Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Engineered Saccharomyces cerevisiae that produces 1,3‐propanediol from d‐glucose

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. tumefaciens‐mediated transformation. This study might lead to a safe and cost‐efficient method for industrial production of 1,3‐propanediol.     

Production of 1,3-propanediol from Glycerol by Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.     

Identification and utilization of a 1,3-propanediol oxidoreductase isoenzyme for production of 1,3-propanediol from glycerol in Klebsiella pneumoniae

In a previous study, we showed that 1,3-propanediol (1,3-PD) was still produced from glycerol by the Klebsiella pneumoniae mutant strain defective in 1,3-PD oxidoreductase (DhaT), although the production level was lower compared to the parent strain. As a potential candidate for another putative 1,3-PD oxidoreductase, we identified and characterized a homolog of Escherichia coli yqhD (88% homology in amino acid sequence), which encodes an alcohol dehydrogenase and is well known to replace the function of DhaT in E. coli. Introduction of multiple copies of the yqhD homolog restored 1,3-PD production in the mutant K. pneumoniae strain defective in DhaT. In addition, by-product formation was still eliminated in the recombinant strain due to the elimination of the glycerol oxidative pathway. An increase in NADP-dependent 1,3-PD oxidoreductase activity was observed in the recombinant strain harboring multiple copies of the yqhD homolog. The level of 1,3-PD production during batch fermentation in the recombinant strain was comparable to that of the parent strain; further engineering can generate an industrial strain producing 1,3-propanediol.     

Construction and Characterization of a 1,3-Propanediol Operon

The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions. These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter. The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications. In a fed-batch cofermentation of glycerol and glucose, Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter. The fermentation had two distinct phases. In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate. In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate. The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B₁₂, which must be provided in E. coli fermentations. However, the amount of coenzyme B₁₂ needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations. 1,3-PD is a useful intermediate in the production of polyesters. The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.     

Construction of stress-induced metabolic pathway from glucose to 1,3-propanediol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

1,3-Propanediol is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive synthesis. The aim of this study was to engineer an Escherichia coli strain that can produce 1,3-propanediol directly from glucose. We successfully constructed a stress-induced metabolic pathway from glucose to 1,3-propanediol in recombinant E. coli by the expression of gpd1 and gpp2 genes from Saccharomyces cerevisiae and dha operon from Klebsiella pneumoniae, respectively. Batch cultivation of the recombinant E. coli showed that 12.1 g/L 1,3-propanediol was accumulated in the culture without using any inducer.     

Effect of aeration strategy on the metabolic flux of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> producing 1,3-propanediol in continuous cultures at different glycerol concentrations

The microbial production of 1,3-propaneidol (1,3-PD) by Klebsiella pneumoniae in continuous fermentation was investigated under low, medium and high glycerol concentrations in the absence and presence of oxygen. The production of 1,3-PD increased with increasing glycerol concentrations, reaching a maximum (266 mmol l⁻¹) under high glycerol concentration (760 mmol l⁻¹) with air sparging at 0.04 vvm. The yield of 1,3-PD, however, decreased gradually with increasing glycerol concentrations, with the highest yield (0.52 mol mol⁻¹) obtained for low glycerol concentration (270 mmol l⁻¹) under anaerobic condition. Enzyme activity assays showed that the specific activity of glycerol dehydratase was highest (0.04 U mg⁻¹) for culture sparged with 0.04 vvm air under high glycerol concentration. The specific activities of glycerol dehydrogenase and 1,3-propanediol oxidoreductase were also improved for all glycerol concentrations and in the presence of oxygen, implying that the dha operon was not repressed under microaerobic conditions. Analysis of metabolic fluxes showed that more carbon flux was shifted to the oxidative pathway with increasing glycerol concentrations, resulting in a reduced flux to 1,3-PD formation. However, the increases in carbon fluxes were not evenly distributed among the oxidative branches of the pathway. Furthermore, ethanol and acetic acid levels were slightly increased whereas 2,3-butanediol and lactic levels were greatly enhanced.     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

1,3-Propandiol production by engineered Hansenula polymorpha expressing dha genes from Klebsiella pneumoniae

Currently, 1,3-propanediol (1,3-PD) is an important chemical widely used in polymer production, but its availability is being restricted owing to its expensive chemical synthesis. A methylotrophic yeast Hansenula polymorpha was engineered by expression of dhaB1, dhaB2, dhaB3, dhaB _RA1 and dhaB _RA2 encoding glycerol dehydratase complex and dhaT encoding 1,3-PD oxidoreductase from Klebsiella pneumoniae under direction of promoter of glyceraldehyde-3 phosphate dehydrogenase (GAPDH). The engineered recombinant yeast strain can produce 1,3-PD from glucose (2.4 g L⁻¹) as well as glycerol (0.8 g L⁻¹), which might lead to a safe and cost-effective method for industrial production of 1,3-PD from various biomass resources.     

