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Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Engineered Saccharomyces cerevisiae that produces 1,3-propanediol from D-glucose Z. Rao, Z. Ma, W. Shen, H. Fang, J. Zhuge and X. Wang The Key Laboratory of Industrial Biotechnology of Ministry of Education, Research Center of Industrial Microbiology, School of Biotechnology and State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, P.R. China

Keywords Agrobacterium tumefaciens, dhaB, glycerol, 1,3-propanediol, Saccharomyces cerevisiae, yqhD. Correspondence Zhiming Rao and Xiaoyuan Wang, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province 214122, P.R. China. E-mail: [email protected] and [email protected]

2007 ⁄ 1840: received 15 November 2007, revised 24 March 2008 and accepted 26 March 2008 doi:10.1111/j.1365-2672.2008.03868.x

Abstract Aims: Saccharomyces cerevisiae is a safe micro-organism used in fermentation industry. 1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensively chemical synthesis. The aim of this study is to engineer a S. cerevisiae strain that can produce 1,3-propanediol at low cost. Methods and Results: By using d-glucose as a feedstock, S. cerevisiae could produce glycerol, but not 1,3-propanediol. In this study, we have cloned two genes yqhD and dhaB required for the production of 1,3-propanediol from glycerol, and integrated them into the chromosome of S. cerevisiae W303-1A by Agrobacterium tumefaciens-mediated transformation. Both genes yqhD and dhaB functioned in the engineered S. cerevisiae and led to the production of 1,3-propanediol from d-glucose. Conclusion: Saccharomyces cerevisiae can be engineered to produce 1,3-propanediol from low-cost feedstock d-glucose. Significance and Impact of the Study: To our knowledge, this is the first report on developing S. cerevisiae to produce 1,3-propanediol by using A. tumefaciensmediated transformation. This study might lead to a safe and cost-efficient method for industrial production of 1,3-propanediol.

Introduction 1,3-Propanediol has been widely used in polymers, cosmetics, foods, lubricants and medicines, but its use is being restricted by its high cost. For example, one of the most successful applications for 1,3-propanediol has been in the synthesis of polytrimethylene terephthalate that is widely used in the manufacture of carpet and textile fibres (Biebl et al. 1999). As fine polymers, polytrimethylene terephthalate is rapidly extending its application in industry, but its production is restricted by the high cost and limited availability of 1,3-propanediol. 1,3-Propanediol has mainly been manufactured by high-cost chemical synthesis. There is an urgent need to develop a new and cost-efficient method to produce 1,3-propanediol. Comparing with the chemical synthesis, fermentation method has the advantage of mass production at low cost, and has been used in the production of various industrial 1768

chemicals. The ideal fermentation production needs a safe micro-organism that can produce the desired chemicals from renewable resources. In nature, a few micro-organisms, including Klebsiella (Huang et al. 2002), Citrobacters (Boenigk et al. 1993) and Clostridium (Saint-amans et al. 2001) can produce 1,3-propanediol, but they all use an expensive resource glycerol. In Klebsiella pneumoniae, genes involved in the biosynthesis of 1,3-propanediol are dhaB and dhaT (Fig. 1b). DhaB is a B12-dependent dehydratase that converts glycerol into 3-hydroxypropionaldehyde, while DhaT is an NADH-dependent oxidoreductase that reduces 3-hydroxypropionaldehyde into 1,3-propanediol (Skraly et al. 1998). As Escherichia coli can produce glycerol from d-glucose, genes dhaB and dhaT had been cloned into E. coli to produce 1,3-propanediol from d-glucose, but the yield was low (Laffend et al. 1997). It turned out that the low yield of 1,3-propanediol was the result of the low activity of DhaT, which led to the accumulation

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(London, UK). Southern blot kit was obtained from Roche Diagnostics.

