新金色分枝杆菌ksdD基因的敲除及其对菌株AD(D)合成的影响

新金色分枝杆菌(Mycobacterium neoaurum)能将植物甾醇转化为药物中间体4-烯-雄甾-3,17-二酮(AD)和1,4-二烯-雄甾-3,17-二酮(ADD),其中3-甾酮-△1-脱氢酶(KSDD)是AD转化为ADD的关键酶.本实验室在筛菌过程中筛选到一株能将甾醇转化为AD(D)的菌株,经鉴定为M.neo...     

Mycobacterium sp．高效转化植物甾醇为雄甾-1，4-二烯-3，17-二酮的诱变选育

采用紫外线、亚硝基胍复合诱变雄甾-4-烯-3,17-二酮（AD）和雄甾-1,4-二烯-3,17-二酮（ADD）的转化产生菌Mycobacterium sp．,结合平板筛选,获得一株遗传性状稳定单产ADD的突变菌株Mycobacterium sp．-11,其ADD质量浓度达到1246ms／L,比原始菌株（484mg／L）提高了150％,经初步优化后发酵液中ADD最高达到1430mg／L,发酵液中ADD质量占ADD、AD两产物质量总和的比例由70％提高到99．1％。     

植物甾醇微生物转化制备甾体药物中间体的研究进展*

微生物选择性降解植物甾醇侧链获取甾体药物合成的重要中间体雄甾-4-烯-3,17-二酮(4-AD)和雄甾-1,4-二烯-3,17-二酮(ADD)对于我国制药行业具有重要意义。现存文献资料对该领域缺乏全面系统的分析总结,从甾醇侧链微生物转化的机理、途径及其收率的影响因素等几个方面综述了近几年的研究进展,并对此领域的发展趋势进行了展望。     

植物甾醇微生物转化制备甾体药物中间体的研究进展

微生物选择性降解植物甾醇侧链获取甾体药物合成的重要中间体雄甾-4-烯-3,17-二酮（4-AD）和雄甾-1,4-二烯-3,17-二酮（ADD）对于我国制药行业具有重要意义。现存文献资料对该领域缺乏全面系统的分析总结,从甾醇侧链微生物转化的机理、途径及其收率的影响因素等几个方面综述了近几年的研究进展,并对此领域的发展趋势进行了展望。     

微生物发酵降解植物甾醇侧链生产17-酮甾体研究进展

微生物发酵降解植物甾醇侧链,生产雄甾-4-烯-3,17-二酮(AD),雄甾-1,4-二烯-3,17-二酮(ADD),和9α-羟基-AD甾体药物中间体的工业生物技术对改变制造甾体激素药物半合成原料薯蓣皂素短缺的现状,实现甾体激素药物半合成原料多元化,合理利用我国甾体植物资源具有重要意义。重点评述了近期微生物法断植物甾醇侧链制AD、ADD和9α-羟基-AD的研究现状,内容包括:1)微生物菌种选育;2)菌种相关的细胞生理,酶学性质和生物催化过程;3)相关酶的细胞定位及生物反应器;4)发酵工艺选择和甾醇原料的合理利用。     

一株高效转化Phytosterol为雄甾烯酮菌株的筛选与鉴定

从保藏的200多株菌中筛选出1株高效转化植物甾醇为4-烯-雄甾-3,17-二酮和1,4-二烯-雄甾-3,17-二酮的菌株,并对该菌进行了形态、生理生化的研究。结果发现菌株ST06可以利用多种碳源,可以水解淀粉,但不利用纤微素。用16SrDNA的方法对其进行鉴定,发现与Bacillus属Bacillus amyloiquefaciens的相似性最高,达到99．9％,将该菌株命名为Bacillus amyloiquefaciens ST06。该菌在培养温度30℃,pH7．0,转速220r／min,转化时间7d,底物添加量为0．3％时,ADD与AD的总得率高达40％以上,此时底物转化率高达93．7％。     

一株分枝杆菌降解胆固醇制备AD(D)的转化条件

通过分枝杆菌(Mycobacteriumsp.)M3限制性降解胆固醇侧链获得了产物雄甾-4-烯-3,17-二酮(AD)和雄甾-1,4-二烯-3,17-二酮(ADD)。优化了胆固醇的投料时间、投料方式、培养基初始pH和葡萄糖浓度等工艺参数。将羟丙基-β-环糊精(HP-β-CD)应用于转化反应中,确定了HP-β-CD的最佳添加时间和添加量,使AD(D)生成率由初始对照的30%提高到60%,转化至72 h时AD(D)生成率达48%,是同期对照的4.0倍,生成率与生成速率均得到显著提高。在添加HP-β-CD的最佳转化条件下,AD(D)生成率达到70%,是初始对照的2.3倍。     

