【作者】 姚抗；

【导师】 魏东芝；

【作者基本信息】 华东理工大学 ， 生物化工， 2014， 博士

【摘要】 类固醇激素,又称甾体激素,是人体一种重要的生命物质,常作为信号分子通过结合受体蛋白,微观调控特定基因的转录与细胞的正常生理功能。当其分泌失常或功能性不足时,会明显引起多种生命体征的失衡,需人为摄入甾类药物用以调节维稳,因此,甾体类药物(甾药)市场需求极大。目前,甾药的主流生产方式为,天然甾类物质的化学／生物法改造制备甾药前体,再由药性修饰获得药物原料,其中微生物法制备甾药前体的工艺因其环境污染小,反应步骤少,产品损失低、收率高等突出优势,受到新型甾药生产企业的青睐。该法获得的重要前体雄甾烯酮,经过化学修饰可用于合成近乎所有重要的临床甾体药物,其地位在当今的甾药加工行业中日益突出。但由于存在技术门槛高,微生物资源稀缺,生物技术手段落后、微生物代谢机制认知匮乏的现实问题,我国微生物转化生产的方式整体上仍处于较低水平,这严重的制约了甾药产业绿色转型的步伐。在这样的背景下,本论文运用先进的生物技术手段,初始于对一株甾类降解分枝杆菌的反应机制的探索,着眼于其代谢途径的人为可控改造,致力于开发先进的甾药微生物资源,系统的阐释了如何通过分子技术手段实现特定、多样代谢中间物的高纯度高产量高效积累。在此,将具体工作展示如下：1、胆固醇氧化酶的代谢解析与应用改造本文首次报道并证实了在生物转化媒介中,催化甾体氧化第一步反应的酶——胆固醇氧化酶ChO,是影响底物分子摄取与跨膜转运的关键因素。克服微生物底物转化力的瓶颈,毋庸置疑,应对ChO以及由其指导的底物代谢途径加以改造。文章鉴定了Mycobacterium neoaurum ATCC25795中具备ChO活性的两种同工酶ChoM1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。进一步的,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。以胞外分泌形式存在的ChoM2是影响ChO代谢底物最为关键因子之一,而以膜结合方式作用的ChoM1则从旁辅助,协同合作,有效加快代谢速率。于是,我们尝试强化关键因子ChoM2的表达,增进现有甾药中间体生产菌摄取与转化底物的效率,改善发酵高浓度甾醇时产率低下的现状。这样一来,在15g/L植物甾醇转化时,重要中间体雄甾-4-烯-3,17-二酮(AD)和雄甾-1,4-二烯-3,17-二酮(ADD)产量较改造前提高达51.2%和40.0%,进一步凸显了该类生物媒介在甾药中间体生产中的应用潜质。2、3-甾酮-△1-脱氢酶的功能阐释与活性缺失9α-羟基雄甾4-烯-3,17-二酮(9-OHAD)是一类重要的甾药前体,其结构中特殊的α-构型羟基,是获得卤代皮质类激素的重要前提。文章充分考虑到现有9-OHAD工业微生物的稀缺,创新的提出通过“开源节流”的技术手段,由野生型分枝杆菌M. neoaurum ATCC25795出发,人工改造其代谢通路,实现目标产物9-OHAD的高纯度积累。分析认为,作为微生物降解甾醇关键酶之一的3-甾酮-△1-脱氢酶(KstD),却是不利于9-OHAD稳定积累的重要诱因,必须将其活性彻底缺失。M. neoaurum中共存在3种KstD的同工酶(MN-KstD1-3),其中MN-KstD1与MN-KstD2为膜蛋白,MN-KstD3是胞内蛋白。三个KstD同工酶分别以9-OHAD, T, AD为最适底物,且MN-KstD1与MN-KstD3特定的参与9-OHAD与AD的代谢,构成甾醇代谢的两条主要代谢流通路。当阻断9-OHAD的分解代谢,缺失各路KstD活性时,缺陷型MutkstD(1&z&3)发酵15g/L甾醇可制9-OHAD5.17-5.42g L-1,但也混杂有1.04-1.55g L-1的AD与0.12-0.24g L-1的4-BNA (22-hydroxy-23,24-bisnorchol-4-en-3-one)等一类9-OHAD的前体代谢物。可见,9-OHAD的高纯度积累不仅需“节流”其降解代谢,还应注重“开源”9α-羟基化过程的代谢通量。3、3-甾酮-9α-羟基化酶的分子改造与功能强化KSH酶为母核代谢的另一关键因素,但其催化的9a-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。KSH为双组份酶(KshA+KshB),而M. neoaurum同时具有2种MN-KshA同工酶以及1种MN-KshB活性。其中MN-KshA1位于甾体降解基因簇内,受到甾醇等的强烈调控,可显著影响中间代谢物AD(D)等的积累,被证实是M. neoaurum内KSH酶活性的重要支柱。此外,MN-KshA1B (KshA1+KshB)可催化多种3-酮基甾体的底物,以1,4-BNC(3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid)为最适反应底物,在甾体催化活性上,远超另一同工酶MN-KshA2。因此,MN-KshA1具备分子改造的重要价值与意义。我们拟定,高KshA活性的分子改造策略,可以包括酶反应中心的置换研究与氨基酸关键残基的定点突变。最终,实验鉴定到位于酶活性中心入口处的一处突变(V202T),可显著提高KshA的酶活,定义为突变体MN-KshAV202T’可用于分枝杆菌KSH的功能强化。符合预期的,过表达有MN-KshAV202T的MutMN-kstD(1&2&3)(NwIB-V)菌株,15g/L甾醇发酵可制9-OHAD5.77-6.13g L-1,无杂产物AD,4-BNA的生成。然而除此之外,代谢产物中却鉴定到另一种副产物9-OH-BNA (0.95-1.26g L-1)的积累。分析表明,9-OH-BNA来自于甾体侧链的不完全代谢,因此只有阐释侧链降解的发生机制,尤其是有关9-OH-BNA的形成与代谢机理,才是最终达到9-OHAD高纯度积累的关键。4、甾醇侧链降解机制的揭示与关键酶的功能放大我们筛查了甾体代谢基因簇内的各有关侧链降解的基因,设定了一系列重要的研究对象,利用基因敲除兼代谢物鉴定的主要技术手段,构建了多达20株不同的基因缺陷株,研究基因对象达到近30个,主要解决了以下两个主要问题：①M.neoaurum侧链代谢的分子机制；②产生副产物9-OH-BNA积累的关键因素。在我们的发现中,脱氢酶FadE26-27,水合酶Hsd4B,硫解酶Ltp3-4,KstR2调控区等不会显著影响侧链代谢,而另一脱氢酶Hsd4A与硫解酶FadA5的敲除却带来重要产物1,4-BNA(22-hydroxy-23,24-bisnorchola-1,4-diene-3-one)的大量积累,证实Hsd4A与FadA5才是引起4-BNA,9-OH-BNA等C22中间体降解代谢的关键因素。于是,通过Hsd4A与MN-KshAlV202T的共强化,工程菌株NwIB-V2产9-OHAD得率为66.2-70.1%,副产物<3%,9-OHAD的产物纯度达88.3-93.1%,较NwIB-V在9-OHAD的发酵纯度更上一层台阶。综上所述,本文通过分子生物学、代谢工程等的研究手段,详尽揭示了微生物甾醇降解分子机制,分别实现了C19类甾药中间体AD、ADD的高效生产,9-OHAD的高纯度高产量积累,同时开发了先进的可有效产C22类中间体1,4-BNA的工程菌株,由此大大丰富了现有的甾药工业微生物资源,并创建了一整套新型、稳定、高效的甾药工程菌开发平台。更多还原

