Xinyao Lu

Bioconversion of L-phenylalanine to 2-phenylethanol by the novel stress-tolerant yeast Candida glycerinogenes WL2002-5

ABSTRACT 2-Phenylethanol (2-PE) is a high value aromatic alcohol with a rose-like odor that is utilized in the cosmetics and other industries. Although the chemical routes of 2-PE production have been altered by some microbial transformation processes, the poor tolerance to organic solvents of these microorganisms has limited the 2-PE yield. In this study, the stress-tolerant yeast Candida glycerinogenes WL2002-5 showed a 2-PE tolerance to 4 g/l, which is the highest reported to date. Moreover, the 2-PE titer in a batch fermentation from L-phenylalanine reached 5g/l, which is the highest level achieved by fermentation without in situ product recovery. These results suggest C. glycerinogenes WL2002-5 is a robust strain for the bioproduction of 2-PE with potential for commercial exploitation.

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of Candida glycerinogenes

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.

Role of CgHOG1 in Stress Responses and Glycerol Overproduction of Candida glycerinogenes

Candida glycerinogenes, the glycerol producer with excellent multi-stress tolerances, is considered to be a potential biotechnological host used in the production of glycerol and its derivatives under extreme fermentation conditions. In this study, to evaluate the multiple roles of mitogen-activated protein kinase CgHOG1, we constructed a gene disruption system in the diploid C. glycerinogenes to obtain CgHOG1 null mutant. Pseudohyphae generation of the CgHOG1 mutant under non-inducing condition indicated a repressor role in morphological transitions. Disruption of CgHOG1 resulted in increased sensitivities to osmotic, acetic acid, and oxidative stress but not involved in thermotolerance. In the CgHOG1 mutant, NaCl shock failed to stimulate the accumulation of intracellular glycerol and was fatal. In addition, the CgHOG1 mutant displayed a significant prolonged growth lag phase in YPD medium with no decrease in glycerol production, whereas the mutant cannot grow under hyperosmotic condition with no detectable glycerol in broth. These results suggested that CgHOG1 plays important roles in morphogenesis and multi-stress tolerance. The growth and glycerol overproduction under osmotic stress are heavily dependent on CgHOG1 kinase.

Enhanced 1,3-propanediol production in Klebsiella pneumoniae by a combined strategy of strengthening the TCA cycle and weakening the glucose effect

This study aimed to strengthen the reducing equivalent generation in Klebsiella pneumoniae for improving 1,3‐propanediol (PDO) production.

Effects of xylitol dehydrogenase (XYL2) on xylose fermentation by engineered Candida glycerinogenes

Efficient bioconversion of d‐xylose into various biochemicals is critical for lignocelluloses application. Candida glycerinogenes, expressing the xylitol dehydrogenase (XYL2) gene, has the ability to produce glycerol from xylose entered into pentose phosphate pathway. In this study, we demonstrate that low expression levels of the XYL2 gene derived from Scheffersomyces stipitis in C. glycerinogenes is a major bottleneck in efficient xylose fermentation. Through a metabolic engineering approach using an integrative expression, XYL2 was identified as an overexpression target for improving xylose metabolism. Two recombinant strains with XYL2 overexpression were constructed to ferment a mixture of glucose and xylose simultaneously in batch fermentation. Compared with C. glycerinogenes (wild type), glycerol production from xylose by C. glycerinogenes (PURGAPX2) and C. glycerinogenes (PURGPDX2) was increased by 94.5% and 103.3%, respectively. It was also found that additional overexpression of XYL2 under the control of strong promoters in a xylose‐fermenting strain not only reduced xylitol accumulation but also increased glycerol yields. As the expression levels of XYL2 increased, the glycerol yields gradually improved from 30.6 to 63.3 g/L, whereas the xylitol yields significantly decreased from 38.7 to 19.9 g/L. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose‐fermenting strains.

A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes

ABSTRACT The tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hybrid promoter pGUKd displayed dramatically increased fluorescence intensity at pH 2.5, thus verifying that pGUKd is a low-pH inducible promoter. The promoter pGUKd was then used to express the D-xylose dehydrogenase gene xylB, resulting in increased expression levels of xylB at low pH. The recombinant protein exhibited higher specific activities under lower pH conditions and produced 38 g/l D-xylonate at pH 2.5. This rate is much higher than that produced by fermentation at pH 5.5. These results suggest that the novel hybrid promoter pGUKd functions to direct the production of D-xylonate at low pH, and we provide a candidate genetic tool for the stress tolerant yeast C. glycerinogenes.

