PDF(1995 KB)
ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD
MAOLiangyang, LINa, GEJingping
PDF(1995 KB)
PDF(1995 KB)
ptsG Knockout: The Impact on CCR Effect of Klebsiella pneumoniae and Its Fermentation Production of 2,3-BD
The objective is to address the issue of Carbon Catabolite Repression (CCR) in Klebsiella pneumoniae during mixed carbon source fermentation for 2,3-butanediol (2,3-BD) production, which leads to a decrease in production efficiency. In this study, a Cm resistance gene was used as a marker, and the ptsG gene deletion strain of K. pneumoniae HD79-N was successfully constructed using the λRed homologous recombination technique. Furthermore, the fermentation results using a mixed carbon source of glucose and xylose (glucose:xylose=2:1) demonstrated that the K. pneumoniae HD79-N strain, with the ptsG gene deletion, significantly alleviated CCR, enabling simultaneous utilization of glucose and xylose for 2,3-BD production with a final yield of 9.81±0.38 g/L. Moreover, the xylose utilization rate of K. pneumoniae HD79-N strain [0.23±0.01 g/(L·h)] was also increased by 57.82% compared to that of K. pneumoniae HD79 strain [0.15±0.00 g/(L·h)]. The findings of this study provide technical insights into alleviating the CCR effect caused by the ptsG gene in K. pneumoniae strains and enhancing the production of 2,3-BD.
2,3-butanediol / gene knockout / ptsG gene / homologous recombination / Klebsiella pneumonia {{custom_keyword}} /
表1 试验菌株 |
| 序号 | 名称 | 菌株用途 | 菌株特点 | 菌株来源 |
|---|---|---|---|---|
| 1 | K. pneumoniae HD79 | 出发菌株 | 野生型 | 黑龙江大学微生物重点实验室保存 |
| 2 | E. coli DH5α | 克隆和转化 | ΔlacU169, Rˉ, Mˉ, Ampr | |
| 3 | K. pneumoniae HD79-N | 用于本探究 | Cmr, ΔptsG, Cmr | 自行构建 |
表2 试验质粒 |
| 序号 | 名称 | 质粒特点 | 质粒用途 | 质粒来源 |
|---|---|---|---|---|
| 1 | pMD18-T | Ampr, lacZ, 2692 bp | 克隆载体 | 大连宝生物有限公司 |
| 2 | pKD46 | Ampr, Exo, Beta, Gam, 6329 bp | 同源重组 | 北京鼎国生物科技有限公司 |
| 3 | pLysS | Cmr, T7 lysozyme, 4886 bp | 克隆cm基因 |
表3 引物序列 |
| 引物名称 | 引物序列(5’-3’) | 用途 |
|---|---|---|
| ptsG1-F | CCGTACTGCCTATCGCAGGTATC | 克隆ptsG1上游片段594 bp |
| ptsG1-R | gacaccaggCGTTCCGATGTGATGCAGACC | |
| ptsG2-F | tggtgctacgcGCGATTTGGCACTCTGCTAAAC | 克隆ptsG2下游片段579 bp |
| ptsG2-R | GTACGCCAGAACCTGCCACTAC | |
| cmr-F | GATCATCTGAACGCGGCACGTAAGAGGTTCC | 克隆cmr序列888 bp |
| cmr-R | CACGCCATTCCCCGCTTATTATCACTTATTCA | |
| pKD46-F | CCCAGCCCTGTGTATAACTCAC | 验证转化结果 1387 bp |
| pKD46-R | CAACTCGGTCGCCGCATACA | |
| ptsG-F | AGCCAAAGCGAGTAAAGTTCAC | 验证重组结果2507 bp |
| ptsG-R | GCGGACTGTTTTCAGGGTTAT | |
| M13R | GTAAAACGACGGCCAGT | 验证重组质粒 |
| M13F | TGTAAAACGACGGCCAGT | |
| ptsG-up | CGTGATCCTCTCCTTCATTTG | qRT-PCR检测扩增ptsG |
| ptsG-down | tAAGCCGCCAGACAGTTTG | |
| xylA-up | CGGGCGAAGTTATTGCTG | qRT-PCR检测扩增xylA |
| xylA-down | GGCTTTCCGTCATTACAACC | |
| xylB-up | CCGCCGATAAGCGTGAT | qRT-PCR检测扩增xylB |
| xylB-down | GGTGTGGTTCCTGCCCTAT | |
| xylE-up | TTCCGCACCGAACATCC | qRT-PCR检测扩增xylE |
| xylE-down | GCCCGCTTTACATTGCC | |
| xylF-up | ACCTTCCTGCTTGGCTTCT | qRT-PCR检测扩增xylF |
| xylF-down | aCTGGCAGAAAGACCGTGAT | |
| xylG-up | GCAGTTCGGATGAAATGACA | qRT-PCR检测扩增xylG |
| xylG-down | tCCAGCAAAAGGCGATTC | |
| xylH-up | ACCCACCATCCGTTCCA | qRT-PCR检测扩增xylH |
| xylH-down | GCCATCGTCGTCATTATGC | |
| 16S rDNA-up | TGTGTAGCCCTGGTCGTAAG | qRT-PCR检测扩增内参基因 |
| 16S rDNA-down | CGGACATCCACAGAACTTAGC |
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旨在应用自杀质粒重组技术,构建阴沟肠杆菌乳酸脱氢酶突变株,为进一步提高乙偶姻的产量和扩大菌株选择范围奠定基础。利用双酶切的方法将同源片段插入到自杀质粒pKR6K中,构建出ldh基因敲除质粒,然后利用细菌接合的方法敲除E. cloacae的ldh基因。成功克隆出两段E. cloacae乳酸脱氢酶基因的同源序列,长度分别为526 bp,通过序列比对分析,E. cloacae乳酸脱氢酶基因序列相似性为100%。通过对E. cloacae进行乳酸脱氢酶基因的敲除,成功构建一株ldh缺失重组菌株E. cloacae△ldh,同时2,3-丁二醇提高6.8%,乙酸提高了24.3%。E. cloacae乳酸脱氢酶缺失工程菌株构建成功,对利用微生物法工业化生产乙偶姻奠定基础。
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There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.
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PDF(1995 KB)
Collection(s)
表1 试验菌株表2 试验质粒表3 引物序列
图1 同源重组片段ptsG1-cmr-ptsG2构建策略图2 同源重组基因敲除构建策略图3 同源重组片段ptsG1-cmr-ptsG2的构建图4 重组载体pT-ptsG1-cmr-ptsG2构建图5 △ptsG基因敲除菌株的构建图6 K. pneumoniae HD79与HD79-N发酵过程中OD600和pH变化情况图7 K. pneumoniae HD79与HD79-N发酵过程中糖和2,3-BD的浓度变化情况图8 ptsG、xylA、xylB、xylF、xylG、xylH和xylE基因的转录水平/
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