葡萄糖-木糖共利用对重组大肠杆菌合成D-1,2,4-丁三醇的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0033

摘要/Abstract

摘要：

研究共利用葡萄糖、木糖对重组大肠杆菌合成 D-1,2,4-丁三醇（BT）的影响,并优化混合底物时重组菌株合成丁三醇的培养条件。通过敲除影响磷酸转移酶系统（PTS）运行的基因,构建共利用葡萄糖-木糖的合成 BT 重组菌株,考察了 mtfA 敲除及 mlc 过表达菌株 MJ133k-ΔmtfA-PTMXM 的摇瓶培养条件。结果显示：分别敲除基因 ptsG、mtfA 和 pgi,可使菌株能同时利用木糖及葡萄糖并促进 BT 合成。菌株 MJ133k-ΔmtfA-PTMXM 合成 BT 的培养基及培养条件为：采用 1.5×LB 培养基,加入 10 g/L CaCO₃ 以控制 pH,发酵温度 33℃,装液量为 60 mL/250 mL;6 h 时分别加入葡萄糖 5 g/L及木糖 20 g/L,24 h 再次加入 5 g/L 葡萄糖,BT 合成量达 3.36 g/L,是未修饰菌株培养条件优化前BT 合成量的 4.15 倍。表明葡萄糖、木糖共利用有效促进了重组大肠杆菌合成 BT。

关键词: D-1,2,4-丁三醇, 生物合成, 混合碳源, 发酵条件优化

Abstract:

The purpose of this study is to investigate the effects of co-utilization of glucose and xylose on D-1,2,4-butanetriol（BT）biosynthesis by recombinant Escherichia coli,and to optimize the culture condition for BT production when glucose and xylose were used as mixed carbon source. By knocking out the genes affecting glucose phosphotransferase system（PTS）,a series of BT-synthesized E. coli that could co-utilize a mixture of glucose and xylose were constructed,the shake-flask culture conditions of mtfA knockout and mlc overexpression strain MJ133k-ΔmtfA-PTMXM were optimized. The results showed that the strains with the genes of ptsG,mtfA and pgi deleted respectively used glucose and xylose simultaneously,and BT biosynthesis was improved. The culture conditions of strain MJ133k-ΔmtfA-PTMXM synthesizing BT was as follows：1.5 × LB was used as culture medium,in which 1% CaCO₃ was added to maintain pH stable,culture temperature was kept at 33℃,medium volume of 60 mL was used in 250 mL flask. Twenty gram per liter xylose was added to the medium at 6 h and 5 g/L glucose was added twice at 6 h and 24 h respectively. The yield of BT reached 3.36 g/L,which was 4.15 times higher compared to that by original strain. In conclusion,co-utilization of glucose and xylose effectively improves BT production by the recombinant E. coli.

Key words: D-1,2,4-butanetriol, biosynthesis, mixed carbon source, optimization of culture conditions

刘雪丹, 杨萌, 张静, 赵东旭. 葡萄糖-木糖共利用对重组大肠杆菌合成D-1,2,4-丁三醇的影响[J]. 生物技术通报, 2021, 37(9): 171-179.

LIU Xue-dan, YANG Meng, ZHANG Jing, ZHAO Dong-xu. Effects of Glucose-xylose Co-utilization on the Synthesis of D-1,2,4-Butanetriol by Recombinant Escherichia coli[J]. Biotechnology Bulletin, 2021, 37(9): 171-179.

图/表 7

参考文献 37

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菌株及质粒Strains and plasmids	相关特性Related characteristics	来源Source
MG1655	wild type	Lab stock
MJ133k	MG1655△yjhH△yagE△xylA∷Kan^r	[8]
MJ133k-Δmlc	MJ133k△mlc∷Kanr	This work
MJ133k-Δpgi	MJ133k△pgi∷Kanr	This work
MJ134k	MJ133k△ptsG∷Kan^r	This work
MJ133k-ΔmtfA	MJ133k△mtfA∷Kanr	This work
MJ133k-1	MJ133k carrying pTMX	[8]
MJ134k-1	MJ133k△ptsG∷Kan^r/ pTMX	This work
MJ133k-Δpgi-1	MJ133k△pgi∷Kan^r/pTMX	This work
MJ133k-Δmlc-1	MJ133k△mlc∷Kan^r/pTMX	This work
MJ133k-ΔmtfA-1	MJ133k△mtfA∷Kan^r/pTMX	This work
MJ133k-G-1	MJ133k carrying PTMXG	This work
MJ133k-P-1	MJ133k carrying PTMXP	This work
MJ133k-A-1	MJ133k carrying PTMXA	This work
MJ133k-ΔmtfA-PTMXM	MJ133k△mtfA∷Kan^r/PTMXM	This work
pTMX	pTrc99a∷mdlC-xdh,Ap^r	[8]
pTMXG	pTrc99a∷mdlC-xdh-ptsG Ap^r	This work
PTMXP	pTrc99a∷mdlC-xdh-pgi Apr	This work
PTMXM	pTrc99a∷mdlC-xdh-mlc Apr	This work
PTMXA	pTrc99a∷mdlC-xdh-mtfA Apr	This work