γ-aminobutyric acid accumulation enhances the cell growth of Candida glycerinogenes under hyperosmotic conditions

γ-aminobutyric acid (GABA) is an important non-protein amino acid involved in the response to various environmental stresses in plant cells. The objectives of this study was to test the hypothesis that intracellular accumulation of GABA improves osmotic tolerance in the unconventional yeast Candida glycerinogenes. In C. glycerinogenes, the expression of UGA4 encoding GABA-specific permease is highly induced by hyperosmotic stress. Exogenous GABA application enhanced intracellular GABA accumulation and promoted cell growth under hyperosmotic conditions. Overexpression of the glutamate decarboxylase gene GAD1 resulted in an increased intracellular GABA and improvement in cell growth under hyperosmotic conditions. These results indicated that improving intracellular GABA accumulation of C. glycerinogenes, either through exogenous application or cellular synthesis, is available for improving the tolerance to hyperosmotic stress. We demonstrate that GABA accumulation plays an important role in osmotic stress resistance of the unconventional yeast C. glycerinogenes.

Functional and expression studies of two novel STL1 genes of the osmotolerant and glycerol utilization yeast Candida glycerinogenes

Candida glycerinogenes is an osmotolerant yeast used for commercial glycerol production, as well as a glycerol utilization yeast which produces high biomass on glycerol medium. In the present study, two STL1 homologues CgSTL1 and CgSTL2 encoding the putative glycerol transporters were identified, and their products were found to be localized to plasma membranes by tagging GFP protein. The functions of CgSTL1 and CgSTL2 on glycerol transport were confirmed by their expression in S. cerevisiae STL1 null mutant and simultaneous deletion in C. glycerinogenes. The expression of CgSTL1 were osmotic-induced, whereas that of CgSTL2 was constitutive. Over-expression of CgSTL1 and CgSTL2 in C. glycerinogenes resulted in improved glycerol consumption rate and cell growth. Our study provided more details on the glycerol transporter of C. glycerinogenes, the potential cell factory for using glycerol as a carbon source.

The role of budABC on 1,3-propanediol and 2,3-butanediol production from glycerol in Klebsiella pneumoniae CICIM B0057.

ABSTRACT 1,3-propanediol (1,3-PD) is an important compound from which many others can be synthesized. 2,3-butanediol (BDO) is the key by-product in the biosynthesis of 1,3-PD from glycerol, but it impedes its downstream purification. In Klebsiella, the budA, budB and budC genes encode enzymes that are responsible for the synthesis of BDO. In this study, 3 individual antisense RNAs were designed to repress the expression and hence activity of BudA-C. Compared with the parent strains, the activities of BudB and BudC were reduced by 60.5% and 70.5%, respectively, and the mRNA level of budA was reduced by 70%. Decreased BudC activity had no effect on cell growth or carbon distribution. However, reduced BudA and BudB activity decreased the BDO concentration by 35% and led to a 10% increase in the yield of 1,3-PD. This result suggests the activities of BudA and BudB could be key factors in the production of BDO from glycerol in Klebsiella. This study provides a deeper understanding of the role of budABC in glycerol metabolism in Klebsiella.

Characterization of a novel cytochrome P450 from Amycolatopsis sp. CGMCC1149 for hydroxylation of lovastatin

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated to 3‐hydroxymethyl lovastatin (product I) by a hydroxylase. In the current study, a novel hydroxylase gene p450lov was isolated from Amycolatopsis sp. CGMCC1149 by degenerate PCR and self‐formed adaptor PCR and expressed in Escherichia coli. The gene encodes a 403‐amino‐acid protein with a molecular weight of 44.8 kDa and was designated as a new member of cytochrome P450 (CYP) 105 family, CYP105A44. Meanwhile, a lovastatin catalytic in vitro system was established, and an optimal hydroxylation reaction system contained 30 µM lovastatin, 600 µM NADH, 120 µM ferredoxin, 0.04 U ferredoxin−nicotinamide adenine dinucleotide phosphate reductase, and 100 µM CYP105A44 in a final volume of 200 µL Tris HCl buffer (50 mM, pH 7.4). These would be helpful for further studies on the hydroxylation of statins.