Wei Wei, Fan Shu-Yue, Wang Feng-Qing, Wei Dong-Zhi
Newworld Institute of Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
World J Microbiol Biotechnol. 2014 Jul;30(7):1947-54. doi: 10.1007/s11274-014-1614-3. Epub 2014 Feb 8.
Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ(1)-dehydrogenase genes (kstD M and kstD(A)) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD(M) and kstD(A) were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD M/kstD(A) overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60%, respectively. This study on the manipulation of the main kstDM gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity.
新金色分枝杆菌NwIB-01具有强大的裂解大豆植物甾醇侧链的能力，可积累4-雄烯-3,17-二酮（AD）和1,4-雄二烯-3,17-二酮（ADD）。从AD中分离ADD的困难是该行业中植物甾醇微生物转化的关键瓶颈之一。为了提高产品中ADD的产量，分别从新金色分枝杆菌NwIB-01和简单节杆菌中获得了3-酮甾体Δ(1)-脱氢酶基因（kstD M和kstD(A)）。利用复制载体pMV261，kstD(M)和kstD(A)在新金色分枝杆菌NwIB-01中过表达。为了使外源基因稳定表达，使用整合载体pMV306进行kstD M/kstD(A)的过表达，并通过PCR扩增启动子和卡那霉素抗生素抗性基因序列的相关序列以验证质粒完整性。对所得质粒和突变菌株进行了验证，kstD增强突变体是良好的ADD生产菌株。NwIB-04和NwIB-05的ADD生产能力分别为0.1401和0.1740 g/l（在含有0.4 g/l植物甾醇的摇瓶中培养），产品中ADD的摩尔比分别为98.34%和98.60%。这项对分枝杆菌属中主要kstDM基因的操作研究为获得具有高产品纯度的优良植物甾醇转化菌株提供了一条可行的途径。
