BackgroundCurrent methods of ethanol production from lignocelluloses generate a mixture of sugars, primarily glucose and xylose; the fermentation cells are always exposed to stresses like high temperature and low nutritional conditions that affect their growth and productivity. Stress-tolerant strains capable of using both glucose and xylose to produce ethanol with high yield are highly desirable.ResultsA recombinant Zymomonas mobilis (Z. mobilis) designated as HYMX was constructed by integrating seven genes (Pfu-sHSP, yfdZ, metB, xylA, xylB, tktA and talB) into the genome of Z. mobilis CP4 (CP4) via Tn5 transposon in the present study. The small heat shock protein gene (Pfu-sHSP) from Pyrococcus furious (P. furious) was used to increase the heat-tolerance, the yfdZ and metB genes from E. coli were used to decrease the nutritional requirement. To overcome the bottleneck of CP4 being unable to use pentose, xylose catabolic genes (xylA, xylB, tktA and talB) from E. coli were integrated into CP4 also for construction of the xylose utilizing metabolic pathway.ConclusionsThe genomic integration confers on Z. mobilis the ability to grow in medium containing xylose as the only carbon source, and to grow in simple chemical defined medium without addition of amino acid. The HYMX demonstrated not only the high tolerance to unfavorable stresses like high temperature and low nutrient, but also the capability of converting both glucose and xylose to ethanol with high yield at high temperature. What’s more, these genetic characteristics were stable up to 100 generations on nonselective medium. Although significant improvements were achieved, yeast extract is needed for ethanol production.
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.
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