耐高渗压高产甘油的一个假丝酵母新种——产甘油假丝酵母

从自然标本中分离获得一高产甘油的菌株WL20025,仅发酵葡萄糖及微发酵蔗糖,能利用葡萄糖、蔗糖、乙醇生长,微利用甘油和柠檬酸,不利用肌醇、硝酸盐、赤藓醇、阿拉伯醇、甘露醇,与DBB显色反应为阴性,可在含500g/L葡萄糖或100mL/L醋酸的培养基中及40℃下生长,可在水活度为0890～0900的培养基中生长,出芽生殖,易形成“假丝酵母菌型”假菌丝,不进行有性生殖,线粒体DNA的分子量为20kb,是假丝酵母属的一个新种,定名为产甘油假丝酵母(Candida glycerolgenesis Zhuge[WTBZ] sp.nov.)。     

玉米浆在产甘油假丝酵母甘油发酵中的作用机理

以复合培养基和合成培养基进行比较发酵,研究了玉米浆在产甘油假丝酵母甘油发酵过程中的作用机理。结果表明:玉米浆中的磷、氮和微量元素是影响产甘油假丝酵母甘油发酵的3个关键因素。当玉米浆磷浓度为121·75mg/L(玉米浆浓度为14g/L),最大甘油转化率达到53·44%。玉米浆磷可以调节EMP途径与HMP途径之间碳架代谢流的分布,随着玉米浆浓度进一步增加,过量磷能抑制HMP途径而激活EMP途径,因而复合培养基各项发酵参数的变化非常显著。玉米浆氮对磷的调节功能有协同作用,但并不是产甘油假丝酵母甘油发酵的理想氮源。玉米浆中的微量元素能够显著提高葡萄糖的消耗速率、促进菌体的生长和增加甘油的产量。     

耐高渗压是产甘油假丝酵母甘油高产的基础

运用化学诱变法从产甘油假丝酵母获得１９株渗透压突变株。对其生理及发酵性能的研究结果显示,所有渗透压敏感突变株的甘油产率皆显著降低并巨大多数渗透压敏感突变株的糖利用速度减慢．耐渗透压性能提高突变株的糖利用率几乎与亲株相同而甘油发酵性能改变呈现多样性,突变株如ＷＬ－ＯｓｍＤ、ＷＬ－ＯｓｍＦ以及ＷＬ-ＯｓｍＨ甘油转化率极度降低而突变株ＷＬ－ＯｓｍＢ的甘油耗糖转化率接近亲株。     

产甘油假丝酵母甘油代谢关键酶的研究

本文对产甘油假丝酵母的甘油代谢关键酶进行了研究,发现产甘油假丝酵母同化甘油能力极弱,少量葡萄糖明显改善其同化甘油的能力;线粒体3磷酸甘油脱氢酶受3磷酸甘油的强烈诱导,受葡萄糖代谢的阻遏。在甘油发酵过程中,产甘油假丝酵母胞浆3磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰,其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平,成为甘油高速积累期(18～48h)甘油合成的关键性的限速酶。在甘油发酵18～48h内,3磷酸甘油酯酶的酶活处于高水平,并在36h时出现酶活峰值;处于缓慢甘油积累阶段的48～72h间,3磷酸甘油酯酶已处于低水平表达,此时,3磷酸甘油酯酶则成为甘油合成的限速酶。产甘油假丝酵母稳定并高表达其胞浆3磷酸甘油脱氢酶基因并且其所表达的3磷酸甘油酯酶酶活远高于胞浆3磷酸甘油脱氢酶这一特征是其高产甘油根本所在。     

耐高渗压是产甘油假丝酵母甘油高产的基础*

运用化学诱变法从产甘油假丝酵母获得１９株渗透压突变株。对其生理及发酵性能的研究结果显示,所有渗透压敏感突变株的甘油产率皆显著降低并巨大多数渗透压敏感突变株的糖利用速度减慢．耐渗透压性能提高突变株的糖利用率几乎与亲株相同而甘油发酵性能改变呈现多样性,突变株如ＷＬ－ＯｓｍＤ、ＷＬ－ＯｓｍＦ以及ＷＬ-ＯｓｍＨ甘油转化率极度降低而突变株ＷＬ－ＯｓｍＢ的甘油耗糖转化率接近亲株。     

