【作者】 陈献忠；

【导师】 诸葛健；

【作者基本信息】 江南大学 ， 发酵工程， 2008， 博士

【摘要】 产甘油假丝酵母(Candida glycerinogenes)是优良的甘油生产菌株,并且已成功的应用于工业生产中。研究者在产甘油假丝酵母菌株的生理生化、发酵工艺优化、代谢机理方面开展了卓有成效的研究。但是与酿酒酵母(Saccharomyces cerevisiae)等模式菌株相比,该工业菌株的遗传和分子生物学信息比较匮乏。在C. glycerinogenes中,甘油合成途径的关键酶—胞浆3-磷酸甘油脱氢酶的编码基因以及该基因在细胞中的具体的生理功能是未知的。为此,本文从产甘油假丝酵母基因组中克隆出甘油合成的关键酶基因,并对其进行了详细的功能鉴定,最后通过在C. glycerinogenes过表达和缺失研究进一步确定该基因在细胞过量合成甘油中的作用。本文主要研究成果概括如下:(1)利用PCR方法,克隆了C. glycerinogenes的胞浆NAD~+依赖的3-磷酸甘油脱氢酶编码基因(CgGPD)。1167 nt的开放阅读框编码一个分子量为43 kDa,包含388个氨基酸的蛋白,氨基酸水平上该基因与具有安格斯毕赤酵母相似性最高,为70.9%,与粟酒裂殖酵母相对较低,仅为46.7%。并且发现在产甘油假丝酵母基因组中仅有一个CgGPD基因,不存第二个同功异构酶编码基因。(2) CgGPD基因在gpd1/gpd2和gpd1酿酒酵母突变株中表达能够显著提高细胞耐渗透压能力和甘油合成能力,表明CgGPD基因能够完全功能互补酿酒酵母GPD1基因的缺失。渗透压诱导实验表明CgGPD基因的表达受到渗透压的生理调控。在野生型酿酒酵母中过量表达CgGPD基因能够大幅度提高细胞的甘油合成能力,同时结果表明CgGPD基因自身上游调控序列能够有效调控基因表达。(3) CgGPD和GPD2基因能够提高hog1细胞的耐渗性,而GPD1基因的表达对突变株的耐高渗透压能力并没有显著提高;在pbs2突变株中分别过量表达CgGPD、GPD1和GPD2基因都能够提高转化子在高渗压条件下的生长性能;过量表达CgGPD和GPD1能够使gpd1/gpd2突变株降低对渗透压的敏感性,GPD2基因的表达则不具这种显著的这种功能;在hog1和pbs2突变株中分别过量表达CgGPD、GPD1和GPD2基因均能使转化子细胞在渗透压刺激下诱导胞内甘油合成和积累;GPD1基因的过量表达仅能一定程度上互补gpd1/gpd2突变株细胞在厌氧环境中GPD2基因的功能,而过量表达CgGPD基因则能够完全功能互补GPD2基因的缺失。(4)根据同源重组原理,以含有腐草霉素(Zeocin)抗性基因的pGAPZb质粒为基本构架,以CgURA3基因作为整合位点构建了用于转化产甘油假丝的酵母的整合载体;分别优化了物理参数和细胞生理参数对电转化效率的影响。结果表明,对数生长中期的细胞经DTT或LiAc预处理后在2×10~9个/mL的细胞浓度下加入200μg的线性整合载体,在1600V电压下,200Ω电阻、25μF电容进行电击,获得的转化效率可达到394个转化子/μg DNA。(5)以腐草霉素抗性基因作为选择标记,分别构建CgGPD基因敲除载体pUC-gpd-Zeoin和同源表达载体pGA-rDNA-CgGPD,导入产甘油假丝酵母细胞,获得稳定的缺失突变株和过表达转化子。甘油发酵实验表明CgGPD基因缺失显著降低了细胞的甘油合成能力,同时增加了细胞的产乙醇能力,而细胞的EMP代谢途径活力的降低导致细胞量显著下降,使得细胞在高浓度葡萄糖下的生长能力受损;过量表达CgGPD基因能够加速葡萄糖的消耗,缩短发酵周期,从而提高甘油的产率和得率,同时胞内副产物丙酮酸、乙酸和乳酸等有一定的提高,而乙醇的生成则受到抑制。更多还原

【Abstract】 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.更多还原