School of Engineering, Department of Chemical and Life Science Engineering West Hall, Virginia Commonwealth University, Room 422, 601 West Main Street, P.O. Box 843028, Richmond, VA, 23284-3028, USA.
Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA, USA.
Appl Microbiol Biotechnol. 2017 Oct;101(20):7567-7578. doi: 10.1007/s00253-017-8488-x. Epub 2017 Sep 8.
Microbial utilization of chitin, a potential renewable biomass feedstock, is being pursued as a means of developing novel consolidated bioprocessing for the production of chemicals. Serratia marcescens is a gram-negative bacterium that is known for its chitinolytic capability and as a native 2,3-butanediol producer. In S. marcescens, ChiR has been suggested to be a positive regulator of chitinase production. In this study, we aim to understand the effect of ChiR in regulating nine chitinase-related genes in S. marcescens Db11 and demonstrate manipulation of chiR as a useful and efficient genetic target to enhance chitin utilization. First, a chiR overexpression (chiROE) strain and a chiR deletion (ΔchiR) strain were generated and characterized in terms of cellular growth, chitinase activity, and total secreted protein. Compared to the wild-type Db11 strain, the S. marcescens chiROE strain showed an increase in chitinase activity (2.14- to 6.31-fold increase). Increased transcriptional expression of chitinase-related genes was measured using real-time PCR, showing 2.12- to 10.93-fold increases. The S. marcescens ΔchiR strain showed decreases in chitinase activity (4.5- to 25-fold decrease), confirming ChiR's role as a positive regulator of chitinase expression. Finally, chiR overexpression was investigated as a means of increasing biochemical production (2,3-butanediol) from crystal chitin. The chiROE strain produced 1.13 ± 0.08 g/L 2,3-butanediol from 2% crystal chitin, a 2.83-fold improvement from the wild-type strain, indicating ChiR is an important and useful genetic engineering target for enhancing chitin utilization in S. marcescens.
微生物利用几丁质（一种有潜力的可再生生物质原料），被视为开发新型综合生物加工技术以生产化学品的一种手段。粘质沙雷氏菌是一种革兰氏阴性菌，以其几丁质分解能力和作为天然 2,3-丁二醇生产菌而闻名。在粘质沙雷氏菌中，ChiR 被认为是几丁质酶产生的正调控因子。在这项研究中，我们旨在了解 ChiR 对粘质沙雷氏菌 Db11 中 9 种几丁质酶相关基因的调控作用，并证明操纵 chiR 作为增强几丁质利用的有用和有效遗传靶标。首先，生成并表征了 chiR 过表达（chiROE）菌株和 chiR 缺失（ΔchiR）菌株，从细胞生长、几丁质酶活性和总分泌蛋白方面进行了评估。与野生型 Db11 菌株相比，粘质沙雷氏菌 chiROE 菌株的几丁质酶活性增加了（2.14-6.31 倍）。使用实时 PCR 测量了几丁质酶相关基因的转录表达，显示出 2.12-10.93 倍的增加。粘质沙雷氏菌 ΔchiR 菌株的几丁质酶活性降低（4.5-25 倍），证实了 ChiR 作为几丁质酶表达的正调控因子的作用。最后，研究了 chiR 过表达作为从晶体几丁质增加生化产物（2,3-丁二醇）的方法。chiROE 菌株从 2%晶体几丁质中生产 1.13±0.08 g/L 2,3-丁二醇，比野生型菌株提高了 2.83 倍，表明 ChiR 是增强粘质沙雷氏菌几丁质利用的重要和有用的遗传工程靶标。
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