Quantitative analysis on inactivation and reactivation of recombinant glycerol dehydratase from Klebsiella pneumoniae XJPD-Li

The genes encoding glycerol dehydratase were cloned and characterized by genomic DNA from Klebsiella pneumoniae XJPD-Li, and the assigned accession number EF634063 was available from the GenBank database. The DNA sequence analysis showed that the clone included three ORFs (dhaB, dhaC and dhaE, encoding α, β and γ subunit of glycerol dehydratase, respectively). Among three subunits of glycerol dehydratase, amino acid residues H₁₃, S₁₉₃, N₃₅₉, E₄₀₇, and M₅₁₅ of α subunit, N₄₇, L₁₅₀, V₁₈₉ of β subunit are different with what had been reported. Subsequently, the expression vector was constructed and transformed into E. coli BL21, and the colony carried genes of glycerol dehydratase were selected. SDS-PAGE examination showed that the three subunits were well expressed. The specific activity of recombined glycerol dehydratase reached to 0.299 U mg^?1, which was about 3 times comparing with that of the wild strain. The research also displayed that both glycerol and O₂ could inactive the glycerol dehydratase expressed in E. coli quickly in 10 min. The inactivated glycerol dehydratase could be effectively reactivated under the system as follows: the concentration of ATP, Mg²⁺ and coenzyme B₁₂ were 50 mM, 10 mM and 3 μM, respectively, when the ratio (W/W) of glycerol dehydratase to reactivation factor was 4:1. The O₂-inactivated and glycerol-inactivated dehydratase could be reactivated to 97.3% and 98.9% of initial activity in 10 min in above-mentioned conditions, respectively. The reactivation factor together with ATP was considered as the “ON/OFF” reactivating condition.     

Molecular Cloning, Co-expression, and Characterization of Glycerol Dehydratase and 1,3-Propanediol Dehydrogenase from Citrobacter freundii

1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.     

Stimulation of reductive glycerol metabolism by overexpression of an aldehyde dehydrogenase in a recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strain defective in the oxidative pathway

Previously, we constructed a glycerol oxidative pathway-deficient mutant strain of Klebsiella pneumoniae by inactivation of glycerol dehydrogenase (dhaD) to eliminate by-product synthesis during production of 1,3-propanediol (1,3-PD) from glycerol. Although by-product formation was successfully blocked in the resultant strain, the yield of 1,3-PD was not enhanced, probably because dhaD disruption resulted in insufficient regeneration of the cofactor NADH essential for the activity of 1,3-PD oxidoreductase (DhaT). To improve cofactor regeneration, in the present study we overexpressed an NAD⁺-dependent aldehyde dehydrogenase in the recombinant strain. To this end, an aldehyde dehydrogenase AldHk homologous to E. coli AldH but with NAD⁺-dependent propionaldehyde dehydrogenase activity was identified in K. pneumoniae. Functional analysis revealed that the substrate specificity of AldHk embraced various aldehydes including propionaldehyde, and that NAD⁺ was preferred over NADP⁺ as a cofactor. Overexpression of AldHk in the glycerol oxidative pathway-deficient mutant AK/pVOTHk resulted in a 3.6-fold increase (0.57 g l⁻¹ to 2.07 g l⁻¹) in the production of 3-hydroxypropionic acid (3-HP), and a 1.1-fold enhancement (8.43 g l⁻¹ to 9.65 g l⁻¹) of 1,3-PD synthesis, when glycerol was provided as the carbon source, compared to the levels synthesized by the control strain (AK/pVOT). Batch fermentation using AK/pVOTHk showed a significant increase (to 70%, w/w) in conversion of glycerol to the reductive metabolites, 1,3-PD and 3-HP, with no production of by-products except acetate.     

High-level expression of the 1,3-propanediol oxidoreductase from klebsiella pneumoniae in escherichia coli

As one of four key enzymes in glycerol dismutation process, 1,3-propanediol oxidoreductase (EC.1.1.1.202) is important in converting glycerol to 1,3-propanediol in Klebsiella pneumoniae. The dhaT gene encoding 1,3-propanediol oxidoreductase was amplified by polymerase chain reaction (PCR) using the genome DNA of K. pneumoniae as template, and then cloned into cloning vector pMD18-T. After DNA sequence was determined, the dhaT gene was subcloned into Escherichia coli expression vector pET-22b (+) and pET-28a (+). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both the recombinant E. coli BL21 (DE3) (pET-22b (+)-dhaT) and E. coli BL21(DE3)(pET-28a (+)-dhaT) expressed predicted 42-kDa 1,3-propanediol oxidoreductase after induced by isopropyl-β-d-thiogalactopyranoside (IPTG), and the recombinant enzyme of E. coli BL21 (DE3) (pET-28a (+)-dhaT) was mostly in soluble form, and exhibited high activity (96.8 U/mL culture). The recombinant enzyme was purified and biochemically characterized. The apparent K _m values of the enzyme for 1,3-propanediol and NAD⁺ were 8.5 and 0.21 mM, respectively. The enzyme had maximum activity at pH 9.5 and 30°C.     

