Shao M, Sha Z, Zhang X, Rao Z, Xu M, Yang T, Xu Z, Yang S
Laboratory of Applied Microorganisms and Metabolic Engineering, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu Province, China.
Laboratory of Pharmaceutical Engineering, School of Pharmaceutical Sciences, Jiangnan University, Wuxi, Jiangsu Province, China.
J Appl Microbiol. 2017 Jan;122(1):119-128. doi: 10.1111/jam.13336.
3-ketosteroid-Δ -dehydrogenase (KSDD), a flavin adenine dinucleotide (FAD)-dependent enzyme involved in sterol metabolism, specifically catalyses the conversion of androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). However, the low KSDD activity and the toxic effects of hydrogen peroxide (H O ) generated during the biotransformation of AD to ADD with FAD regeneration hinder its application on AD conversion. The aim of this work was to improve KSDD activity and eliminate the toxic effects of the generated H O to enhance ADD production.
The ksdd gene obtained from Mycobacterium neoaurum JC-12 was codon-optimized to increase its expression level in Bacillus subtilis, and the KSDD activity reached 12·3 U mg , which was sevenfold of that of codon-unoptimized gene. To improve AD conversion, catalase was co-expressed with KSDD in B. subtilis 168/pMA5-ksdd -katA to eliminate the toxic effects of H O generated during AD conversion. Finally, under optimized bioconversion conditions, fed-batch strategy was carried out and the ADD yield improved to 8·76 g l .
This work demonstrates the potential to improve enzyme activity by codon-optimization and eliminate the toxic effects of H O by co-expressing catalase.
This study showed the highest ADD productivity ever reported and provides a promising strain for efficient ADD production in the pharmaceutical industry.
3-酮甾体-Δ-脱氢酶（KSDD）是一种参与甾醇代谢的黄素腺嘌呤二核苷酸（FAD）依赖性酶，它特异性催化雄甾-4-烯-3,17-二酮（AD）转化为雄甾-1,4-二烯-3,17-二酮（ADD）。然而，在AD通过FAD再生转化为ADD的生物转化过程中，KSDD活性较低，且产生的过氧化氢（H₂O₂）具有毒性作用，这阻碍了其在AD转化中的应用。本研究的目的是提高KSDD活性并消除所产生H₂O₂的毒性作用，以提高ADD的产量。
对从新金色分枝杆菌JC-12获得的ksdd基因进行密码子优化，以提高其在枯草芽孢杆菌中的表达水平，KSDD活性达到12.3 U mg⁻¹，是未进行密码子优化基因的7倍。为了提高AD转化率，在枯草芽孢杆菌168/pMA5-ksdd -katA中与KSDD共表达过氧化氢酶，以消除AD转化过程中产生的H₂O₂的毒性作用。最后，在优化的生物转化条件下，采用补料分批策略，ADD产量提高到8.76 g l⁻¹。
本研究证明了通过密码子优化提高酶活性以及通过共表达过氧化氢酶消除H₂O₂毒性作用的潜力。
本研究展示了迄今报道的最高ADD生产率，并为制药行业高效生产ADD提供了一种有前景的菌株。
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