基因工程改造促进大肠杆菌发酵纤维素水解液合成1,2,4-丁三醇

doi:10.16085/j.issn.1000-6613.2023-0298

摘要/Abstract

摘要：

1,2,4-丁三醇（BT）是广泛应用于各领域的高附加值化合物之一，利用纤维素水解液发酵生产BT需要一株能够耐受其中抑制物的菌株。在水解液中常见抑制因子糠醛存在下，大肠杆菌的发酵严重受限。本研究首先在含有BT合成途径的大肠杆菌中过表达四种糠醛耐受相关的基因：yghA、thyA、mdtJI和pntAB。在0.4g/L糠醛压力下，所有改造菌株的生长均有不同程度的提升，其中过表达pntAB基因的菌株生长最好，同时有助于BT合成酶的酶活力或转录水平的提高，BT产量达到11.0g/L。当以玉米芯纤维素水解液为底物时，过表达yghA、thyA和pntAB均明显促进了大肠杆菌的生长与BT产量的提高，其中yghA基因表现最好，在摇瓶中获得了3.36g/L的BT，在5L发酵罐中BT产量达到15.3g/L，转化率为63.8%。该策略为玉米芯纤维素水解液发酵生产BT提供了借鉴。

关键词: 大肠杆菌, 玉米芯纤维素水解液, 糠醛, 耐受, 1,2,4-丁三醇

Abstract:

1,2,4-Butanetriol (BT) is one of the high value-added compounds widely used in various fields, and the production of BT by fermentation using cellulose hydrolysate requires a strain that can tolerate the inhibitors present in the hydrolysate. In the presence of furfural, a common inhibitor in hydrolysates, the fermentation of Escherichia coli is severely limited. In this study, four furfural tolerance genes, yghA, thyA, mdtJI, and pntAB were overexpressed in the BT-producingstrain. In the presence of 0.4g/L furfural, all modified strains showed different degrees of growth. Among them, the strain overexpressing pntAB had the best growth, which also contributed to the enzymatic activity or transcriptional level of the enzymes in the BT pathway, yielding 11.0g/L BT. When corncob cellulose hydrolysate was used as the substrate, the strains overexpressing yghA, thyA, and pntAB all significantly improved the growth and BT production. The strain carrying yghA performed the best, yielding 3.36g/L BT in shake flasks and 15.3g/L BT in 5L fermenters, with a conversion of 63.8%. This strategy provided a reference for BT production by fermentation of corncob cellulose hydrolysate.

Key words: Escherichia coli, corncob cellulose hydrolysate, furfural, tolerance, 1,2,4-butanetriol

中图分类号:

佘丹, 王舒婷, 陆信曜, 宗红, 诸葛斌. 基因工程改造促进大肠杆菌发酵纤维素水解液合成1,2,4-丁三醇[J]. 化工进展, 2024, 43(2): 1063-1068.

SHE Dan, WANG Shuting, LU Xinyao, ZONG Hong, ZHUGE Bin. Genetic engineering modification to promote the synthesis of 1,2,4-butanetriol from cellulose hydrolysate of Escherichia coli[J]. Chemical Industry and Engineering Progress, 2024, 43(2): 1063-1068.

图/表 9

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菌株和质粒	相关特点	来源
E. coli KXW3009P0	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-cm	实验室保存
E. coli KXW3009P1	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-yghA	本研究
E. coli KXW3009P2	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-thyA	本研究
E. coli KXW3009P3	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-mdtJ-tac-mdtI	本研究
E. coli KXW3009P4	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-pntA-tac-pntB	本研究
pEtac-kivD-tac-xdh	Kan^r，tac启动子	实验室保存
pRSFDuet-tac-cm	Cm^r，tac启动子	实验室保存
pRSFDuettac-yghA	Cm^r，tac启动子	本研究
pRSFDuettac-thyA	Cm^r，tac启动子	本研究
pRSFDuettac-mdtJ-tac-mdtI	Cm^r，tac启动子	本研究
pRSFDuettac-pntA-tac-pntB	Cm^r，tac启动子	本研究

菌株和质粒	相关特点	来源
E. coli KXW3009P0	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-cm	实验室保存
E. coli KXW3009P1	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-yghA	本研究
E. coli KXW3009P2	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-thyA	本研究
E. coli KXW3009P3	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-mdtJ-tac-mdtI	本研究
E. coli KXW3009P4	E. coli W3110ΔxylABΔyagEΔyjhHΔptsHIΔmgsA, pEtac-kivD-tac-xdh, pRSFDuettac-pntA-tac-pntB	本研究
pEtac-kivD-tac-xdh	Kan^r，tac启动子	实验室保存
pRSFDuet-tac-cm	Cm^r，tac启动子	实验室保存
pRSFDuettac-yghA	Cm^r，tac启动子	本研究
pRSFDuettac-thyA	Cm^r，tac启动子	本研究
pRSFDuettac-mdtJ-tac-mdtI	Cm^r，tac启动子	本研究
pRSFDuettac-pntA-tac-pntB	Cm^r，tac启动子	本研究

菌株	生物量OD₆₀₀	葡萄糖消耗量 /g·L^-1	木糖消耗量 /g·L^-1	BT /g·L^-1
E. coli KXW3009P0	14.2	9.8	27.0	14.8
E. coli KXW3009P1	14.6	9.9	27.5	14.7
E. coli KXW3009P2	14.8	8.9	27.8	13.2
E. coli KXW3009P3	13.4	9.8	26.0	13.0
E. coli KXW3009P4	14.9	9.0	28.1	14.9

菌株	生物量OD₆₀₀	葡萄糖消耗量 /g·L^-1	木糖消耗量 /g·L^-1	BT /g·L^-1
E. coli KXW3009P0	14.2	9.8	27.0	14.8
E. coli KXW3009P1	14.6	9.9	27.5	14.7
E. coli KXW3009P2	14.8	8.9	27.8	13.2
E. coli KXW3009P3	13.4	9.8	26.0	13.0
E. coli KXW3009P4	14.9	9.0	28.1	14.9

菌株	葡萄糖消耗量 /g·L^-1	木糖消耗量 /g·L^-1	木糖转化率/%	单位OD₆₀₀的BT产量/g·L^-1
E. coli KXW3009P0	4.84	15.5	17.2	0.17
E. coli KXW3009P1	7.79	20.4	23.4	0.19
E. coli KXW3009P2	6.77	19.5	17.1	0.14
E. coli KXW3009P3	4.66	14.4	18.7	0.17
E. coli KXW3009P4	8.53	22.4	16.8	0.16