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Figure 1 The proposed biosynthesis pathway of 1,3-propanediol in the engineered Saccharomyces cerevisiae. (a) Wild-type S. cerevisiae W303-1A can only synthesize glycerol from d-glucose. (b) Klebsiella pneumoniae can synthesize 1,3-propanediol only from glycerol. (c) The engineered S. cerevisiae W303-1A-ZR in this study can synthesize 1,3-propanediol from d-glucose. This might lead to a safe and costefficient method for industrial production of 1,3-propanediol.

of 3-hydroxypropionaldehyde in the cell. When the concentration of 3-hydroxypropionaldehyde reached 30 mmol l)1, the activity of DhaB was reduced by feedback inhibition (Barbirato et al. 1996). In 2002, Emptage et al. reported that gene yqhD, an alcohol dehydrogenase in E. coli, has high DhaT activity. By overexpressing dhaB and yqhD in E. coli, the high activity of YqhD in the production of 1,3-propanediol has been confirmed (Zhang et al. 2005; Wang et al. 2007). Saccharomyces cerevisiae has been used in fermentation industry for many years, mainly because it uses the lowcost feedstock d-glucose as the carbon and energy source. Furthermore, S. cerevisiae is considered much safer than E. coli because E. coli produces endotoxins that can cause diseases. Could S. cerevisiae be used to produce 1,3propanediol? This was the question that led to this study. Wild-type S. cerevisiae can only produce glycerol, but has no capacity to produce 1,3-propanediol (Fig. 1a). In this study, we have integrated genes dhaB and yqhD into the chromosome of S. cerevisiae by Agrobacterium tumefaciensmediated transformation, and constructed a new strain W303-1A-ZR. The engineered S. cerevisiae W303-1A-ZR can directly produce 1,3-propanediol from d-glucose (Fig. 1c). This study might lead to a safe and cost-efficient method for industrial production of 1,3-propanediol. Materials and methods Materials The PCR reagents, restriction enzymes and calf intestinal alkaline phosphatase were purchased from TaKaRa Biotechnology Co. Ltd (Shiga, Japan). The Miniprep kit and Gel Extraction kit were purchased from Promega (USA). Nylon membrane was obtained from Amersham

Four media were used in this study. Luria–Bertani (LB) medium was used to grow E. coli and A. tumefaciens cells; it consisted of 1% tryptone, 0Æ5% yeast extract and 1% sodium chloride. Yeast strains were grown on yeastpeptone-dextrose (YPD) medium (1% bacto yeast extract, 2% bacto peptone extract, 2% glucose). B media was used for A. tumefaciens-mediated transformation; they are minimal media supplemented with 20 lg ml)1 uracil, 30 lg ml)1 lysine, 40 lg ml)1 trytophan and 40 lg ml)1 adenine (Bundock et al. 1995). The co-cultivation media (CM) was also used for A. tumefaciens-mediated transformation; they consisted of uracil, lysine, trytophan and adenine at the concentration mentioned earlier for B media, in addition to 50 lg ml)1 kanamycin. Escherichia coli JM109 was cultured in LB media supplemented with 100 lg ml)1 ampicillin or 25 lg ml)1 zeocin. Agrobacterium tumefaciens LBA4404 was grown in LB media supplemented with appropriate antibiotics to maintain the plasmid. The fermentations were carried out by shaking at 250 rev min)1 and at 30C under aerobic conditions. Table 1 describes the various bacterial strains and plasmids used in this study. Cell growth was monitored by using the optical density at 650 nm and converted to dry cell weight (DCW). PCR amplification of DNA fragments Unless otherwise stated, PCR amplification was carried out in a 50-ll reaction mixture containing 1 ng of template, 200 lmol l)1 dNTP, 20 lmol l)1 primers and 1 unit ExTaq DNA polymerase. The reaction was started at 95C for 300 s, followed by 35 cycles of denaturation at 95C for 90 s, annealing at 52C for 120 s, and extension at 72C for 240 s. After the 35th cycle, a 10-min extension at 72C was used. The reaction product was separated on a 0Æ8% agarose gel. The desired band was excised and gel purified. The PCR reaction was performed using an automated thermocycler (Whatman Biometra, Gottingen, Germany). Construction of plasmids pZR1 and pZR2 harbouring genes yqhD and dhaB, respectively Plasmid pZR1 harbouring yqhD was constructed as follows: the yqhD gene was amplified from E. coli genome DNA by using primers P1 and P2 (Table 1), digested with EcoRI and ligated into vector pGAPZB that had been similarly digested and treated with calf intestinal alkaline phosphatase. The ligation mixture was transformed into

