Improved xylose tolerance and 2,3-butanediol production of Klebsiella pneumoniae by directed evolution of rpoD and the mechanisms revealed by transcriptomics

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.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1312-8) contains supplementary material, which is available to authorized users.

Transcriptional factor engineering in microbes for industrial biotechnology

“…Analysis of the differentially expressed genes also enabled the authors to pinpoint the most relevant genes that supported the enhanced yield of 2,3-BD. 24 In another study, the mutant C9 containing three amino acid substitutions and a non-sense mutation resulting in a truncated version of RpoD with only regions 1.1 and 1.2 (roughly corresponding to the region 1 of the ⊞-factor shown in Fig. 1), enabled E. coli JM109 to tolerate high concentration of cyclohexane (69%).…”

“…Sequencing analysis revealed the impacts on the transcriptome‐level of the mutant RpoD: 500 genes were up‐regulated and 174 genes were down‐regulated. Analysis of the differentially expressed genes also enabled the authors to pinpoint the most relevant genes that supported the enhanced yield of 2,3‐BD 24 . In another study, the mutant C9 containing three amino acid substitutions and a non‐sense mutation resulting in a truncated version of RpoD with only regions 1.1 and 1.2 (roughly corresponding to the region 1 of the σ‐factor shown in Fig.…”

Trends in Synthetic Biology in the Bioeconomy of Non-Food-Competing Biofuels

“…Another Gram-negative bacterium with biotechnological potential is Klebsiella pneumoniae. It is widely used in microbial consortia to improve tolerance to xylose [115]. K. pneumoniae is nutritionally versatile with numerous native pathways useful for biofuel production.…”

Engineering of Klebsiella oxytoca for the Production of 2,3-Butanediol from High Concentration of Xylose

“…To the end, the engineered cells rapidly consumed 238.1 g/L xylose for 47 h and produced 57.5 g/L 2,3-BDO with 0.24 g/g yield, resulting in high 2,3-BDO productivity (1.22 g/L/h). 2,3-BDO has been produced from xylose as the sole carbon source in several microorganisms, including Klebsiella species, 47 Enterobacter ludwigii, 48 Saccharomyces cerevisiae, 49,50 and Bacillus species. 51−53 Compared to the above results, KO8S16 exhibits higher productivity (1.22 g/L/h) of 2,3-BDO in batch/fed-batch cultivations by improving the tolerance to high concentration of xylose (Table 3).…”