【Abstract】 Steroid hormones are human bioactive materials. When bound to protein receptors, steroid hormones can be often used as signal molecule to regulate gene transcription and cellular physiological behavior. People have to intake specific steroid drugs to cure the off-balance of internal hormone level and some physiological malfunctions. Thus, demand for hormone drugs keeps rising annually. Currently, the major way to produce these drugs was to firstly prepare some key steroid intermediates in a microbial or chemical way from a variety of natural sterols such as cholesterol, phytosterols or diosgenin, and then chemically modify those molecules into related hormone pharmaceuticals. Among these, the microbial production of steroid metabolites was increasingly highlighted, due to a few of its advantages, such as environment-friendly, simple transformation procedures, relatively low loss but a high yield. However in China, steroid processing industry lacks the core technology and microorganism resources, thus stagnated at a junior stage of application for bio-transformation of sterols into pharmaceutical precursors. In this sense, this study aims at establishing high-efficient microbial producers for steroid precursor supply, through researching and artificially modifying the catabolic pathway of sterols, then redirecting the major metabolic flux to the targeted product in some microbial cells, such as Mycobacterium neoaurum. The elaborate work is represented as follows:1. The metabolic annotation of cholesterol oxidases and their engineeringThis study, for the first time, claimed and demonstrated cholesterol oxidases (ChO) are involved in the initial and rate-limiting step of sterols uptake. Some industrial bacteria were conversionally deficient in the mass transfer of sterol molecules across cell membrane, probably because of a low activity of ChO. There were totally two ChO isoforms identified in M. neoaurum ATCC25795, i.e. ChoM1and ChoM2, which were extracellularly distributed and membrane-associated, respectively. In comparison to ChoMl, ChoM2appeared to function as a main ChO activity, for its inactivation would remarkably attenuated the mutant for the uptake and utilization of cholesterol. Therefore, ChoMl would be regarded as a critical factor to improve the microbial transformation under a high-concentration of phytosterols. Accordingly, we augmented ChoM2in M. neoaurum NwIB-O1MS (producing1,4-androstadiene-3,17-dione, ADD) and M. neoaurum NwIB-R10(producing4-androstene-3,17-dione, AD), then achieved a yield of5.57g/LADD and6.85g/L AD, greatly higher than the original level,3.87g/LADD and4.53g/L AD.2. The functional determination of3-ketosteroid-△’-dehydrogenase and its inactivation for the production of9-OHAD9a-Hydroxyandrost-4-ene-3,17-dione (9-OHAD) is widely considered as a significant assistor for a C9-halogen substitution of corticoids, due to its advantageous conformation of9a-hydroxyl group. This study distinctively proposed a conception of "increase influx and reduce efflux" to over-produce9-OHAD, with metabolic re-construction in a wild-type M. neoaurum ATCC25795. Above all, there are two crucial factors involving in the accumulation of9-OHAD, one of which is S-ketosteroid-A’-dehydrogenase (KstD). KstD would dehydrogenate3-oxosteroids and threaten the integrity of our products; thus removal of total KstD activities should be the precondition to ensure the stable accumulation of9-OHAD. Up to three KstD isoenzymes were identified in M. neoaurum, and two of them (MN-KstD1and MN-KstD2) were located on the membrane. These three KstDs kinetically used9-OHAD, T, AD as their optimum substrates. Moreover, MN-KstD1and MN-KstD3were found to metabolically participate in the9-OHAD-pathway