Wang Miaomiao, Yu Huimin, Shen Zhongyao
Department of Chemical Engineering , Tsinghua University , Beijing 100084 , P. R. China.
Key Laboratory of Industrial Biocatalysis (Tsinghua University) , Ministry of Education , Beijing 100084 , P. R. China.
ACS Synth Biol. 2019 Feb 15;8(2):251-256. doi: 10.1021/acssynbio.8b00459. Epub 2019 Jan 31.
The antisense RNA (asRNA) strategy is commonly used to block protein expression and downregulate the contents of metabolites in several microorganisms. Here, we show that the asRNA strategy can also be used to block gfp expression in Bacillus subtilis TS1726, which could further be utilized in the identification of new genes and functions. Via application of this strategy, biotin carboxylase II encoded by yngH (GeneID 939474) was identified to play a more significant role in maintaining acetyl-CoA carboxylase (ACCase) activity and enhancing surfactin synthesis compared to those of other ACCase subunits. The yngH gene was then overexpressed in the engineered strain B. subtilis TS1726(yngH). The surfactin titer of TS1726(yngH) increased to 13.37 g/L in a flask culture, representing a 43% increase compared to that of parental strain TS1726. This strategy opens the door to achieving large-scale production and broad application of surfactin.
反义RNA（asRNA）策略通常用于阻断几种微生物中的蛋白质表达并下调代谢物含量。在此，我们表明asRNA策略也可用于阻断枯草芽孢杆菌TS1726中的gfp表达，这可进一步用于新基因和功能的鉴定。通过应用该策略，与其他乙酰辅酶A羧化酶（ACCase）亚基相比，发现由yngH（基因ID 939474）编码的生物素羧化酶II在维持ACCase活性和增强表面活性素合成方面发挥更重要的作用。然后在工程菌株枯草芽孢杆菌TS1726（yngH）中过表达yngH基因。在摇瓶培养中，TS1726（yngH）的表面活性素滴度增加到13.37 g/L，与亲本菌株TS1726相比增加了43%。该策略为实现表面活性素的大规模生产和广泛应用打开了大门。