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Table 1 Strains, plasmids and primers used in this study Strains or plasmids or primers Strains E. coli JM109 JM109-ZR1 JM109-ZR2 JM109-ZR3 JM109-ZR4 LBA4404 LBA4404-ZR4 S. cerevisiaeW303-1A W303-1A-zeocin W303-1A-yqhD W303-1A-ZR Plasmids pGAPZB pCAMBIA3300 pCAMBIA3300-zeocin pZR1 pZR2 pZR3 pZR4 Primers P1 P2 P3 P4 P5 P6

Description

Source or reference

recA1, endA1, gyrA96, thi-1, hsd R17(rk)mk+)supE44 JM109 containing plasmid pZR1 JM109 containing plasmid pZR2 JM109 containing plasmid pZR3 JM109 containing plasmid pZR4 Ach5, containing a virulence plasmid LBA4404 containing plasmid pZR4 MATa,leu2-3,ura3-1,trp1-1, his3-11,ade2-1,can1-100 W303-1A with gene zeocin in the chromosome W303-1A with gene yqhD in the chromosome W303-1A with genes yqhD and dhaB in the chromosome

Yanish-Perron et al. 1985 This work This work This work This work Hoekema et al. 1983 This work Thomas and Rothstein 1989 This work This work This work

Harbouring the GAP promoter and AOX1TT terminator Binary vector for LBA4404 Harbouring the zeocin-resistant cassette pGAPZB containing yqhD pGAPZB containing dhaB pCAMBIA3300-zeocin containing yqhD pCAMBIA3300-zeocin containing yqhD and dhaB

Invitrogen Dr. Jefferson RA , CAMBIA, Australia This work This work This work This work This work

5¢-ACCGGAATTCATGAACAACTTTAATCTGC-3¢ (EcoR I) 5¢-ACCGGAATTCTTAGCGGGCG GCTTC-3¢ (EcoR I) 5¢- ACCGTCTAGAATGAAAAGATCAAAACG-3¢ (Xba I) 5¢- ACCGTCTAGATTAGCTTCC TTTACGCAGC -3¢ (Xba I) 5¢-CGCAGATCTTTTTTGTAGAAATG-3¢ (Bgl II) 5¢-CGCAGATCTGCACAAACGAAGGTCTCAC-3¢ (Bgl III)

This This This This This This

work work work work work work

E., Escherichia; S., Saccharomyces; GAP, glyceraldehyde 3-phosphate dehydrogenases.

E. coli JM109 by using the calcium chloride method (Ausubel et al. 1987). The zeocin-resistant colonies were selected on LB plates supplemented with 25 ug ml)1 zeocin, and further purified. The resulted E. coli JM109 containing plasmid pZR1 was designated JM109-ZR1. The plasmid pZR1 was extracted from JM109-ZR1 by using alkaline lysis procedure (Sambrook et al. 2001), and confirmed by digesting with EcoRI and BglI (Figs 2 and 3). A similar procedure was used to construct plasmid pZR2. Gene dhaB was amplified from K. pneumoniae genomic DNA by using primers P3 and P4 (Table 1), digested with XbaI and ligated into vector pGAPZB that had been similarly digested and treated with calf intestinal alkaline phosphatase. The ligation mixture was transformed into E. coli JM109 to form JM109-ZR2. The plasmid pZR2 was purified from JM109-ZR2 and confirmed by digesting with XbaI and BamHI (Figs 2 and 3).

