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Metabolic engineering of Saccharomyces cerevisiae for production of shinorine, a sunscreen material, from xylose Seong-Hee Park, Kyusung Lee, Jae Woo Jang, and Ji-Sook Hahn ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00388 • Publication Date (Web): 26 Dec 2018 Downloaded from http://pubs.acs.org on December 26, 2018

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ACS Synthetic Biology

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Metabolic engineering of Saccharomyces cerevisiae for production of shinorine, a

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sunscreen material, from xylose

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Seong-Hee Park1, Kyusung Lee2, Jae Woo Jang2, and Ji-Sook Hahn1*

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1School

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Gwanak-gu, Seoul 08826, Republic of Korea

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2BIO

of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro,

Research Institute, CJ CheilJedang, Suwon 16495, Republic of Korea

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*Corresponding

author:

Phone: +82-2-880-9228

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Fax: +82-2-888-1604

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e-mail: [email protected]

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ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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For Table of Contents Use Only

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Metabolic engineering of Saccharomyces cerevisiae for production of shinorine, a

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sunscreen material, from xylose

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Seong-Hee Park1, Kyusung Lee2, Jae Woo Jang2, and Ji-Sook Hahn1*

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1School

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Gwanak-gu, Seoul 08826, Republic of Korea

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2BIO

of Chemical and Biological Engineering, Seoul National University, 1 Gwanak-ro,

Research Institute, CJ CheilJedang, Suwon 16495, Republic of Korea

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Abstract

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Shinorine, a mycosporine-like amino acid (MAA), is a small molecule sunscreen produced in

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some bacteria. In this study, by introducing shinorine biosynthetic genes from cyanobacteria

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Nostoc punctiform into Saccharomyces cerevisiae, we successfully constructed yeast strains

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capable of producing shinorine. Sedoheptulose 7-phosphate (S7P), an intermediate of pentose

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phosphate pathway, is a key substrate for shinorine biosynthesis. To increase the S7P pool,

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xylose, which is assimilated via pentose phosphate pathway, was used as a carbon source after

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introducing xylose assimilation genes from Scheffersomyces stipitis into the shinorine-

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producing strain. The resulting xylose-fermenting strain produced a trace amount of shinorine

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when cells were grown in glucose, but shinorine production was dramatically increased by

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adding xylose in the medium. Shinorine production was further improved by modulating

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pentose phosphate pathway through deleting TAL1 and overexpressing STB5 and TKL1. The

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final engineered strain JHYS17-4 produced 31.0 mg/L (9.62 mg/gDCW) of shinorine in the

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optimized medium containing 8 g/L xylose and 12 g/L glucose, demonstrating that S. cerevisiae

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is a promising host to produce this natural sunscreen material.

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Keywords: Pentose phosphate pathway; Saccharomyces cerevisiae; Sedoheptulose 7-

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phosphate; Shinorine; Sunscreen; Xylose

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To prevent skin damage from UV radiation, several chemical and physical sunscreen materials

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such as oxybenzone, ZnO, and TiO2 are widely used as cosmetics additives. However, due to

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the concerns about their negative effects on human health and environment, there is an

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increasing demand for safer bio-based sunscreen compounds1. Mycosporines and

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mycosporine-like amino acids (MAAs) are UV-absorbing natural products synthesized in

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various prokaryotic and eukaryotic organisms such as cyanobacteria, fungi, and algae2.

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Mycosporines, mainly produced in fungi, have cyclohexenone ring with the nitrogen

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substituent of an amino acid or an imino alcohol at C3 position (Supporting Information Figure

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S1)3. MAAs have an additional nitrogen substituent conjugated via imine linkage, forming the

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cyclohexenimine core structure. So far, approximately 30 MAAs have been identified, of which

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shinorine, containing glycine and serine substituents (Supporting Information Figure S1), is

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the most attractive sunscreen molecule. Shinorine has a high molar extinction coefficient

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(ε=28100-50000M-1cm-1) and its maximal absorbance wavelengths (310-365 nm) cover UV-A

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that is more abundant in sunlight and more penetrating than UV-B4-6. Shinorine is

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commercially used as a sunscreen material, but considering the low production yield (3.27

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mg/g DCW) of its natural producer, red alga Porphyra umbilicalis, shinorine production using

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heterologous microbial host might be a promising alternative7.

