| 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. |
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