顶头孢霉转化系统的构建与应用

【作者】 张丕燕；

【导师】 朱春宝；

【作者基本信息】 上海医药工业研究院 ， 微生物与生化药学， 2003， 博士

【摘要】 过去的50年中，β-内酰胺类抗生素的工业发酵生产成为生物技术应用最成功的范例。目前，β-内酰胺类抗生素的全球销量已达到150亿美元，占整个抗生素市场的65％。在增加产量和大幅度地降低生产成本方面，最主要的β-内酰胺抗生素生产菌，产黄青霉(Penicillium chrysogenum)和顶头孢霉(Acremonium chrysogenum or Cephalosporium acremonium)的菌种改良和发酵工艺的优化得到卓有成效的发展。传统的突变和选择、理性化筛选技术和原生质体融合技术最大可能地提高了β-内酰胺类抗生素生产菌的发酵产量；如今，现代生物技术的迅速发展正在成为菌种改良的又一有效工具。 丝状真菌顶头孢霉转化系统的建立是应用分子遗传方法研究顶头孢霉最为重要的先决条件之一。过去的一些研究表明质粒对顶头孢霉的转化率太低，限制了许多分子生物学技术的应用，如利用互补突变株从顶头孢霉的DNA文库筛选和鉴别基因、基因敲除或者基因替换(gene replacements)。而一个有效的转化载体在一定程度上取决于载体上选择标记上游的启动子的强弱，因此，在构建一个真菌的转化载体时，克隆合适的启动子序列是必需的。 酵母-大肠杆菌穿梭质粒pGBT9上的Trp1基因互补了色氨酸营养缺陷型啤酒酵母Y153(Saccharomyces cerevisiae Y153)的色氨酸合成能力。利用质粒pGBT9构建了一个用于筛选具有真菌启动子功能的DNA片段的捕获载体pGBT14。该质粒上的两个Trp1基因“头对头”相连，中间有一个BamHI单克隆位点但没有启动子序列，携带pGBT14的啤酒酵母Y153不能在无色氨酸的完全极限省却培养基(complete minimal dropout medium)上生长。利用pGBT14在大肠杆菌中构建了带顶头孢霉0.5～2Kb片段的染色体DNA文库，将分离到的质粒导入啤酒酵母Y153中进行筛选鉴别，获得了24个能使TrpⅠ表达而互补色氨酸缺陷的顶头孢霉的染色体DNA片段。在啤酒酵母Y153中，来自顶头孢霉的这些外源DNA片段具有不同的启动Trp1基因表达的能力。 根据已发表的pcbAB-pcbC双向启动子序列设计了一对引物，从顶头孢霉染色体DNA中扩增出1.3Kb的DNA片段，序列测定及比对表明该DNA片段的两端序列与发表的pcbAB-pcbC双向启动子序列完全相同。利用启动子筛选质粒pUT715构建了两个转化载体pYG8和pYG9并转化顶头孢霉，根据其启动博莱霉素抗性基因的表达能力验证了这个pcbAB-pcbC双向启动子的强弱，证明pcbC的启动能力强于pcbAB。利用克隆的pcbAB-pcbC的双向启动子构建了带VHb基因的转化载体，它对顶头孢霉的转化率平均为9.8个／μg质粒DNA，与本实验室先前利用构巢曲霉的TrpC基因的启动子构建的顶头孢霉转化质粒pYG715／Vgb的平均0.5个转化子／μg质粒DNA相比，转化率有了显著的提高。勿 摘要 利用 PCR技术从顶头跑霉染色体DNA中扩增了编码已驶COA：DAC乙酸转移酶的CofEF 基因。与已发表的序列进行比对发现有三个鹏发生了改变：77 T—C、451C—A、803 G—C， 导致了三个密码子的改变，它们分别是GTC－－＋GCC、GAT、AAT、CGG、CCG．其中，第一 个突变是沉默突变，后两个突变分别是Asp－－＋Asn，Afg、Pro．这些贻的突变是由于PCR 扩增的错误所致，还是本研究所用的工业菌株经长期菌种选育而产生的有益突变，有待进一 步证实。 以温控诱导表达载体 pws构建了 Cofsr基因的大肠杆菌表达载体 pixsB经高温诱导 后，SDS－PAGE电泳显示，在4lm：附近有明显的蛋白质条带，与已报道的乙酸COA：DAC．乙酸转移酶的大小一致。 CofEF基困编码的DAOC合成购羟化酶和CofG编码的DAC G＆转移娜化头抱菌素C 生物合成的限速步骤．利用扩增的pCM8pC比双向启动子和博莱霉素抗性基因，构建了含 CofEF基因的载体质粒 pYG20，转化顶头跑霉时，转化率可达 14＊个个g质粒 DNA．另外， 以宿主菌为对照分别对10个重组菌株进行了发酵试验，初步结果表明宿主菌的最高产量为 4．24 mg／ffil，重组菌的最高产量为 4．20 mg／ffil． 还尝试了根癌农杆菌介导的顶头跑霉的转化，每个转化平板可获得30－50个转化子，与 PEG介导的顶头抱霉的转化相比，每次转化试验能够获得更多的转化子，表明该方法是目前． 最高效的转化顶头抱霉的方法．更多还原

