不同启动子过表达xylR基因提高大肠杆菌的葡萄糖-木糖共利用能力

doi:10.13560/j.cnki.biotech.bull.1985.2025-0794

摘要/Abstract

摘要：

目的为削弱碳代谢抑制效应（carbon catabolite repression, CCR），提高大肠杆菌对葡萄糖-木糖共利用能力。方法以产D-乳酸的大肠杆菌工程菌JH15为出发菌，利用带有4种启动子（PcysG、PrrsA、PJ23119和PpflBp6）的pBR322质粒过表达木糖调控因子的编码基因xylR，构建重组菌株CX-A15、RX-A15、JX-A15和PX-A15。通过qPCR检测和发酵实验比较各菌株在30 g/L葡萄糖+20 g/L木糖的碳源条件下木糖转运与代谢基因的转录水平与发酵性能的变化。结果过表达xylR后，各重组菌株的木糖代谢关键基因xylA和xylB，以及木糖转运相关基因xylF、xylG和xylH的转录水平均有提高。相较于出发菌株JH15，重组菌株CX-A15、RX-A15、JX-A15、PX-A15木糖消耗速率分别提高了22%、84%、206%、209%；D-乳酸生产速率分别提高了8%、27%、97%、98%；其中JX-A15，PX-A15菌株在48 h内可完全消耗完碳源，D-乳酸产量达到最大，发酵周期较JH15的84 h缩短了43%。结论通过启动子PJ23119和PpflBp6过表达基因xylR，能有效降低CCR效应，在保证D-乳酸产量的同时，显著提高D-乳酸工程菌对葡萄糖和木糖的共利用，提高生产强度，缩短发酵周期，为促进利用木质纤维素等廉价发酵原料进行工业化发酵提供技术支持。

关键词: 大肠杆菌, 混合糖, 木糖, D-乳酸, 启动子

Abstract:

Objective To mitigate the Carbon Catabolite Repression (CCR) effect and enhance the co-utilization capacity of glucose and xylose by Escherichia coli (E. coli). Method Using the D-lactic acid-producing engineered E. coli strain JH15 as the starting strain, the xylR gene encoding the xylose regulatory factor was overexpressed via the pBR322 plasmid carrying four different promoters (PcysG, PrrsA, PJ23119, and PpflBp6). Four recombinant strains, namely CX-A15, RX-A15, JX-A15, and PX-A15, were constructed. Quantitative real-time polymerase chain reaction (qPCR) analysis and fermentation experiments were further performed to compare the transcription levels of xylose transport and metabolism genes, as well as changes in fermentation performance, among all strains under the carbon source condition of 30 g/L glucose + 20 g/L xylose. Result After xylR overexpresed, the transcription levels of key xylose metabolism genes (xylA and xylB) and xylose transport-related genes (xylF, xylG, and xylH) increased in all recombinant strains. Compared with the parent strain JH15: 1) The xylose consumption rates of CX-A15, RX-A15, JX-A15, and PX-A15 increased by 22%, 84%, 206%, and 209%, respectively; 2) the D-lactic acid production rates increased by 8%, 27%, 97%, and 98%, respectively. Among the recombinant strains, JX-A15 and PX-A15 completely consumed the carbon source within 48 h and achieved the maximum D-lactic acid yield. Their fermentation cycle was shortened by 43% compared with the 84-h cycle of JH15. Conclusion The overexpression of the xylR gene driven by promoters PJ23119 and PpflBp6 can effectively reduce the CCR effect. While ensuring D-lactic acid yield, this strategy significantly improves the co-utilization of glucose and xylose by the D-lactic acid-producing engineered bacteria, enhances production intensity, and shortens the fermentation cycle. It provides technical support for promoting industrial fermentation using low-cost raw materials such as lignocellulose.

Key words: Escherichia coli, mixed sugars, xylose, D-lactic acid, promoter

李正, 邱炜玥, 孙瑞雪, 赵筱. 不同启动子过表达xylR基因提高大肠杆菌的葡萄糖-木糖共利用能力[J]. 生物技术通报, 2026, 42(1): 329-337.

LI Zheng, QIU Wei-yue, SUN Rui-xue, ZHAO Xiao. Overexpression of the xylR Gene Driven by Different Promoters Enhances Glucose-xylose Co-utilization Capability in Escherichia coli[J]. Biotechnology Bulletin, 2026, 42(1): 329-337.