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Two alternative biosynthetic pathways of MAAs have been suggested, of which one

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uses 3-dehydroquinate (DHQ) and the other uses sedoheptulose 7-phosphate (S7P) as

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intermediates. DHQ, an intermediate of shikimate pathway, was suggested as a substrate for

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fungal mycosporine and cyanobacterial MAA biosynthesis based on feeding studies using

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radiolabeled DHQ8. In addition, an inhibition of shinorine synthesis was observed when

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cyanobacteria Nostoc commune and Anabaena variabilis, and the coral Stylophora pistillata

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where treated with the shikimate pathway inhibitor, glyphosate9. In contrast, S7P was identified 4


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as an intermediate for shinorine biosynthesis based on the biochemical characterization of

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shinorine biosynthetic gene clusters from the cyanobacteria Nostoc punctiforme ATCC 29133

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and Anabaena variabilis ATCC 2941310. S7P, an intermediate of pentose phosphate pathway,

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is converted to shinorine via four enzymatic steps. First, S7P is converted to 4-deoxygadusol

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(4-DG) by 2-demethyl 4-deoxygadusol synthase (DDGS) and O-methyltransferase (O-MT)11.

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Then glycine is conjugated to 4-DG by ATP-grasp ligase to form mycosporine-glycine (MG).

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Finally, serine is attached to MG either by non-ribosomal peptides synthetase (NRPS)-like

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enzyme in A. variabilis or by D-ala-D-ala ligase in N. punctiforme, generating shinorine.

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Homologous gene clusters have been identified in other cyanobacteria and also in

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actinobacteria10.

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To date, several efforts have been made to produce shinorine by introducing the

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biosynthetic genes into heterologous hosts including cyanobacteria, Streptomyces, Escherichia

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coli, and Corynebacterium10,

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cyanobacterium Fischerella sp. PCC9229 were expressed in unicellular model cyanobacterium

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Synechocystis sp. PCC6803, leading to a production of 2.37 mg/gDCW (0.71 mg/L) of

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shinorine via improving gene expression levels by using multiple promoters12. Expression of

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MAA biosynthetic gene cluster from actinobacterium Actinosynnema mirum DSM 43827 in

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Streptomyces avermitilis SUKA22 resulted in 13.9 mg MAAs/gDCW (154 mg/L shinorine and

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188 mg/L total MAA)13. However, when the same biosynthetic genes from A. mirum DSM

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43827 were expressed in Corynebacterium glutamicum, 19 mg/L of shinorine was produced14.

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Furthermore, expression of shinorine biosynthetic gene cluster from A. variabilis in E. coli led

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to a very low amount of shinorine production around 0.15 mg/L10.

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Shinorine biosynthetic genes from the filamentous

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Saccharomyces cerevisiae is a promising host for production of various natural

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products because of its safety and well-studied metabolic pathways15,

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production in S. cerevisiae has not yet been reported. In this study, we developed S. cerevisiae

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strains capable of producing shinorine by introducing shinorine biosynthetic pathway from N.

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punctiforme. Furthermore, as an effort to increase S7P pool available for shinorine production,

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xylose, which is assimilated via pentose phosphate pathway, was used as a carbon source by

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introducing the xylose assimilation pathway.

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but shinorine

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Results and discussion

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Construction of shinorine biosynthetic pathway in S. cerevisiae

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To produce shinorine in S. cerevisiae, heterologous shinorine biosynthetic pathway genes from

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cyanobacteria N. punctiforme, consisting of genes encoding DDGS (NpR5600), O-MT

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(NpR5599), ATP-grasp ligase (NpR5598), and D-ala-D-ala ligase (NpR5597), were introduced

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into CEN.PK2-1C strain (Figure 1B). S7P is sequentially converted to DDG, 4-DG, MG, and

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shinorine by NpR5600, NpR5599, NpR5598, and NpR5597, respectively (Figure 1A). The four

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shinorine biosynthetic genes were cloned into a multigene-expression vector17 under the

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control of strong constitutive promoters, PTDH3 or PTEF1, generating a plasmid coex413-NpR4.

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S. cerevisiae harboring coex413-NpR4 (JHYS10) produced a trace amount of shinorine (0.46

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mg/L and 0.085 mg/gDCW) when the cell extracts were analyzed by HPLC (Figure 2). The

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identity of shinorine in the cell extracts was confirmed by tandem mass spectrometry analysis

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(Supporting Information Figure S2)18. On the other hand, the control strain harboring p413GPD

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plasmid (WT-1) did not produce any shinorine, confirming that the biosynthetic genes from N.

