Abstract
BackgroundThe biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear.ResultsIn this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae.ConclusionsThese results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.
Highlights
Renewable resources have attracted great attention due to the growing demand for energy and chemicals [1, 2]
Examination of the xylose tolerance of the kpG strain The kpG was cultured at xylose concentrations of 75 100, 125, 150, and 175 g/L in a shake flask, and the concentrations of the carbon source and metabolite were detected every 24 h
The results showed that for kpG, the threshold value of xylose tolerance was 75 g/L
Summary
Renewable resources have attracted great attention due to the growing demand for energy and chemicals [1, 2]. Xylose is the most abundant sugar in lignocellulosic materials, except in glucose [3]. Its efficient utilization is crucial for the utilization of the lignocellulosic biomass, an abundant, geographically ubiquitous, and potentially cheap renewable resource, for the enhanced production of fuels and chemicals [4]. The strain with high tolerance to substrate, production, and toxic materials is quite essential for biotransformation. The K. pneumoniae is among the microbes that can metabolize xylose naturally [8,9,10]. K. pneumoniae has been used to produce 2,3-butanediol in glucose medium, but xylose has rarely been reported. K. pneumoniae cannot fully metabolize 2,3-butanediol by fermenting xylose at a high concentration (exceeding 70 g/L). The biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. Whether it can regulate the tolerance in K. pneumoniae remains unclear
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