【作者】 魏巍；

【导师】 魏东芝；

【作者基本信息】 华东理工大学 ， 生物化工， 2010， 博士

【摘要】 甾体类药物具有重要的生理活性,在临床上有广泛的应用,是仅次于抗生素的第二大类药物。目前甾体药物生产中常用的方法有化学合成和微生物转化两种,其中微生物转化法显示出其巨大的优势。分枝杆菌可降解植物甾醇生成系列代谢中间体,例如雄甾-4-烯-3,17-二酮(AD)、雄甾-1,4-二烯-3,17-二酮(ADD)、9a-羟基雄甾-4-烯-3,17-二酮(9α-OH-AD)等,这些产物可作为前体用于制备临床甾体药物。国内对生产甾体菌种的研究仅限于简单筛选诱变,并未对菌种在分子水平上进行深入的探索,这也限制了国内甾体药物生产的进一步发展,而在国外,上述研究已经展开并进展迅速。随着分子生物学的发展和人们对甾体代谢机制研究的深入,基因工程在甾体药物生产中必将得到广泛的应用。本课题针对本研究室新筛选的一株具有应用前景的新金分枝杆菌NwIB-01 (Mycobacterium neoaurum NwIB-01),从基因水平上对甾体转化过程中的关键酶进行研究并在此基础上通过基因操作对筛选菌种的生产性状进行改良。NwIB-01能够转化植物甾醇同时生成AD与ADD,但AD与ADD结构相似,在工业生产中很难进行分离,这限制了该菌的进一步应用。基于此考虑,我们从NwIB-01中克隆出AD(D)相互转化并积累的关键酶基因,3-甾酮-△’-脱氢酶(KsdD)及3-甾酮-9α-羟基化酶(KSH)基因,进而,通过基因工程的方法对这些关键酶进行了基因敲除强化等分子操作并初步构建了具有应用前景积累ADD的基因工程菌,上述基因都是在新金分枝杆菌中的首次报道。具体而言,本论文主要做了以下工作：1.菌种的筛选及性状研究鉴定出一株新金分枝杆菌(Mycobacterium neoaurum NwIB-01),在3.7-L发酵罐中NwIB-01可以转化15g/l的植物甾醇生成1.76g/l的AD及4.23 g/1 ADD,底物的摩尔转化率为57.8%。2.关键基因3-甾酮-△1-脱氢酶基因(ksdDM)的研究首次在新金分枝杆菌中克隆出3-甾酮-△1-脱氢酶的全序列。分析表明,ksdDM大小1701 bp,以GTG为起始密码子,可能的核糖体结合位点为其上游的GAAAGG序列,该基因与Mycobacterium smegmatis str. MC2155 (Genbank CP000480) ksdD的核苷酸序列一致性为82%(NCBI最高序列一致性)。通过pET系列质粒实现了ksdDM在大肠杆菌BL21(DE3)中的异源表达,pET-22b所表达的KsdDM酶活为0.8 U／mg蛋白。3.关键基因3-甾酮-9α-羟基化酶基因(kshA)及3-甾酮-9α-羟基化酶还原酶基因(kshB)的研究通过设计简并引物和基因走读的方法,分别得到了kshA及kshB基因的全长序列。kshA大小1188 bp,以GTG为起始密码子,其核糖体结合位点为以其上游的GGGAGG序列,这是kshA在新金分枝杆菌中的首次报道。kshA与NCBI中Mycobacterium smegmatis str. MC2155 (Genbank ACV87349)的核苷酸序列一致性为85%(NCBI最高序列一致性)。利用大肠杆菌表达载体pET-22b对kshA进行了克隆表达及全菌转化实验,同时将基因kshA及kshB在载体pET-22b中进行了共表达。4.3-甾酮-△1-脱氢酶基因(ksdDM)的无标记敲除利用分枝杆菌基因敲除质粒p2NIL和pGOAL19构建ksdDM自杀质粒,通过双交换基因重组技术实现目的基因的无标记敲除。ksdDM基因敲除菌(重组分枝杆菌NwIB-02)主要积累AD,在含0.1 g/L底物的摇瓶实验中,发酵96 h后,产物中AD和ADD的摩尔比为11.5：1,而野生菌主要积累ADD (AD:ADD=1:2.4)。5.3-甾酮-／△1-脱氢酶基因(ksdDM)的强化表达和产ADD基因工程菌的构建利用分枝杆菌复制型质粒pMV261及整合型质粒pMV306,将ksdDM在新金分枝杆菌NwIB-01中进行强化表达,构建分枝杆菌强化表达菌株NwIB-03及NwIB-04。发酵实验表明,NwIB-04在3.7-L发酵罐上产物中ADD的产量在加入底物96 h后达到最大值4.94g/l,此时AD的含量只有0.096g/l,产物中AD与ADD的摩尔比为AD:ADD=1:51.5。最终产物中ADD的纯度超过95%。6.甾体降解基因簇研究利用质粒pCC2FOS,构建新金分枝杆菌NwIB-01的Fosmid基因组文库,通过筛选及测序,得到了大约81 kb的基因簇序列并找到了多个甾体代谢关键酶。综上所述,本论文首次克隆出新金分枝杆菌中负责甾体代谢的关键基因ksdDM、kshA及kshB,并通过基因工程的方法改良了菌株的生产特性,得到了生产ADD的基因工程菌,这为甾体生产菌株的基因改良及甾体药物中间体的制备奠定了基础。更多还原