菌株及质粒Strains and plasmids	相关特性Related characteristics	来源Source
MG1655	wild type	Lab stock
MJ133k	MG1655△yjhH△yagE△xylA∷Kan^r	[8]
MJ133k-Δmlc	MJ133k△mlc∷Kanr	This work
MJ133k-Δpgi	MJ133k△pgi∷Kanr	This work
MJ134k	MJ133k△ptsG∷Kan^r	This work
MJ133k-ΔmtfA	MJ133k△mtfA∷Kanr	This work
MJ133k-1	MJ133k carrying pTMX	[8]
MJ134k-1	MJ133k△ptsG∷Kan^r/ pTMX	This work
MJ133k-Δpgi-1	MJ133k△pgi∷Kan^r/pTMX	This work
MJ133k-Δmlc-1	MJ133k△mlc∷Kan^r/pTMX	This work
MJ133k-ΔmtfA-1	MJ133k△mtfA∷Kan^r/pTMX	This work
MJ133k-G-1	MJ133k carrying PTMXG	This work
MJ133k-P-1	MJ133k carrying PTMXP	This work
MJ133k-A-1	MJ133k carrying PTMXA	This work
MJ133k-ΔmtfA-PTMXM	MJ133k△mtfA∷Kan^r/PTMXM	This work
pTMX	pTrc99a∷mdlC-xdh,Ap^r	[8]
pTMXG	pTrc99a∷mdlC-xdh-ptsG Ap^r	This work
PTMXP	pTrc99a∷mdlC-xdh-pgi Apr	This work
PTMXM	pTrc99a∷mdlC-xdh-mlc Apr	This work
PTMXA	pTrc99a∷mdlC-xdh-mtfA Apr	This work

名称Name	引物序列Primer sequence（5'-3'）	用途Usage
ptsG-knock-s	gcgtgagaacgtaaaaaaagcacccatactcaggagcactctcaattgtgtaggctggagctgcttc	Forward primer for ptsG deletion
ptsG-knock-an	aaaaggcagccatctggctgccttagtctccccaacgtcttacggattaatgggaattagccatggtcc	Reverse primer for ptsG deletion
pgi-knock-s	atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgatg	Forward primer for pgi deletion
pgi-knock-an	cgtcggtgctatcgagctggttaccagactaattggcgatatttcgcaccgcgccaatt	Reverse primer for pgi deletion
mlc-knock-s	gtggttgctgaaaaccagcctgggcacattgatcaaataaagcagaccaacgcgggcgcg	Forward primer for mlc deletion
mlc-knock-an	gaccattttctgcgctacatattgccaagaaacaactaagcagacaacgtcccaatt	Reverse primer for mlc deletion
mtfA-knock-s	atgattaagtggccctggaaagtacaagaatcagcacatcaaactgcccttccctggca	Forward primer for mtfA deletion
mtfA-knock-an	cgtctctgacgtagtgcgattactatgtctgagcaaaagccgctgcttacaagtaatt	Reverse primer for mtfA deletion
ptsG-F	cgcttcccgccttcaatcc	Verification primer for ptsG deletion
ptsG-R	gacgccgtatggcaccttccg	Verification primer for ptsG deletion
pgi-F	accgccaaatttggagacaacaatttcaga	Verification primer for pgi deletion
pgi-R	aggtgcagcccacgcagaagtcgccgcaag	Verification primer for pgi deletion
mlc-F	ttatggcgatgatcccgcacgatcat	Verification primer for mlc deletion
mlc-R	gcagttgttcctgcactacccattcagaga	Verification primer for mlc deletion
mtfA-F	ggctttaatctgacggccgcgttccttttt	Verification primer for mtfA deletion
mtfA-R	ttcgtagccatgcttaccttccctgaacga	Verification primer for mtfA deletion
p-ptsG-F	acacaggaaacagaccatggtcatgtacaccgttggtgactacctg	Amplification of the ptsG gene
p-ptsG-R	ttaaacaaaattattaagatttgttctgttcagcga	Amplification of the ptsG gene
p-pgi-F	ctttaagtaaggaggatatattatgtcctcagccatctatccc	Amplification of the pgi gene
p-pgi-R	atccgaaagaggagaaatatgaacaactttaatctgcacacc	Amplification of the pgi gene
p-mlc-F	atatacgaagccgcccgctaatcacacaggaaagatgtctaaccgcacgccc	Amplification of the mlc gene
p-mlc-R	tcatccgccaaaacagccaagcttttcgcatcagtggttgtgg	Amplification of the mlc gene
p-mtfA-F	cctcgaggtcgacggtatcgataagcttgatgtctaaccgcacgcccc	Amplification of the mtfA gene
p-mtfA-R	ggcggccgctctagaactagtggatcc tcagtggttgtggcgggg	Amplification of the mtfA gene
pkd13-k1-s	aggctattcggctatgactg	Verification primer for gene deletion
pkd13-k1-an	ttgtcaagaccgacctgtcc	Verification primer for gene deletion

