Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system

Jing-Li Wang, Bo Wu, Han Qin, Yang You, Song Liu, Zong-Xia Shui, Fu-Rong Tan, Yan-Wei Wang, Qi-Li Zhu, Yan-Bin Li, Zhi-Yong Ruan, Ke-Dong Ma, Li-Chun Dai, Guo-Quan Hu, Ming-Xiong He

The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.