【Abstract】 The industrial production of p-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples for the application of biotechnology in fermentation industry. Today, the P-lactam antibiotics, particularly penicillins and cephalosporins, represent the major products of antibiotics with worldwide sales for approximately 15 billion dollars and account for approximately 65% shares of the total world market for antibiotics. Traditional natural isolation, mutation and selection, rational screening and protoplast fusion have made a great progress in the improvement of both Penicillium chrysogenum and Cephalosporium acremonium, two main industrial strains for production of P-lactam antibiotics. Rapid developments in molecular biology have become another powerful tool for strain breeding.Establishment of genetic transformation system is an important prerequisite for application of molecular genetics to extensive study of C. acremonium. However, low frequency of integrative genetic transformation is a major obstacle to gene cloning in Cephalosporium acremonium, for example, screening and identification of genes from a DNA bank by complementation of blocked mutants, knock out or replacement of genes. An effective vector for transformation will to some extent depend on the promoter function located in the upstream of selective marker. It is necessary to clone an appropriate promoter when a vector for transformation is constructed. Trp1 gene in an E. coli.-yeast shuttling plasmid pGBT9 complements the synthesis of tryptophan in an auxotroph of Saccharomyces cerevisiae Y153. A promoter-trapping vector pGBT14 containing two Trpl genes linked by head to head but without promoter region was constructed in order to select DNA segments with promoter activity. Saccharomyces cerevisiae Y153 containing pGBTH could not grow up on a complete minimal dropout medium without tryptophan. Using this vector, a 0.5-2.0Kb chromosomal DNA library of C. acremonium was constructed, and twenty four DNA fragments exhibiting promoter activity in Saccharomyces cerevisiae Y153 were selected by expressing the DNA library. It was demonstrated that the promoter activity for those DNA fragments was different in Saccharomyces cerevisiae Y153. A pair of primers was designed based on the published pcbAB-pcbC promoter sequence. 1.3Kb DNA fragment containing the full-length bidirectional pcbAB-pcbC promoter region was amplified by PCR method from chromosomal DNA isolated from filamentous fungus C. acremonium. DNA sequencing for the amplified fragment was determined and sequence alignment between amplified and known fragments was analyzed by Vector NTI software. Two plasmid vectors designated as pYG8 and pYG9 were constructed, which contain a bleomycin resistant gene downstream pcbAB promoter or pcbC promoters respectively. It was found thatthe activity of pcbC was much stronger than that of pcbAB depending on the resistance of their separate transformants to bleomycin. A plasmid vector pYG13 was constructed by inserting a VHb gene downstream pcbC promoter. It was demonstrated that the frequency of transformation was significantly increased as compared with plasmid pYG715/Vgb containing TrpC gene promoter from Aspergillus nidulans and could reach 9.8 transformants per ug DNA when Cephalosporium acremonium was transformed with pYG13. Southern blotting and Carbon monoxide-binding analysis reveal that vgb gene was integrated into the genomic DNA of C. acremonium and functional vgb gene was expressed in C. acremonium.CefEF gene encoding DAOC synthetase/hydroxylase was cloned with PCR technique. The alignment of sequences revealed that three base mutation exist in the amplified cefEF gene, that is T77→C77; G451→A451; G803→C803, respectively, which resulted in the changes of three codons: GTC→GCC; GAT→AAT and CGG→CCG. The first one is silence mutation, the others are Asp→Asn, Arg→Pro. It is unknown whether such changes were caused by PCR error or real mutation resulting from the processes of breeding performed in thi更多还原