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punctiforme are functional in producing shinorine in S. cerevisiae. WT-1 and JHYS10 showed

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the same growth rates, indicating that overexpression of the shinorine biosynthetic genes did

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not affect cell growth (Supporting Information Figure S3). This is the first demonstration of

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the functional expression of the shinorine biosynthetic genes from N. punctiforme in a

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heterologous host.

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Generation of shinorine-producing S. cerevisiae strains by random multi-copy delta-

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integration of the biosynthetic genes

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The S. cerevisiae genome contains hundreds of retrotransposon Ty1 long terminal repeats 7



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(LTR) sequences known as delta-sequences19, 20. Homologous recombination using these delta

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sites allows random multi-copy integration of target genes21, 22. Therefore, to improve shinorine

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production, we integrated the four biosynthetic genes into the delta-sequences. Two delta-

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integration cassettes containing either NpR5597 and NpR5600 genes or NpR5598 and NpR5599

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genes were transformed together into S. cerevisiae CEN.PK2-1C, and the transformants were

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selected on the medium containing 2 mg/L of G418 (Figure 3A). Among 18 transformants,

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JHYS12 and JHYS13 were selected based on better shinorine production compared with cells

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harboring coex413-NpR4 (JHYS10) (Figure 2B). Strain JHYS13 produced higher level of

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shinorine (0.67 mg/L and 0.125 mg/gDCW) than JHYS12 (0.51 mg/L and 0.106 mg/gDCW)

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(Figure 3B). The copy numbers of the integrated genes were determined by qPCR. Results

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indicated that JHYS12 genome contained 5 copies of NpR5600 and NpR5597 and 2 copies of

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NpR5599 and NpR5598, whereas JHYS13 contained 4 copies of NpR5600 and NpR5597 and

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9 copies of NpR5599 and NpR5598 compared with the control strain JHYS11 integrated with

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only one copy of each NpR gene at HIS3 locus (Figure 3C).

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Using xylose as a carbon source for shinorine production by introducing xylose

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assimilation pathway

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Although multiple integration of biosynthetic genes demonstrated an improved production of

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shinorine, the production levels were still very low, which might be due to a limited supply of

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S7P, the substrate for the first enzyme DDGS in the shinorine biosynthetic pathway. Since S7P

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is an intermediate of the pentose phosphate pathway, increasing the carbon flux toward the

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pentose phosphate pathway might improve shinorine production.

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Although S. cerevisiae cannot assimilate xylose naturally, S. cerevisiae can grow on 8


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xylose by introducing heterologous pathways consisting of a xylose isomerase (XI) or xylose

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reductase/xylitol dehydrogenase (XR/XDH)23. XI is an enzyme present in most xylose-

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consuming bacteria and has the advantage of eliminating cofactor imbalance during xylose

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fermentation24. However, because of low enzymatic activity of XI when expressed in yeast,

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XR/XDH pathway has been used more frequently for xylose fermentation in S. cerevisiae23, 25.

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Xylose reductase (XR) and xylitol dehydrogenase (XDH) serially convert xylose to xylitol and

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xylitol to xylulose26,

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xylulokinase (XK) Xks1, then xylulose-5-phosphate enters the pentose phosphate pathway

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(Figure 1). Therefore, using xylose as a carbon source was thought to increase the S7P pool via

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pentose phosphate pathway. Previously, it has been shown that efficient xylose-fermenting S.

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cerevisiae strains can be generated by expressing xylose assimilation genes encoding XR

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(XYL1), XDH (XYL2), and XK (XYL3) from Scheffersomyces stiptis28. Therefore, to generate

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a xylose-fermenting strain, the coex416-XYL plasmid containing the XYL1, XYL2, and XYL3

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genes under the control of a strong constitutive promoter, PTDH3, was introduced into the strain

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JHYS13, and then the cells were cultivated in four different media containing different ratios

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of glucose to xylose (Figure 4). The control JHYS13 strain harboring the empty p416GPD

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vector (JHYS13-1) and JHYS13 strain harboring the coex416-XYL plasmid (JHYS13-2)

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showed similar growth rates in the medium containing only glucose (20 g/L), producing low

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level (

Metabolic engineering of Saccharomyces cerevisiae for production of

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