pZR1 was digested with BglII and BamHI. The DNA fragment pGAP-yqhD-AOX1TT containing the glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter, yqhD and terminator AOX1TT was purified from the digestion and ligated into pCAMBIA3300-zeocin which was digested with BamHI and dephosphorylated by calf intestinal alkaline phosphatase (Fig. 2). The ligation mixture was transformed into E. coli JM109 to form JM109-ZR3. The plasmid pZR3 was purified from JM109-ZR3 and confirmed by PCR amplification of yqhD using the primers P1 and P2 (Fig. 3). Next, the DNA fragment pGAP-dhaB-AOX1TT containing the promoter GAP, dhaB and terminator AOX1TT was amplified by using primers P5 and P6 (Table 1) from plasmid pZR2, digested with BglII, purified, and ligated into pZR3 which was digested with BamHI. Then the ligation mixture was transformed into E. coli JM109 to form JM109-ZR4. The plasmid pZR4 was purified from JM109ZR4 and confirmed by digesting with XbaI (Figs 2 and 3).

Construction of the plasmids pZR4 harbouring both yqhD and dhaB

Agrobacterium tumefaciens-mediated transformation

Genes yqhD and dhaB were integrated into vector pCAMBIA3300-zeocin by the following steps. First, the plasmid

Agrobacterium tumefaciens is a Gram-negative soil bacterium that can transfer a piece of its Ti plasmid DNA

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Xba I (563)

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Figure 2 Maps of plasmids constructed in this study. The arrows indicate the flowing direction during construction of the key plasmid pZR4.

(T-DNA) into the chromosome of plant cells or S. cerevisiae cells (Bundock et al. 1995; Piers et al. 1996; Sugui et al. 2005). In this study, genes yqhD and dhaB were integrated into the chromosome of S. cerevisiae by A. tumefaciens-mediated transformation. First, plasmid pZR4 was transformed into A. tumefaciens LBA4404 by electroporation (Mark et al. 1990) to form LBA4404-ZR4. Agrobacterium tumefaciens LBA4404ZR4 was grown overnight in B media containing zeocin, harvested, resuspended in B media (with or without 100 lmol l)1 acetosyringone) at a final concentration of 1 · 1011 cells ml)1, and incubated for additional 6 h. Meanwhile, S. cerevisiae W303-1A was grown overnight at 30C with shaking, was diluted 20-fold into fresh YPD media and grown for additional 6 h. The cells were harvested by centrifugation, washed with B liquid media without acetosyringone (Bundock et al. 1995), and resuspended in the same media at a final concentration of 1 · 109 cells ml)1.

Xba I (14772) T-Border(right) yqh D Xba I (13066) pGAP

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Subsequently, 50 ll of S. cerevisiae W303-1A and 50 ll of A. tumefaciens LBA4404-ZR4 prepared earlier were mixed together, deposited onto an autoclaved cellophane paper (25-mm diameter) on top of the solid CM media supplemented with acetosyringone, and incubated at 28C for 72 h. Then, the cellophane paper was moved onto the solid YDP media supplemented with 150 lg ml)1 zeocin, incubated at 30C for 3–4 days. To prevent the growth of the donor A. tumefaciens, 200 lg ml)1 of cefotaxime was included in the media. Recombinant S. cerevisiae, designated W303-1A-ZR, was further purified at 30C in YEPD media supplemented with 150 lg ml)1 zeocin. PCR and Southern blot analysis of yqhD and dhaB in the chromosome of Saccharomyces cerevisiae Genomic DNA was isolated from the recombinant S. cerevisiae W303-1A-ZR (Ausubel et al. 1987). The

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existence of yqhD in the chromosome was confirmed by PCR amplification of yqhD gene using primers P1 and P2 (Table 1). PCR amplification was started at 94C for 5 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing at 56C for 1 min and elongation at 72C for 2 min. The presence of dhaB in the chromosome was confirmed by PCR amplification of dhaB using primers P3 and P4. PCR amplification was started at 94C for 5 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing at 58C for 2 min and elongation at 72C for 4 min. For Southern blot analysis, the genomic DNA was digested with XbaI and EcoRI and separated on 0Æ8% agarose gels by field-inversion gel electrophoresis. DNA was denatured with alkali and washed with the washing buffer, and transferred to positively charged nylon membrane (Santangelo et al. 1995). Dig-High labelled probes were synthesized from the dhaB fragment in pZR2 and yqhD fragment in pZR1, respectively. Southern hybridization was carried out by using DIG-High prime DNA labelling and Detection starter kit according to manufacturer’s instructions. 1772