黑曲霉催化雄甾-4-烯-3,17-二酮16β-羟基化

为进一步确定黑曲霉菌株TCCC41650的生物转化能力,以雄甾-4-烯-3,17-二酮(Androstenedione)为底物,利用黑曲霉菌株TCCC41650进行催化,产物经纯化、重结晶后,通过单晶衍射鉴定为16β-羟基雄甾-4-烯-3,17-二酮。转化条件为:培养液pH 6.0,乙醇添加量为2%,投料浓度为1‰时,72 h转化率为85.8%。目前甾体研究领域对于C16β-羟基化的微生物转化未见报道,研究结果为C16β-羟基甾体药物的研发奠定了基础。     

雄甾-4-烯-3,17-二酮(4-AD)转化菌种的筛选及鉴定

从土壤中筛选能将植物甾醇转化为雄甾-4-烯-3,17-二酮(4-AD)的菌种。采用富集培养基富集能降解植物甾醇的菌种、采用摇瓶进行发酵、采用薄层层析的方法对发酵产物进行检测、采用高效液相方法测定发酵液中4-AD的含量、采用PCR方法扩增菌种的16S rDNA序列。筛选出了一株转化能力最强的菌株,命名为:3-12。将这株菌的16S rDNA序列与GeneBank中收载的序列进行比对,3-12号菌株为分支杆菌。     

总状毛霉对4-烯-3-酮甾体的生物转化研究

从土样中筛选到一株能转化甾体的菌株,经形态观察,鉴定为总状毛霉(Mucor racemosus)。首次利用该菌株对4-烯-3-酮类甾体衍生物进行生物转化,目的是合成具有潜在活性的羟基类4-烯-3-酮衍生物。转化条件为27℃,220r/min振荡培养4d。转化产物经乙酸乙酯萃取,用硅胶柱层析法分离,通过红外、质谱和核磁分析确定了甾体转化产物的化学结构。黄体酮生物转化得到的产物是14α-羟基-4-孕甾烯-3,20-二酮和7α,14α-二羟基-4-孕甾烯-3,20-二酮;4-雄烯二酮的转化产物是14α-羟基-雄甾-4-烯-3,17-二酮1、4α,17β-二羟基-雄甾-4-烯-3-酮和6α,17β-二羟基-雄甾-4-烯-3-酮。研究结果表明总状毛霉具有转化甾体的能力,对4-烯-3-酮类甾体进行生物转化的主要产物是14α-羟基甾体衍生物。     

Enhanced Production of Androst-1,4-Diene-3,17-Dione by Mycobacterium neoaurum JC-12 Using Three-Stage Fermentation Strategy

To improve the androst-1,4-diene-3,17-dione (ADD) production from phytosterol by Mycobacterium neoaurum JC-12, fructose was firstly found favorable as the initial carbon source to increase the biomass and eliminate the lag phase of M. neoaurum JC-12 in the phytosterol transformation process. Based on this phenomenon, two-stage fermentation by using fructose as the initial carbon source and feeding glucose to maintain strain metabolism was designed. By applying this strategy, the fermentation duration was decreased from 168 h to 120 h with the ADD productivity increased from 0.071 g/(L·h) to 0.108 g/(L·h). Further, three-stage fermentation by adding phytosterol to improve ADD production at the end of the two-stage fermentation was carried out and the final ADD production reached 18.6 g/L, which is the highest reported ADD production using phytosterol as substrate. Thus, this strategy provides a possible way in enhancing the ADD production in pharmaceutical industry.     

Isolation of a biodegradable sterol-rich fraction from industrial wastes

Several industrial waste materials were screened for their sterol content. The possibility of using these industrial by-products as sterol sources for the microbiological production of 4-androsten-3,17-dione (AD) and 1,4-androsta-diene-3,17-dione (ADD) was investigated. Two methods of obtaining the sterol fraction from wastes were developed. Sterol-rich (96-98%) fractions were isolated in a yield above 70%, from a tall-oil effluent of a paper pulp industry and from edible-oil deodorizates. These fractions were subsequently used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD.     

Growth and cholesterol oxidation by Mycobacterium species in Tween 80 medium.

