基于Fosmid基因组文库及转录组分析的甾体降解基因簇的挖掘与表达调控

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. Although，steroidal degradation mechanism has been researched using Mycobacterium tuberculosis as the mode bacteria, the detailed mechanism of sterol metabolism of key enzyme reactions and related regulation mechanism remains to be in-depth study. The gene responsible for the steroidal degradation general arranged as a gene cluster with the length about 100 kbp, and presently only a few steroidal degradation gene cluster were repoerted. This increases the difficulty to study the metabolism of the steroidal degradation gene cluster from the gene cluster level. In abroad, the key technologies of steroidal drugs production was monopolized by some large multinational pharmaceutical companies. As the main saponin resources and low-end steroidal drugs producing country, although steroidal pharmaceutical industry is a large scale, the profits is meager. 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. To solve the scientific issues, this application research the key gene in steroidal degradation gene cluster and the molecular mechanisms of the key gene in-depth research. Then, we try to get the gene cluster sequences using the method of building genomic fosmid library. And the genetic information of steroidal degradation gene cluster was researched using the methods of transcriptome analysis. In summary，this study not only research the function and regulation mechanism of steroidal degradation gene cluster but also provide a feasible way to achieve excellent phytosterol-transforming strains with high product purity. 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 and promote the development of domestic steroidal pharmaceutical industries.

甾体类药物是应用广泛，仅次于抗生素的第二大药物。工业上利用微生物转化法生产甾体药物在经济、生态环保方面有巨大优势。虽然近两年以结核分枝杆菌为模型，研究了甾体代谢相关机理，但对具体酶反应详细机制、基因簇调控等尚待深入研究。负责甾体降解的基因一般呈基因簇（约100kb）形式排列且国际上已得到的基因簇很少，这增加了从基因簇水平研究甾体代谢的难度。目前甾体药物生产关键技术被一些大型跨国制药公司垄断。我国作为皂素资源与中低端甾体药物生产大国，虽工业规模大，但利润微薄，这严重制约行业的发展。本申请首先研究甾体代谢关键基因的表征及其分子机理，进而试图利用Fosmid文库得到基因簇全序列，并利用转录组分析挖掘基因簇上的基因信息，解析未知基因的功能。综上，本申请不仅在科学问题上研究甾体代谢基因簇的功能和调控机理，更为基因工程菌株的构建及基因工程在甾体药物生产中的应用乃至国内甾体医药工业发展及打下一定的基础。

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。工业上利用微生物转化法生产甾体药物在经济、生态环保方面有巨大优势。分枝杆菌可降解植物甾醇生成系列代谢中间体，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，并在甾体代谢基因簇层面进行深入研究。负责甾体降解的基因一般呈基因簇（约100kb）形式排列且国际上已得到的基因簇很少，这增加了从基因簇水平研究甾体代谢的难度。.本课题主要进展如下：.1. 新金分枝杆菌NwIB-01基因文库构建：本研究利用质粒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。并解析部分未知基因的功能。.2. 甾体代谢关键基因3-甾酮-9α-羟基化酶的表征及分子改造：KSH酶为母核代谢的一关键因素，但其催化的9α-羟基化反应同时也是生成9-OHAD的必经之路，因此KSH酶的高活性是高产9-OHAD的重要保障。本研究发现了M. neoaurum中的2种KshA同工酶以及1种KshB活性。.3. 甾体代谢关键基因胆固醇氧化酶的表征及代谢解析：鉴定了 NwIB-01中具备ChO活性的两种同工酶ChoMl与ChoM2，通过酶催化反应动力学的表征，揭示了其催化甾醇反应的分子机制。基于基因敲除的代谢解析，我们首次阐释了 ChO在甾醇生物催化过程中的代谢功能，同时指明了其因不同的细胞定位而各自独特的代谢方式。.综上所述，本论文首次得到了81 kb的新金分枝杆菌NwIB-01 （Mycobacterium neoaurum NwIB-01）基因簇序列，并对其基因的功能进行了解析，为后续研究奠定了良好的基础。