Figure 3 Identification of four plasmids constructed in this study. Plasmids were digested, or the genes in the plasmids were PCR amplified. The DNA fragments from the digestion and the PCR products were separated on agarose gel, and the sizes of the DNA fragments were analysed according to the maps shown in Fig. 2. Two DNA markers (M1 and M2) were used. (a) Identification of plasmid pZR1. Lane 1, plasmid pGAPZB digested with EcoRI; lane 2, PCR product of gene yqhD digested with EcoRI; lane 3, plasmid pZR1 digested with EcoRI; lane 4, plasmid pZR1 digested with BglI. (b) Identification of plasmid pZR2. Lane 1, plasmid pGAPZB digested with XbaI; lane 2, PCR product of gene dhaB digested with XbaI; lane 3, plasmid pZR2 digested with XbaI; lane 4, pZR2 digested with BamHI. (c) Identification of plasmid pZR3. Lane 1, plasmid pCAMBIA3300-zeocin digested with BamHI; lane 2, plasmid pZR3 digested with BamHI; lane 3, PCR product of the pZR3 using primer P1 and P2; lane 4, PCR product of the control. (d) Identification of plasmid pZR4. Lane 1, plasmid pZR3 digested with HindIII; lane 2, plasmid pZR4 digested with HindIII; lane 3, plasmid pZR3 digested with XbaI; lane 4, plasmid pZR4 digested with XbaI.

Enzyme assays Crude cell extracts were prepared by sonication of cell pastes and subsequent centrifugation. Cell pastes were obtained by centrifugation of fermentation broth at 6000 rev min)1 for 10 min at 4C. The pastes were washed in 20-mmol l)1 Tris-HCl buffer (pH 8Æ0) or 50 mmol l)1 potassium phosphate buffer (pH 8Æ0), centrifuged as described earlier, and resuspended in a small amount of the appropriate assay resuspension buffer. The cells were then disrupted by sonication for 5 min on ice at a duty cycle of 60% with 1-s cycles. Cell debris was removed by centrifugation at 12 000 rev min)1 for 10 min at 4C in a microcentrifuge. The glycerol dehydratase activity was estimated by using the 3-methyl-2-benzothiazolinon method (Ahrens et al. 1998) with a correction factor of 1Æ41. The activity of 1,3propanediol oxidoreductase isoenzyme was determined using the reverse reaction (conversion of 1,3-propanediol to 3-hydroxypropionaldehyde) owing to the instability of the 3-hydroxypropionaldehyde. The concentration of 1,3-propanediol oxidoreductase isoenzyme was determined