限磷对产甘油假丝酵母甘油合成与胞内磷积累的影响

研究了磷酸盐限量对产甘油假丝酵母甘油合成与胞内磷积累的影响。结果表明, 当酵母细胞从适磷或富磷培养基转接入低磷培养基时, 发酵过程中胞内积累的磷逐渐减少; 而当菌体从低磷培养基转接入适磷或富磷培养基时, 发酵过程中胞内聚磷酸盐的积累量迅速增加。当细胞在第14小时和第38小时从适磷培养基转接入低磷培养基时甘油得率分别高达60.9%和61.4%, 而甘油产率则分别为2.03 g/(L·h)和2.23 g/(L·h)。这些现象说明限制发酵培养基中的磷浓度是产甘油假丝酵母高产甘油的必要条件, 并为其反复分批发酵法生产甘油提供了重要依据。     

运用穿梭载体建立产甘油假丝酵母质粒基因文库

产甘油假丝酵母WL2002─5既具有耐高渗压性能,又能以葡萄糖为原材料高产甘油,且耐高渗压性能与高产甘油的能力相关。本研究以大肠杆菌—酿酒酵母穿梭质粒YEp51为载体,通过对产甘油假丝酵母WL2002─5高分子量染色体DNA的提取、Sau3AI部分酶切、体外重组等建立起产甘油假丝酵母WL2002—5的质粒基因文库,库容为5．3×105,已满足对WL2002—5某一特定基因的克隆。     

一些氨基酸对产甘油假丝酵母甘油生产的促进作用

以EMP途径与TCA循环中间代谢物的添加为对照,研究在尿素为氮源的产甘油假丝酵母发酵过程中添加氨基酸对甘油产量的影响。结果表明:对甘油产量有强促进作用的氨基酸有谷氨酸、谷氨酰胺、天冬氨酸、天冬酰胺、甘氨酸、赖氨酸、酪氨酸、脯氨酸、组氨酸和丝氨酸,其最适添加浓度在0.26～0.45g/L之间,丙酮酸、α_酮戊二酸、草酰乙酸、柠檬酸和琥珀酸的最适添加浓度在0.24～0.42g/L之间;赖氨酸最适于在0h添加,丙酮酸和草酰乙酸在第14h,谷氨酸、谷氨酰胺、组氨酸、脯氨酸、天冬氨酸、酪氨酸、甘氨酸、α_酮戊二酸和琥珀酸在第30h,天冬酰胺、丝氨酸和柠檬酸在第48h;在最适条件下添加这些促进剂,甘油产量均呈显著增加趋势,转化率和增加率分别达到60%和16%以上。氨基酸的作用机理为其脱氨形成的碳骨架经特定的分解代谢途径进入TCA循环,使其强化,导致碳代谢流在3_磷酸甘油醛节点处发生转移,使甘油合成途径的代谢流增加。     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。     

REMI介导电转化产甘油假丝酵母

为了分离耐高渗和甘油代谢相关基因,以Zeocin为选择标记,利用REMI技术电转化产甘油假丝酵母Candida glycerinogenes。考察了7种限制性内切酶对转化的影响,选择HindIII进一步优化了转化的几个条件。结果表明,在OD600≈1.3时收集细胞,在1.5kV电压下,感受态细胞浓度为2.0×109个细胞/mL,100U Hind III时,能获得129个转化子/μgDNA的较高转化率,58%的转化子稳定,表明REMI技术适合于产甘油假丝酵母的转化。     