Expression of 1,3-propanediol oxidoreductase and its isoenzyme in Klebsiella pneumoniae for bioconversion of glycerol into 1,3-propanediol

In the Klebsiella pneumoniae reduction pathway for 1,3-propanediol (1,3-PD) synthesis, glycerol is first dehydrated to 3-hydroxypropionaldehyde (3-HPA) and then reduced to 1,3-PD with NADH consumption. Rapid conversion of 3-HPA to 1,3-PD is one of the ways to improve the yield of 1,3-PD from glycerol and to avoid 3-HPA accumulation, which depends on enzyme activity of the reaction and the amount of reducing equivalents available from the oxidative pathway of glycerol. In the present study, the yqhD gene, encoding 3-propanediol oxidoreductase isoenzyme from Escherichia coli and the dhaT gene, encoding 3-propanediol oxidoreductase from K. pneumoniae were expressed individually and co-expressed in K. pneumoniae using the double tac promoter expression plasmid pEtac-dhaT-tac-yqhD. The three resultant recombinant strains (K. pneumoniae/pEtac-yqhD, K. pneumoniae/pEtac-dhaT, and K. pneumoniae/pEtac-dhaT-tac-yqhD) were used for fermentation studies. Experimental results showed that the peak values for 3-HPA production in broth of the three recombinant strains were less than 25% of that of the parent strain. Expression of dhaT reduced formation of by-products (ethanol and lactic acid) and increased molar yield of 1,3-PD slightly, while expression of yqhD did not enhance molar yield of 1,3-PD, but increased ethanol concentration in broth as NADPH participation in transforming 3-HPA to 1,3-PD allowed more cellular NADH to be used to produce ethanol. Co-expression of both genes therefore decreased by-products and increased the molar yield of 1,3-PD by 11.8%, by catalyzing 3-HPA conversion to 1,3-propanediol using two cofactors (NADH and NADPH). These results have important implications for further studies involving use of YqhD and DhaT for bioconversion of glycerol into 1,3-PD.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Production of 1,3-Propanediol from Glucose by Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

A range of recombinant strains of Escherichia coli were developed to produce 1,3-propanediol (1,3-PDO), an important C3 diol, from glucose. Two modules, the glycerol-producing pathway converting dihydroxyacetone phosphate to glycerol and the 1,3-PDO-producing pathway converting glycerol to 1,3-PDO, were introduced into E. coli. In addition, to avoid oxidative assimilation of the produced glycerol, glycerol oxidative pathway was deleted. Furthermore, to enhance the carbon flow to the Embden- Meyerhof-Parnas pathway, the Entner-Doudoroff pathway was disrupted by deleting 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. Finally, the acetate production pathway was removed to minimize the production of acetate, a major and toxic by-product. Flask experiments were carried out to examine the performance of the developed recombinant E. coli. The best strain could produce 1,3-PDO with a yield of 0.47 mol/mol glucose. Along with 1,3-PDO, glycerol was produced with a yield of 0.33 mol/mol glucose.     

Development of a Saccharomyces cerevisiae strain for the production of 1,2-propanediol by gene manipulation

The main goal of this research was to achieve a more efficient production of 1,2-propanediol (1,2-PD) using mutated Saccharomyces cerevisiae. 1,2-PD cannot be produced by wild type S. cerevisiae. To develop a S. cerevisiae mutant that could produce 1,2-PD, the mgs gene of E. coli-K12 MG1655 and the dhaD gene of Citrobacter freundii were inserted into yeast expression vectors such as pESC-URA and pESC-TRP and transformed into the wild type of S. cerevisiae. As a result, the batch fermentation of S. cerevisiae YPH500, harboring an mgs gene inserted pJES27 vector, resulted in a yield of 0.17 g/L. On the other hand, the methylglyoxal synthase of the recombinant S. cerevisiae YPH500, harboring a dhaD gene inserted pJES29 vector, was inactive and produced no detectable amount of 1,2-PD. Therefore, in order to achieve a maximum yield of 1,2-PD, we selected the pESC-TRP vector that is able to co-express the dhaD gene with the pJES27 vector. By inserting the dhaD gene into the pESC-TRP vector, the pJES30 vector was constructed. The maximal yield of 1,2-PD achieved in a 1% galactose batch fermentation by pJES27 and pJES30 harboring S. cerevisiae was 0.45 g/L.     

Inactivation of <Emphasis Type="Italic">dhaD</Emphasis> and <Emphasis Type="Italic">dhaK</Emphasis> abolishes by-product accumulation during 1,3-propanediol production in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose P_BAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.