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by the method of Gerlind et al. (2004). All enzyme assays were performed at 30C. One unit of enzyme activity is defined as the amount of enzyme needed to catalyse the conversion of 1 lmol of substrate per min at 30C. Specific enzyme activity is indicated as unit per mg protein. All assays were performed in duplicate; the reported values are the average from two assays. The protein concentration of the cell extracts was determined by the method of Bradford. Determination of 1,3-propanediol by gas chromatography 1,3-Propanediol and glycerol were determined using a gas chromatograph (Shimazu GC-14B, FID-detector). The column used in the study was 2 m · 5 mm stainless steel column packed with Chromosorb 101. N2 gas was used as a carrier at the flow rate of 40 ml min)1. The detector temperature was set at 220C and column temperature was set at 210C. Results Construction of key plasmids needed for engineering Saccharomyces cerevisiae To produce 1,3-propanediol from d-glucose in S. cerevisiae, we planned to integrate the heterogeneous genes yqhD and dhaB into the chromosome of S. cerevisiae by A. tumefaciens-mediated transformation (Piers et al. 1996). To this end, we had to integrate the two genes yqhD and dhaB into the vector pCAMBIA3300-zeocin which can transfer its T-DNA to S. cerevisiae. First, the gene yqhD encoding for 1,3-propanediol oxidoreductase isoenzyme was isolated from E. coli, and the gene dhaB encoding for glycerol dehydratase from K. pneumoniae. Both genes were cloned into the vector pGAPZB between the promoter GAP and AOX1TT terminator to form plasmids pZR1 and pZR2, respectively (Fig. 2). The GAP promoter and AOX1TT terminator would help the expression of heterogeneous gene in S. cerevisiae. The plasmid pZR1 containing the gene yqhD was digested with EcoRI, and the DNA fragments was analysed as shown in lane 3 of Fig. 3a. There were only two fragments in the digestion: one had the same size as empty plasmid pGAPZB digested with EcoRI (Fig. 3a, lane 1) and the other had the same size as the gene yqhD (Fig. 3a, lane 2). Digesting pZR1 with BglI yielded three DNA fragments (Fig. 3a, lane 4), which are consistent with the map of pZR1 (Fig. 2). Plasmid pZR1 contains four Bg I sites that could divide the plasmid into three major DNA fragments of 1960, 1211 and 897 bp, respectively, and a small DNA fragment of size 32 bp.

1,3-Propanediol production in S. cerevisiae

The plasmid pZR2 containing the gene dhaB was digested with XbaI, and the DNA fragments were analysed as shown in Fig. 3b. The size of empty plasmid pGAPZB (Fig. 3b, lane 1) is very close to the size of the gene dhaB (Fig. 3b, lane 2). Digesting pZR2 with XbaI yielded two DNA fragments with similar sizes around 2800 bp (Fig. 3a, lane 3). Digesting pZR2 with BamHI yielded two major DNA fragments which are consistent with the map of pZR2 (Fig. 2). Plasmid pZR2 contains three BamHI sites that can yield three major DNA fragments with sizes of 3000, 2300 and 300 bp. Next, the DNA fragment pGAP-yqhD-AOX1TT was digested from plasmid pZR1, purified, and ligated into pCAMBIA3300-zeocin to form pZR3. The plasmid pZR3 was digested with BamHI. As expected, the digested DNA fragment from pZR3 was bigger in size than the digested empty vector (Fig. 3c, lane 2 vs. lane 1). The integration of gene yqhD in the plasmid pZR3 was further confirmed by PCR amplification using primers P1 and P2 (Table 1). The PCR products had the same size as the gene yqhD (Fig. 3C, lane 3 vs. Fig. 3a, lane 2). Finally, the DNA fragment pGAP-dhaB-AOX1TT was digested from plasmid pZR2, purified, and ligated into pZR3 to form pZR4. The plasmid pZR4 was digested with HindIII. As expected, the digested DNA fragment from pZR4 was bigger in size than the digested pZR3 (Fig. 3d, lane 2 vs. lane 1). The integration of both genes yqhD and dhaB in the plasmid pZR4 was further confirmed by digesting with XbaI. The digested pZR3 contained two DNA fragments, which is consistent with the two XbaI sites in pZR3 (Fig. 3d, lane 3), while the digested pZR4 contained four DNA fragments, consistent with the four XbaI sites in pZR4 (Fig. 3d, lane 4). Thus, the key plasmid pZR4 needed for engineering S. cerevisiae was constructed. Integration of genes yqhD and dhaB into the chromosome of Saccharomyces cerevisiae Plasmid pZR4 containing both genes yqhD and dhaB was transformed into A. tumefaciens LBA4404 to form LBA4404-ZR4. Incubation of S. cerevisiae W303-1A cells with A. tumefaciens LBA4404-ZR4 led to the formation of zeocin-resistant colonies on media containing acetosyringone, indicating that the T-DNA of pZR4 in A. tumefaciens has transferred into the chromosome of S. cerevisiae W303-1A. The engineered S. cerevisiae strain was designated W303-1A-ZR. Stability of the zeocin-resistant phenotype of W303-1A-ZR was confirmed by growing the cells on fresh YDP media containing 150 lg ml)1 zeocin (Fig. 4). To confirm the presence of the yqhD and dhaB in the chromosome, gene yqhD was amplified from genomic