名称Name	引物序列Primer sequence（5'-3'）	用途Usage
ptsG-knock-s	gcgtgagaacgtaaaaaaagcacccatactcaggagcactctcaattgtgtaggctggagctgcttc	Forward primer for ptsG deletion
ptsG-knock-an	aaaaggcagccatctggctgccttagtctccccaacgtcttacggattaatgggaattagccatggtcc	Reverse primer for ptsG deletion
pgi-knock-s	atgaaaaacatcaatccaacgcagaccgctgcctggcaggcactacagaaacacttcgatg	Forward primer for pgi deletion
pgi-knock-an	cgtcggtgctatcgagctggttaccagactaattggcgatatttcgcaccgcgccaatt	Reverse primer for pgi deletion
mlc-knock-s	gtggttgctgaaaaccagcctgggcacattgatcaaataaagcagaccaacgcgggcgcg	Forward primer for mlc deletion
mlc-knock-an	gaccattttctgcgctacatattgccaagaaacaactaagcagacaacgtcccaatt	Reverse primer for mlc deletion
mtfA-knock-s	atgattaagtggccctggaaagtacaagaatcagcacatcaaactgcccttccctggca	Forward primer for mtfA deletion
mtfA-knock-an	cgtctctgacgtagtgcgattactatgtctgagcaaaagccgctgcttacaagtaatt	Reverse primer for mtfA deletion
ptsG-F	cgcttcccgccttcaatcc	Verification primer for ptsG deletion
ptsG-R	gacgccgtatggcaccttccg	Verification primer for ptsG deletion
pgi-F	accgccaaatttggagacaacaatttcaga	Verification primer for pgi deletion
pgi-R	aggtgcagcccacgcagaagtcgccgcaag	Verification primer for pgi deletion
mlc-F	ttatggcgatgatcccgcacgatcat	Verification primer for mlc deletion
mlc-R	gcagttgttcctgcactacccattcagaga	Verification primer for mlc deletion
mtfA-F	ggctttaatctgacggccgcgttccttttt	Verification primer for mtfA deletion
mtfA-R	ttcgtagccatgcttaccttccctgaacga	Verification primer for mtfA deletion
p-ptsG-F	acacaggaaacagaccatggtcatgtacaccgttggtgactacctg	Amplification of the ptsG gene
p-ptsG-R	ttaaacaaaattattaagatttgttctgttcagcga	Amplification of the ptsG gene
p-pgi-F	ctttaagtaaggaggatatattatgtcctcagccatctatccc	Amplification of the pgi gene
p-pgi-R	atccgaaagaggagaaatatgaacaactttaatctgcacacc	Amplification of the pgi gene
p-mlc-F	atatacgaagccgcccgctaatcacacaggaaagatgtctaaccgcacgccc	Amplification of the mlc gene
p-mlc-R	tcatccgccaaaacagccaagcttttcgcatcagtggttgtgg	Amplification of the mlc gene
p-mtfA-F	cctcgaggtcgacggtatcgataagcttgatgtctaaccgcacgcccc	Amplification of the mtfA gene
p-mtfA-R	ggcggccgctctagaactagtggatcc tcagtggttgtggcgggg	Amplification of the mtfA gene
pkd13-k1-s	aggctattcggctatgactg	Verification primer for gene deletion
pkd13-k1-an	ttgtcaagaccgacctgtcc	Verification primer for gene deletion