Mycobacterium strain DP was isolated from marine coastal sediment and tested for its ability to oxidize cholesterol in Tween 80-cholesterol (2.59 mM) medium. Strain DP degraded cholesterol to 4-cholesten-3-one (cholestenone), 4-androsten-3,17-dione (AD), 1,4-androstadien-3,17-dione (ADD), testosterone, and 1-dehydrotestosterone (DHT). Cholesterol disappeared in about 4 days. Cholestenone, AD, testosterone, and DHT accumulations were transient with peak concentrations of 300, 600, 30 to 40, and 21 microM. ADD production peaked after 6 days with a concentration of 1,100 microM. Peak ADD concentrations and production rates compared well with those reported for strain NRRL B3683 on cyclodextrin medium. Tween 80 medium was superior to finely dispersed cholesterol particles for both strains. In comparison, NRRL B3683 (patented for its ability to accumulate AD and ADD) on Tween 80 medium transiently accumulated more AD (approximately 1,000 microM) than did strain DP, but ADD accumulations (200 microM) were significantly lower than those for strain DP. Strain DP could be adapted to grow on ADD, which was initially inhibitory at 3.25 mM. ADD-adapted strain DP cultures produced approximately four times as much DHT from ADD than unadapted cultures did from cholesterol, showing that additional manipulation might enhance testosterone production. We believe that ADD toxicity might account for the low ADD accumulations by NRRL B3683 in Tween 80 medium.     

Steroid bioconversion in water-insoluble organic solvents: Δ1-Dehydrogenation by free microbial cells and by cells entrapped in hydrophilic or lipophilic gels

A cell suspension in a water-insoluble organic solvent (benzene: n-heptane, 1 : 1 by volume) of Nocardia rhodocrous (previously induced to synthesize steroid Δ¹dehydrogenase) rapidly catalyzed the stoichiometric oxidation of 4-androstene-3,17-dione (4-AD) to androst-l,4-diene-3,17-dione (ADD) in the presence of phenazine methosulfate (PMS). High levels of 4-AD or PMS reduced the conversion rates. No appreciable decrease in the conversion rate was observed on adding aqueous buffer solution to the thawed ceils (up to 9.4 g water/g dry cell). The whole cells were immobilized by entrapment in a hydrophilic gel (H-gel) or a lipophilic gel (L-gel) by use of a water-soluble or water-insoluble photocrosslinkable prepolymer. The reticula of H- and L-gel matrices were impregnated with water and organic solvent, respectively. Both the H- and L-gels could convert 4-AD to ADD in the presence of PMS, the L-gel showing a slightly higher conversion rate. Various lines of evidence indicate that the limiting factor is the penetration rate of 4-AD into gel particles for the H-gel, and the penetration rate of PMS for the L-gel. The catalytic activities decreased considerably after several successive runs with the free cell suspension system, while the immobilized cells were more stable, the stability of H-gel and L-gel being almost the same.     

表达3-甾酮-△1-脱氢酶降解植物甾醇合成雄甾-1,4-二烯-3,17-二酮

构建分枝杆菌表达载体pMTac并在分枝杆菌Mycobacterium neoaurum JC-12中加强表达甾醇降解过程中的关键酶3-甾酮-△1-脱氢酶(KSDD)以提高雄甾-1,4-二烯-3,17-二铜(ADD)的产量。将p MF41的启动子pACE替换成tac启动子构建载体pMTac,在分枝杆菌中分别表达报告基因绿色荧光蛋白(GFP)和关键酶KSDD,通过GFP亮度和KSDD酶活验证tac启动子在M.neoaurum JC-12中的效果,并发酵验证加强表达KSDD对产物ADD的影响。荧光显微照片表明两个载体均能在M.neoaurum JC-12表达GFP,但tac启动子的效果比pACE强。酶活测定结果为重组菌M.neoaurum JC-12/pMTac-ksdd破碎细胞上清液中KSDD酶活比原始菌提高了6.53倍,比M.neoaurum JC-12/pMF41-ksdd提高了4.36倍。摇瓶发酵显示重组菌M.neoaurum JC-12/pMTac-ksdd ADD的产量比原始菌提高了22.2%,由4.86 g/L提高到5.94 g/L,而AD的产量由0.92 g/L减少到0.17 g/L,降低了81.5%;与M.neoaurum JC-12/p MF41-ksdd比,ADD产量提高了12.7%,AD降低了71.2%。以20 g/L植物甾醇为底物,5 L发酵罐中重组菌M.neoaurum JC-12/pMTac-ksdd的ADD产量达到10.28 g/L。结果表明,构建的新型表达载体pMTac适用于在M.neoaurum JC-12中加强表达关键酶KSDD,而且在M.neoaurum JC-12中过量表达KSDD有助于ADD产量的提高,为目前报道的发酵法利用新金色分枝杆菌降解植物甾醇合成ADD的最高水平。     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.