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Figure 5 (a) PCR amplification of yqhD and dhaB from the chromosome of W303-1A-ZR. Lane 1, the gene yqhD with a size of 1Æ2 kb; lane 2, the gene dhaB with a size of 2Æ7 kb; lane 3, PCR product of yqhD from W303-1A; lane 4, PCR product of yqhD from W303-1AZR; lane 5, PCR product of gene dhaB from W303-1A; lane 6, PCR product of gene dhaB from W303-1A-ZR. (b) Southern hybridization of two randomly recombinant S. cerevisiaeW303-1A genomic DNA with Dig-high labelled probes synthesized from the 2Æ7 kb XbaI fragment dhaB of pGAPZB-dhaB and from the 1Æ2 kb EcoRI fragment yqhD of pGAPZB-yqhD. Lane 1, the negative control; lane 2, the positive control; lane 3, DNA markers; lanes 4 and 5, Southern blot analysis of two independent samples.

The engineered Saccahromyces cerevisiae W303-1A-ZR can produce 1,3-propanediol from D-glucose

Zeocin+ Figure 4 The engineered Saccharomyces cerevisiae W303-1A-ZR was resistant to zeocin, while the wild-type S. cerevisiae W303-1A was not. (a) Both W303-1A and W303-1A-ZR could grow on the yeastpeptone-dextrose (YPD) media. (b) W303-1A-ZR grew on YPD media containing 150 ug ml)1 zeocin, while W303-1A did not.

DNA isolated from W303-1A-ZR by using primers P1 and P2, and the gene dhaB was amplified using primers P3 and P4. Genomic DNA isolated from W303-1A was used as an empty control. As shown in Fig. 5a, genes yqhD and dhaB could be amplified from W303-1A-ZR (Fig. 5, lanes 4 and 6), but not from W303-1A (Fig. 5, lanes 3 and 5), indicating that both genes yqhD and dhaB were present in the chromosome of W303-1A-ZR, but not in W303-1A. The integration of both genes yqhD and dhaB in the chromosome of W303-1A-ZR was further confirmed by Southern blot analysis (Fig. 5b). Except for genes yqhD and dhaB, another DNA fragment with higher molecular weight could also be observed on the Southern blot, indicating that the T-DNA had integrated into the chromosome of S. cerevisiae. 1774

The engineered W303-1A-ZR grew normally as the wild-type strain W303-1A (Fig. 6). Both W303-1A and W303-1A-ZR were grown to log phase, and the cell extracts were prepared. The activity of glycerol dehydratase and 1,3-propanediol oxidoreductase isoenzyme in the cell extracts were measured by the standard assay conditions. Both specific activities were detected in the cell extracts of S. cerevisiae W303-1A-ZR, but not in S. cerevisiae W303-1A (Table 2). This indicates that genes dhaB and yqhD were expressed in W303-1A-ZR. To test whether the engineered S. cerevisiae W3031A-ZR could produce 1,3-propanediol from d-glucose, both W303-1A and W303-1A-ZR were grown aerobically, and the concentration of 1,3-propanediol was measured. As we expected, W303-1A-ZR could produce 1,3propanediol, but wild-type W303-1A could not (Table 2). This suggests that genes dhaB and yqhD not only can be expressed, but also function in the engineered S. cerevisiae W303-1A-ZR. To check the stability of genes dhaB and yqhD in the chromosome, the aforementioned assays were performed with samples taken from several continuous fermentations of W303-1A-ZR. All the assays showed similar specific activities of the dehydratase and the oxidoreductase (data not shown), suggesting the stable integration of both genes in the chromosome of W303-1A-ZR.

