 100 μL of cell culture was taken for measuring OD600 by microplate reader, and 10 μL hexadecane layer was sampled and diluted in 490 μL ethyl acetate for the determination of product levels by gas chromatography-flame ionization detector (GCFID). During the GC-FID analysis, 1 μL of diluted sample was injected into Agilent 7890A system equipped with a HP-5 column (Agilent Technologies, USA). Helium was used as a carrier gas at a flow rate of 1.0 mL/min. For the styrene detection, the temperature program



was set as following: initial temperature at 70°C, increase 25°C/min until it reaches 200°C; subsequently increase to 250°C at 50°C/min, then hold for 1 minute; finally increase to 270°C at 20°C/min. Authentic styrene standard from Sigma-Aldrich was used for determining the retention time and plotting the standard curve for quantitation. Phenylethanediol production under shake-flasks. For (S)-phenylethanediol production in yeast cells, the engineered yeast BY2M was transformed with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/SpEH. For (R)-phenylethanediol production in yeast cells, the engineered yeast BY2M was transformed with plasmid P426-PAL2/PHA2, P425-PAL2/FDC1 and P423-SMO/StEH instead. Strains used in the present study are listed in Table 2. Experiments were carried out in 250 mL shake-flasks. Flasks containing 50 mL SC-Leu-Ura-His medium supplemented with 1.8% (w/v) galactose + 0.2% (w/v) glucose were inoculated to an initial OD600 of 0.1 with overnight cultures. Each time, 100 μL of cell culture was taken for measuring OD600 by microplate reader, and 200 μL cell broth was sampled and diluted in 800 μL ultrapure water for determining the product levels. For the reverse phase HPLC analysis of phenylethanediol, experiments were carried out as below. The HPLC system (Agilent 1260 Infinity II System) was equipped with Agilent Poroshell 120 EC-C18 (150 mm x 4.6 mm x 2.7 µm). Mobile phase: 30% acetonitrile and 70% water. Flow rate: 0.5 mL/min. Column temperature: 40°C. The photodiode array detector (DAD) was set at wavelength 210 nm. During the GC-MS analysis, the cell cultures were extracted with ethyl acetate and 1 μL of


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organic layer was injected into Agilent 7890A system equipped with a DB-5ms column (Agilent Technologies, USA). Helium was used as a carrier gas at a flow rate of 1.0 mL/min. The temperature program was set as following: initial temperature at 70°C, increase 25°C/min until it reaches 200°C; subsequently increase to 250°C at 50°C/min, then hold for 1 min; finally increase to 270°C at 20°C/min. Chiral HPLC for enantiopurity analysis After 5 days of cultivation, samples were taken and centrifuged for 10 mins (1000 g). The aqueous phase was collected, extracted with ethyl acetate (1:1, v/v), and dried through evaporation. The dried sample was collected and dissolved in 10% isopropanol and 90% n-hexane for the chiral HPLC analysis. The Shimadzu LC-20A system was equipped with Daicel Chiralpak AS-H column (250 x 4.6 mm x 5 um). Mobile Phase: 10% Isopropanol and 90% n-hexane. Flow rate: 1 mL/min. Column temperature: 25°C. Retention Time: 10.9 min for (S)-phenylethanediol and 11.5 min for (R)-phenylethanediol. Acknowledgements This research was financially supported by National Research Foundation (NRF) Singapore by through AME IRG Grant (Project iGrants number A1783c0014; Project No. 279-000-511-305). Author contributions J.Y. and Z.L. conceived the project. J.Y. designed and performed the experiments. J.Y. and B.R.L. collected and interpreted the data. J.Y. and Z.L. wrote the manuscript.



Conflict of interest The authors declare no conflict of interests. Supporting information This material is available free of charge via the Internet at http://pubs.acs.org.

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De Novo Biosynthesis of (S)- and (R ... - ACS Publications

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