Cloning, Expression And Characterization Of Glycerol 3-phosphate Dehydrogenase From Candida Glycerinogenes

In recent years, Candida. glycerinogenes, a novel osmotolerant yeast, was isolated from glazed fruit in Southern China and has been commercially exploited to produce glycerol. Compared to other yeasts, C. glycerinogenes has several useful properties, such as tolerance to high concentration glucose, rapid growth and excellent glycerol production in aerobic fermentation. These good properties are supposed to be due to the fact that this yeast possesses several specific genes involved in glycerol biosynthesis. Although the physiological and fermentation properties of C. glycerinogenes have been investigated, little molecular studies on glycerol biosynthesis and osmoregulation have been done. Our knowledge of cell properties at the molecular level and genetic background lags far behind those of model yeasts such as S. cerevisiae because of lack of effective genetic manipulation tool. The significant production and accumulation of glycerol in C. glycerinogenes poses the questions whether a biochemical pathway similar to other yeasts or an alternative pathway for glycerol formation exists in this yeast and CgGPD is the target of HOG pathway. In this dissertation, CgGPD encoding cytol NAD~+-glycerol 3-phospahte dehydrogenase was cloned from C. glycerinogenes and characterized by heterologous expression in S. cerevisiae. The effects of deletion and/or overexpression of CgGPD in C. glycerinogenes for glycerol production were investigated. The main contents of this dissertation follow:(1) A 4900-bp genomic fragment containing a GPD gene encoding a glycerol-3-phosphate dehydrogenase from C. glycerinogenes homologous to GPD genes in other yeasts was cloned using degenerate primers PCR in conjunction with inverse PCR. This gene was named CgGPD (GenBank Accession No. EU186536). Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42,695 Da. The CgGPD gene consisted of a N-terminal NAD+-binding domain and a central catalytic domain whereas seven STREs were found in the upstream region. Comparison of CgGPD with amino acid sequences revealed that CgGPD showed the highest identity to the GPD of Pichia angusta (70.9%) but only 46.7% to the Schizosaccharomyces pombe gpd1. However, the amino acid sequence is more identical to the GPD1 (60.9%) than the GPD2 (56.2%) in S. cerevisiae. Southern hybridization suggests that GPD gene may exist as a single copy in C. glycerinogenes.(2) Functional analysis revealed that S. cerevisiae gpd1Δand gpd1Δ/gpd2Δosmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD specific activity increased significantly when stressed with NaCl whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. Wild type strain W303A employing CgGPD improved glycerol yield significantly. The results showed that CgGPD containing upstream regulatory sequence is functional in S. cerevisiae. (3) To facilitate the difference among CgGPD, GPD1 and GPD2, functional comparision with three genes was undertaken, using S. cereivisae and isogenic mutants as a heterologous expression system. Expression of CgGPD and GPD2 in hog1 improved osmotolernace and glycerol production, but GPD1 have no similar effect. However, expression of CgGPD, GPD1 and GPD2 in pbs2 enhanced growth ability in high osmolarity conditions. Overexpression CgGPD and GPD1 in gpd1/gpd2 suppressed the osmosensitivity and enhanced glycerol production. However, overexpression GPD2 in gpd1/gpd2 have no phenotype and physiology difference. It was worthy to be mentioned that in anaerobic conditions CgGPD complemental fulfill GPD2 for redox balance regulation in gpd1/gpd2 mutant.(4) The genetic transformation of C. glycerinogenes by electroporation were tested and the physical and biological parameters involved in transformation efficiency were optimized. Pretreatment of yeast cells with either dithiothreitol (DTT) or Lithium acetate (LiAc) enhanced the frequency of transformation markedly by electroporation. The optimized cell concentrations, amount of DNA and pulse amplitude were 2×10~9 cells/mL, 200 ng and 8 kV/cm respectively. Furthermore, significantly higher transformation efficiency was obtained when the electroporated cells were pretreated with Zeocin before plating.(5) To investigate the effect of CgGPD on glycerol biosynthesis in C. glycerinogenes, the CgGPD-disrupted mutant and the overexpressed CgGPD in C. glycerinogenes were constructed and confirmed by diagnositc PCR. The CgGPD-disrupted mutant reduced its osmotolerance, defected its growth profile and resulted in a remarkable decrease of glycerol yield when cultured in the fermentation glycerol medium. On the other hand, the C. glycerinogenes improved its ethanol production by deletion of CgGPD. Hovever, the transformant employing CgGPD accelerated its glucose consumption rate, enhanced its glycerol productivity, increased the specific GPD activity and decreased its ethanol prodution ability.