and AD-pathway during the sterol transformation. Only when all those KstDs were inactivated, a stable yield of9-OHAD (5.17-5.42g L-1) can be obtained, however with two other by-products, i.e.1.04-1.55g L-1of AD and0.12-0.24g L-1of4-BNA (22-hydroxy-23,24-bisnorchol-4-en-3-one). Therefore, further steps of metabolic modification for the high-purity of9-OHAD have to be made.3. Engineering of3-ketosteroid-9a-hydroxylase for the overproduction of9-OHAD3-Ketosteroid-9a-hydroxylase (KSH) acts on degradation of the steroid nucleus with association of KstD, but for the overproduction of9-OHAD, its high activity means much more. KSH is known as a two-component monooxygenase, comprising the terminal oxygenase KshA and ferredoxin reductase KshB. The results showed, there were two KshAhomologs but one form of KshB in M. neoaurum. MN-KshAl was located within the proposed gene cluster of steroid catabolism, strongly induced by cholesterol and outstandingly involved in the formation of steroid metabolites. Further, MN-KshAlB also showed a wide range of substrates and preferably catalyzed such3-oxosteroids as1,4-BNC (3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid). Therefore, engineering of a high-level KSH activity could primarily rely on the MN-KshA1. Mutation analyses demonstrated aβ-sheet structure within the catalytic domain greatly influenced the KSH activity and further a point mutagenesis of V202T at the entrance of channel to the active center substantially improved the performance of9a-hydroxylation (defined as MN-KshA1v202T-By means of MN-KshA1V202T augmentation in MutMN-kstD(1&2&3), we generated NwIB-V and realized an improvement of9-OHAD to5.77-6.13g L-1, without contamination of AD and4-BNA. Unexpectedly, however, another form of by-product,9-OH-BNA (0.95-1.26g I-1) which derived from an incomplete side-chain degradation, occurred and lowed the final purity of9-OHAD.4. Insight into the steroid side-chain degradationThis section screened the putative key factors within the gene cluster of steroid catabolism in M. neoaurum and gained an insight into the mechanism of steroid side-chain degradation. We altogether constructed up to20DCO (double cross-over) mutants covering approximately over30targets of genes, investigated their phenotypes and then solved the following two questions:1) The putative mechanism of steroid side-chain degradation;2) The particular cause to induce the occurrence of9-OH-BNA. In our conclusion, a putative CoA-dehydrogenase FadE26-27, an enoyl-CoA hydratase Hsd4B, a thiolase Ltp3-4and a whole KstR2-regulon were not involved in the rate-limiting step of side-chain oxidation. By contrast, another hydroxyl-CoA dehydrogenase and a thiolase FadA5played a central role in the accumulation of such C22-ketosteroids as1,4-BNA and aroused our special concern. Through co-augmentation of Hsd4A and MN-KshAv202T, the resultant NwIB-V2overcame the catabolic deficiency of side-chain cleavage and thus gave rise to a66.2-70.1%molar yield of9-OHAD with less than3%of9-OH-BNA.From what has been discussed above, this study specified strategies of metabolic engineering, and elaborated the catabolic mechanism of microbial sterol degradation. Eventually, high-efficient industrial bio-producer of such C19-ketosteroids as AD(D),9-OHAD and C22-ketosteroids as1,4-BNA were developed, which enriched the microorganism resources and also served as a novel, stable and promising platform for the future development.更多还原