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1·2

high-cost feedstock glycerol for fermentation. There is an urgent desire to produce 1,3-propanediol from low-cost d-glucose in a single micro-organism (Nakamura and Whited 2003). By expressing K. pneumoniae dha regulon genes, Emptage et al. (2002) have patented a recombinant E. coli that can convert d-glucose to 1,3-propanediol with high titre. In this study, we constructed a recombinant S. cerevisiae strain W303-1A-ZR that can convert d-glucose to 1,3propanediol. We cloned genes yqhD and dhaB required for the production of 1,3-propanediol from glycerol, integrated them in the chromosome of S. cerevisiae W303-1A and demonstrated that the engineered S. cerevisiae could produce 1,3-propanediol from the low-cost feedstock d-glucose. This study also confirmed that A. tumefaciens genetic transfer system is a valuable tool in yeast biotechnology. To our knowledge, this is the first report on engineering S. cerevisiae for the production of 1,3-propanediol by using ATMT (Agrobacterium tumefaciens-mediated transformation) method. The yield of 1,3-propanediol in our engineered S. cerevisiae was about 0Æ4 g l)1. This is reasonable because the wild-type S. cerevisiae W303-1A can only produce 5–6 g glycerol in 1 l of YPD media containing 10% d-glucose. The importance of this work is that it opened a new way to produce 1,3-propanediol from low-cost feedstock. To further increase the yield of 1,3-propanediol in S. cerevisiae, we can either modify the surrounding genes in the glycerol pathway in S. cerevisiae W303-1A-ZR to produce more glycerol, or integrate genes yqhD and dhaB into some yeast strains that can produce high levels of glycerol. For example, we can delete the genes dhaK encoding glycerol kinase and dhaD encoding glycerol dehydrogenase in S. cerevisiae W303-1A-ZR to prevent glycerol from re-entering central carbon metabolism. We can integrate genes yqhD and dhaB in Candida glycerinogenes strain WL2002-5 that can produce 120 g glycerol in 1-l media containing 25% d-glucose (Zhuge et al. 2001). These studies might lead to a safe and cost-efficient method for industrial production of 1,3-propanediol in S. cerevisiae.

1·0

DCW (G/l–1)

0·8

0·6

0·4

0·2

0

0

20

40

80 60 Time (h)

100

120

Figure 6 The growth curves of the W303-1A ( ) and W303-1AZR ( ) showed that the engineered W303-1A-ZR grows normally.

Table 2 Expression of glycerol dehydratase (DhaB) and 1,3-propanediol oxidoreductase isoenzyme (YqhD) and production of 1,3-propanediol in engineered Saccharomyces cerevisiae W303-1A-ZR. The different strains were all cultivated at 30C for 72 h in media containing 100 g l)1 D-glucose

Strains

DhaB (U mg)1 protein)

YqhD (U mg)1 protein)

1,3-Propanediol (g l)1)

W303-1A W303-1A-zeocin W303-1A-yqhD W303-1A-ZR

0 0 0 4Æ1 ± 0Æ2

0 0 2Æ9 ± 0Æ2 3Æ0 ± 0Æ3

0 0 0 0Æ4 ± 0Æ05

Discussion Metabolic engineering is an emerging technique that genetically modifies or designs biochemical pathways in micro-organisms, and has a potential importance in fermentation industry. In this study, we used metabolic engineering to construct S. cerevisiae that can produce 1,3-propanediol. 1,3-Propanediol is a very important chemical that could improve the quality of polymers, food and medicines. However, because it has been synthesized chemically by expensive enzymes at high pressure and high temperature, 1,3-propanediol is expensive. Fermentation method has the advantage of mass production at low cost, but the few naturally available microorganisms producing 1,3-propanediol could only use a

Acknowledgements The authors thank Dr Xiaomei Zhang for her valuable suggestions. This work was supported by the National Natural Science Foundation of China (20676053, 30570142), National Programs for High Technology Research and Development of China (2006AA020103, 2007AA02Z207), Jiangsu Provincial Youth Scientific and Technological Innovation Foundation (BK2006504) (Academic Leader) and Program for Changjiang Scholars and Innovative Research Team in University (IRT0532).

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