Overexpression of the xylR Gene Driven by Different Promoters Enhances Glucose-xylose Co-utilization Capability in Escherichia coli

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

Abstract

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

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.

Figures/Tables 6

References 31

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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

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

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

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