新一代高产甘油重组菌的构建及其初步发酵

产甘油假丝酵母(Candida glycerinogenes WL2002-5)是一株发酵生产甘油的工业化菌株。为进一步提高其产甘油能力,本研究利用前期研究中成功克隆的产甘油假丝酵母中甘油合成关键酶3-磷酸甘油脱氢酶基因CgGPD1,构建根癌农杆菌双元载体pCAM3300-zeocin-CgGPD1后,电击转化根癌农杆菌LBA4404,通过根癌农杆菌介导法(ATMT)转化产甘油假丝酵母,构建了产甘油假丝酵母重组菌。并从中筛选出一株酶活力和产甘油性能较好的产甘油假丝酵母重组菌株C.g-G8。以葡萄糖为底物摇瓶发酵96h后,重组菌C.g-G8的甘油产量比野生型菌株Candida glycerinogene提高18.06%,平均耗糖速率提高12.97%,平均酶活力提高27.55%。本研究成功利用ATMT法转化产甘油假丝酵母构建新一代高产甘油菌株。     

The relation between the assimilation of methanol and glycerol in yeasts

Fifteen yeast strains of the genera Candida, Lodderomyces, Endomycopsis, Saccharomyces, Hansenula, Pichia and Torulopsis were investigated with respect to their ability to grow on methanol, glycerol and glucose as sole carbon and energy source. Eight of them can grow on both methanol and glycerol.Methanol is assimilated via triosephosphate (dihydroxyacetone) pathway. The dihydroxyacetone kinase is a key enzyme in methanol metabolism.The assimilation of glycerol can take place in bacteria via a phosphorylative or/and oxidative pathways. In general, the phosphorylative pathway is found in eucaryotes. In the present paper it is shown that in yeasts, which can utilize methanol and glycerol, too, glycerol is assimilated via an oxidative pathway, Dihydroxyacetone is a central intermediate in the assimilation of methanol as well as glycerol. It is metabolized by means of the dihydroxyacetone kinase.The enzyme formed during growth of Candida methylica on methanol does not differ from that of Candida valida H 122 after growing on glycerol as far as the regulatory properties are concerned.     

Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

产甘油假丝酵母甘油合成关键酶编码基因的克隆

产甘油假丝酵母(Candida glycerinogenes)作为优良的甘油生产菌株已经成功应用于工业化生产。但相对于酿酒酵母, 该菌株的耐高渗机理和甘油代谢的分子机制还不甚清楚。本文根据已公布的3-磷酸甘油脱氢酶基因的序列信息, 设计出一组寡核苷酸, 再运用简并PCR结合反向PCR技术从C. glycerinogenes的基因组DNA中获得了4 900 bp的核苷酸序列, 递交GenBank (No. EU186536)。该序列包含完整的编码胞浆3-磷酸甘油脱氢酶编码基因(CgGPD)开放阅读框及其上、下游调控序列。1 167 bp的开放阅读框编码388个氨基酸残基的蛋白。所演绎出氨基酸序列分析比对结果表明该基因产物的序列具有典型的胞浆3-磷酸甘油脱氢酶结构特征, 但与已鉴定的相关基因存在中等程度的同源性并在相应的辅酶催化位点和底物结合位点区域具有高度的保守性, 在氨基酸水平上与安格斯毕赤酵母的相似性最高, 达到70.9%。该基因在Saccharomyces cerevisiae W303A中异源表达能够显著提高细胞的甘油合成能力。     

An efficient genetic transformation method for glycerol producer Candida glycerinogenes

Transformation techniques generally require development before genetic and molecular studies of industrial yeast strains can commence. Candida glycerinogenes WL2002-5 has been used for industrial-scale glycerol production but has proven difficult to transform for molecular studies following previously published procedures. In the present study, phleomycin was used as drug-resistance marker based on perturbing plasma membranes and the lower minimum inhibitory concentration for C. glycerinogenes. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Pretreatment of yeast cells with either dithiothreitol or lithium acetate enhanced the frequency of transformation markedly by electroporation. Significantly higher transformation efficiency was obtained when the electroporated cells were pretreated with phleomycin before plating. With this method, a maximal transformation frequency of 394+/-50transformants/mug plasmid DNA was obtained. The transformation method will foster genetic manipulation of this industrial strain.     

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。