图/表 6

表1 所用引物及序列

Table 1 Primers used and their sequences

引物名称 Primer name	序列 Sequence （5´-3´）
P1	AGGCCCTTTCGTCTTCAAGACGCATATGCTGGATCCTT
P2	CATACACGGTGCCTGACTGCGCTACAACATGACCTCGCTATTTA
P3	TCTTGAAGACGAAAGGGCCTC
P4	GCAGTCAGGCACCGTGTA
P5	ATGTTTACTAAACGTCACCGCA
P6	CAGGATAACCAACGGTTAATCG
P7	CTGATTCTGTGGATAACCGT
P8	CGGTGACGTTTAGTAAACATACTAGTATTATACCTAGGACTGAGC
P9	AGGCCCTTTCGTCTTCAAGACTGCAGTCCTGAAGCTTG
P10	CATACACGGTGCCTGACTGCCATCGTCAGTATTGACTGCAG
P11	AGTGTGAAAGCTGACAACC
P12	GCACTCGAAGATACGGAT
P13	TGCCGGTTAATTACTAAGGG
P14	GACAGCTAGCTCAGTCCTA
P15	TATAGCGCTAGCAGCACG
QxylA-F	GAAACCGCCTGCTTTGAGAA
QxylA-R	CATTGAAGCTAACCACGCGA
QxylB-F	GATTATTGTGTGGCGTACGC
QxylB-R	TACATAGCTTTTGCCATGCG
QxylR-F	TGGGGATTATTGTGTGGCGT
QxylR-R	CATAGCTTTTGCCATGCGCT
QxylF-F	GGTGTCGATGTTCTTGTCAT
QxylF-R	CTTGGCGTTGTTATCTACCG
QxylG-F	TATGACCTGATGACGCTACG
QxylG-R	TGCTCAGTTAATGAGGCTGT
QxylH-F	TTGCAGCTATCATCGCAATC
QxylH-R	CACAATGATGGTAAGTGGCA
PcysG-F	TGGGCCAGGTAGCGAAATAC
PcysG-R	TAGGCAGAGCAACCAGAAGC

图1 表达质粒的验证电泳图A：质粒CX-322的电泳图（1： JH15； 2：CX-322）；B：质粒RX-322的电泳图（1：JH15；2：RX-322）；C：质粒JX-322的电泳图（1：JH15；2：JX-322）；D：质粒PX-322的电泳图（1：JH15；2：PX-322）。 M：DL5000DNA marker

Fig. 1 Verification electrophoresis profile of the expressing plasmidsA: Electrophoretogram of plasmid CX-322 (1: JH15; 2: CX-322). B: Electrophoretogram of plasmid RX-322 (1: JH15; 2: RX-322). C: Electrophoretogram of plasmid JX-322 (1: JH15; 2: JX-322). D: Electrophoretogram of plasmid PX-322 (1: JH15; 2: PX-322). M: DL5000 DNA marker

图2 重组菌株及出发菌的SDS-PAGE验证电泳图M： 180 protein marker； 1： RX-A15； 2： CX-A15； 3： JX-A15； 4： PX-A15； 5： JH15

Fig. 2 Electrophoretogram of SDS-PAGE verification of recombinant strains and the original strain

图3 木糖转运及代谢基因转录水平差异分析FC>2，视为显著性差异

Fig. 3 Differential analysis of the transcription levels of xylose transport and metabolism genesA: Fold change (FC)>2 is considered as significant difference

图4 出发菌株JH15及重组菌株CX-A15、RX-A15、JX-A15、PX-A15混糖（3%葡萄糖+2%木糖）发酵结果曲线A：不同菌株生长曲线；B：不同菌株葡萄糖消耗曲线；C：不同菌株木糖消耗曲线；D：不同菌株D-乳酸生产曲线

Fig. 4 Fermentation result curves of starting strain JH15 and recombinant strains CX-A15, RX-A15, JX-A15, and PX-A15 in mixed sugars （3% glucose+2% xylose）A: Growth curves of different strains. B: Glucose consumption curves of different strains. C: Xylose consumption curves of different strains. D: D-lactic acid production curves of different strains

表2 出发菌株JH15及重组菌株CX-A15、RX-A15、JX-A15和PX-A15混合糖（3%葡萄糖+2%木糖）发酵结果

Table 2 Fermentation results of the original strain JH15 and recombinant strain CX-A15, RX-A15, JX-A15, PX-A15 in fermentation with mixed-sugars (3% glucose + 2% xylose)

发酵菌株 Fermentative strain	葡萄糖消耗速率 Glucose consumption rate （g/（L·h））	木糖消耗速率Xylose consumption rate （g/（L·h））	D-乳酸生产速率 D-lactic acid production rate （g/（L·h））	糖酸转化率 Sugar-acid conversion rate （%）	最大OD_{600 nm} Maximum OD_{600 nm}	发酵周期 Fermentation cycle （h）
JH15	0.369±0.01^c	0.129±0.01^c	0.368±0^c	64^b	3.77^c	84
CX-A15	0.424±0^c	0.158±0^c	0.399±0^c	64^b	4.18^bc	84
RX-A15	0.493±0.02^b	0.238±0.02^b	0.468±0.02^b	63^b	4.43^b	72
JX-A15	0.630±0.01^a	0.396±0.01^a	0.726±0.01^a	66^a	5.34^a	48
PX-A15	0.624±0.03^a	0.398±0.02^a	0.729±0.03^a	66^a	5.44^a	48

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