Use of an EZ-Tn5-based random mutagenesis system to create a Zymomonas mobilis with significant tolerance to heat stress and malnutrition.

During ethanol production, the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions, which affect their growth and productivity. Stress-tolerant strains with high ethanol yield are highly desirable. Therefore, a recombinant Zymomonas mobilis (Z. mobilis) designated as HYM was constructed by integrating three genes (yfdZ, metB, and Pfu-sHSP) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furiosus (P. furiosus) was used to increase the heat tolerance. The genomic integration of three genes confers on Z. mobilis the ability to grow in simple chemical defined medium without the addition of amino acid. The HYM not only demonstrated the high tolerance to unfavorable lower nutrition stresses but also the capability of converting glucose to ethanol with high yield at higher temperature. What is more, these genetic characteristics were stable up to 100 generations on nonselective medium. The effects of glucose concentration, fermentation temperature, and initial pH on ethanol production of the mutant strain HYM were optimized using a Box-Behnken design (BBD) experiment. The integration of three genes led to a significant increase in ethanol production by 9 % compared with its original Z. mobilis counterpart. The maximum ethanol production of HYM was as high as 105 g/l.