【Abstract】 Due to the significant physiological activity, steroid drugs have been widely used in clinic application as its second place following antibiotic drugs. In contrast to the chemical synthesis, biotransformation provides an alternative method in the production of steroid medicine intermediates and has been used extensively as a common and economical process in the pharmaceutical industry. Phytosterols can be catabolized to a series of steroidal derivatives by Mycobacterium. Among these,4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD) and 9a-hydroxy-4-androstene-3,17-dione (9α-OH-AD) are the major products usually applied in the industry due to their essential value in producting various steroid medications. Present domestic research of steroid production strains is just limited to strain isolation and mutation screening, but not involved in the steroid degradation genes at the molecular level, which hinder the development of domestic steroid drug industry. In abroad, rapid progress in this research has been occured. With the development of molecular biology and in-depth studies on steroid degradation system, genetic engineering is surely to play an important role in the production of steroid drugs.We obtained and investigated the key enzyme genes involved in the steroid degradation and modified Mycobacterium neoaurum NwIB-01 at the genetic level in this study. Consequently, strain NwIB-01 exhibited powerful ability of cleaving the side chain specifically from soybean phytosterols to accumulate AD (4-androstene-3,17-dione) and ADD (1,4-androstadiene-3,17-dione). However, the difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. Due to the structural similarity, the AD and ADD mixture complicates their purification and decreases their yield, which impedes the further commercial application of strain NwIB-01. In this paper, we obtained some genes for key enzyme including 3-ketosteroidΔ1-dehydrogenase and 3-ketosteroid 9a-hydroxylase and this is the first report of the genes in Mycobacterium neoaurum. Moreover, We have tackled the problem via the gene disruption and augmentation of the key genes. The KsdDM augmentation mutant showed to be good 1,4-androstadiene-3,17-dione (ADD)-producing strains respectively. The detailed work was introduced as following:1. Strain screening and research on phenotypeStrain NwIB-01 was selected and identified as Mycobacterium neoaurum. It was showed that when cultured in 15 g/1 phytosterols, the yield of ADD reached 4.23 g/1 while accompanied by 1.76 g/1 AD in 96-h-old culture (the molar yield of AD+ADD is 57.8%). 2. Investigation of the key gene 3-ketosteroidΔ1-dehydrogenase (ksdDM)Complete open reading frame of 3-ketosteroidΔ1-dehydrogenase has been obtained from Mycobacterium neoaurum for the first time. The results show that the full length of ksdDM is 1701 bp with GTG as its starting codon, and the upstream sequence of GAAAGG is probably the ribosome binding site. Homology analysis revealed that ksdDM in NwIB-01 is 82% identical to the ksdD nucleotide sequence (Genbank CP000480) in Mycobacterium smegmatis str. MC2155 (NCBI highest homology). In addition, plasmids of pET series were used to the heterologous expression in E. coli BL21(DE3). The KsdDM activity was assayed by the spectrophotometrical method and the maximum activity of the intracellular soluble parts of KsdDM was 0.8 U/mg.3. Study of the key genes coding for 3-ketosteroid 9a-hydroxylase (kshA) and 3-ketosteroid 9α-hydroxylase reducase (kshB)Complete gene sequences of 3-ketosteroid 9a-hydroxylase (kshA) and 3-ketosteroid 9a-hydroxylase reducase (kshB) have been obtained respectively relying on design of degenerate primers and genome walking. The results show that the full length of kshA is 1188 bp with GTG as its start codon, and a 6 bp sequence GGGAGG acts for the corresponding ribosome biding sites. Homology analysis revealed that kshA in NwIB-01 is 85%identical to the 3-ketosteroid 9a-hydroxylase nucleotide sequence (ACV87349) in Mycobacterium smegmatis str. MC2155 (NCBI highest homology). Subsequently, the whole open reading frame of kshA has been cloned and heterologously expressed using vector pET-22b in E.coli and co-expression system of kshA and kshB through vector pET-22b has been finally achieved.4. Gene knock-out of ksdDMThe suicide plasmid of KsdD was constructed with the plasmids p2NIL and pGOAL19. At 96 h after adding phytosterols, the molar ratio of AD:ADD in products of strain NwIB-01 was 1:2.4, while the molar ratio reached 11.5:1 in products of mutant NwIB-02 (ksdDM knock-out mutant).5. Overexpression of ksdDM and construction of industrial strain producing ADD.Mycobacterial replicating vectors pMV261 and pMV306 were used to over-express ksdDM in NwIB-01 and the mutant NwIB-04 by overexpression ksdDM was constructed. For strain NwIB-04, ADD reached the maximum 4.94 g/1 at 96 h of culture, while AD showed no obvious accumulation during the whole time course and only 0.096 g/1 at 96 h. The molar ratio of AD:ADD is 1:51.5 in products of mutant NwIB-04, and the final purity of ADD exceeded 95%.6. Preliminary research on the steroid degradation gene clusterThe genomic fosmid library was built by using plasmid pCC2FOS and the steroid degradation gene cluster was selected using degenerate primers. And about 81 kb fragment of the steroid degradation gene cluster was obtained. Furthermore, some key enzymes involved in steroid degradation have been discerned in this research.In short, this is the first report of the KsdD and Ksh genes in Mycobacterium neoaurum and the first report of gene-augmentation of a sterol catabolic enzyme to construct a sterol pathway intermediate (ADD)-producing strain. This study provides a feasible way to achieve excellent phytosterol-transforming strains with high product purity.更多还原