大肠杆菌偏利共培养系统合成大豆苷元

doi:10.11949/0438-1157.20220461

摘要/Abstract

摘要：

大豆苷元是一种植物雌激素，具有多种生物活性，但在大肠杆菌中的生物全合成还未见报道。基于大豆苷元合成途径的三个模块（对香豆酸、甘草素和大豆苷元模块），构建大肠杆菌共培养系统从头合成大豆苷元。将对香豆酸和甘草素模块分配到两株大肠杆菌中构建双菌共培养系统，合成甘草素。在此基础上，探索了三种共培养模式合成大豆苷元，结果显示，三菌共培养系统比其他两种双菌共培养系统的产量更高，达到27.8 mg/L。共培养菌株间通过苯丙氨酸的单向流动形成了偏利共生的关系，有助于平衡代谢途径，提高大豆苷元产量。该共培养系统在大肠杆菌中实现大豆苷元的从头合成，为其他黄酮类化合物的生物合成提供了即插即用的平台。

关键词: 合成生物学, 代谢工程, 大豆苷元, 大肠杆菌, 共培养系统, 偏利共生

Abstract:

Daidzein is a phytoestrogen with various biological activities, but its total biosynthesis in Escherichia coli has not been reported. The biosynthetic pathway of daidzein was divided into three modules, namely, the p-coumaric acid, liquiritigenin, and daidzein modules to engineer coculture systems for the biosynthesis of daidzein. First, to synthesize the precursor liquiritigenin, the p-coumaric acid and liquiritigenin modules were distributed into two E. coli strains to construct a two-strain coculture. Based on this, three coculture patterns were explored for the biosynthesis of daidzein. The results showed that the three-strain coculture produced 27.8 mg/L daidzein, which was superior to the other two two-strain coculture systems. Commensalism of the engineered strains was established through a unidirectional flow of phenylalanine, which benefited the pathway balance and daidzein production. The coculture system in this study realized the de novo biosynthesis of daidzein in engineered E. coli, and provided a plug-and-play platform for the biosynthesis of other flavonoids.

Key words: synthetic biology, metabolic engineering, daidzein, Escherichia coli, coculture, commensalism

中图分类号:

Q 819

刘雪, 张莉娟, 赵广荣. 大肠杆菌偏利共培养系统合成大豆苷元[J]. 化工学报, 2022, 73(9): 4015-4024.

Xue LIU, Lijuan ZHANG, Guangrong ZHAO. Commensalistic Escherichia coli coculture for biosynthesis of daidzein[J]. CIESC Journal, 2022, 73(9): 4015-4024.

图/表 9

图1 代谢工程改造大肠杆菌从头合成大豆苷元红叉表示敲除的基因;实线箭头表示一步反应,虚线箭头表示多步反应,黑色箭头表示大肠杆菌内源途径,彩色箭头表示大豆苷元合成途径,灰色箭头表示副产物合成途径;G6P—葡萄糖6-磷酸;E4P—赤藓糖4-磷酸;PEP—磷酸烯醇式丙酮酸;DAHP—3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸

Fig.1 Metabolic engineering E. coli for the de novo biosynthesis of daidzein

表1 本研究所用菌株及质粒

Table 1 Strains and plasmids used in this study

Strains/plasmids	Characteristics	Source
strains
LTR3	E. coli BL21(DE3) ΔptsG ΔtyrR ΔpheA::Kan^R	[19]
LCA2G	LTR3 with pLCA1G and pYBT5C	[10]
LLQ4	BL21(DE3) with pLNR12 and pLLQ4	this study
LLQ4R	BL21(DE3) with pLNR12R and pLLQ4	this study
LLQ5	LTR3 with pLNR13 and pLLQ4	this study
LDZ8	LTR3 with pLDZ1 and pLGN37 and pLLQ4	this study
LDZ10	BL21(DE3) with pLLQ4R, pLGN37K, and pLDZ4	this study
LDZ11	BL21(DE3) with pLGN37 and pLDZ3	this study
LDZ12	LTR3 with pLGN37 and pLDZ5	this study
plasmids
pETDuet-1	pET ori with P_T7; Amp^R	Novagen
pCDFDuet-1	pCDF ori with P_T7; Sm^R	Novagen
pACYCDuet-1	pACYC ori with P_T7; Cm^R	Novagen
pYBT5	pMB1 ori with P_lacUV5 -aroG^fbr -tyrA^fbr -aroE, P_trc-ppsA-tktA-glk; Amp^R	[20]
pYBT5C	pYBT5 with change of Amp^Rto Cm^R	[10]
pLCA1G	pCDF-FjTAL	[19]
pLCA1G	pCDF-FjTAL-EGFP	[10]
pLNR12	pCDFDuet-1 with At4CL, PhCHIL	[10]
pLNR12R	pCDFDuet-1 with At4CL, PhCHIL, and mCherry	[10]
pLNR13	pCDFDuet-1 with FjTAL, At4CL and PhCHIL	[10]
pLLQ4	pETDuet-1 with ErCHI and EbCHS-(GGGGGS)₃-MsCHR	our lab
pLLQ4R	pETDuet-1 with ErCHI and EbCHS-(GGGGGS)₃-MsCHR and mCherry	this study
pLGN37	pACYCDuet-1 with 17A-LjtCPR	[10]
pLGN37K	pLGN37 with exchange of Cm^R to Kan^R	[10]
pLDZ1	pCDFDuet-1 with PhCHIL, GmHID, FjTAL, At4CL and KKK-GetIFS	our lab
pLDZ3	pCDFDuet-1 with KKK-GetIFS and GmHID	this study
pLDZ4	pCDFDuet-1 with PhCHIL, GmHID, At4CL and KKK-GetIFS	our lab
pLDZ5	pCDFDuet-1 with FjTAL, KKK-GetIFS, GmHID	this study

表2 本研究所用引物

Table 2 Primers used in this study

Primers	Sequences(5'-3')
Cherry-CDF-F	GTCATCGTGGCCGGATCTTGCGCAAAAAACCCCTCAAGACC
Cherry-CDF-R	TTCGTTCAAGCCGAGGGGCCTCGAACAGAAAGTAATCGTA
CDF-Cherry-F	ATACGATTACTTTCTGTTCGAGGCCCCTCGGCTTGAACGAA
CDF-Cherry-R	GGTCTTGAGGGGTTTTTTGCGCAAGATCCGGCCACGATGA
(KKK) GetIFS-F	GTATAAGAAGGAGATATACATATGGCGAAGAAGAAGCTGAGCGCAAAAAGCAAAAG
GetIFS-R	CAGCGGTTTCTTTACCAGACTCGAGTTAAGAAGAAAACAGTTTCG
GmHID-F	CATGCCATGGCCAAAGAAATCGTTAAAG
GmHID-R	CCCAAGCTTTTAAACCAGAAAGCTGGCC
BglII-HID-F	CAGATCTCATGGCCAAAGAAATCGTTAAAGA
XhoI-IFS-R	GACTCGAGTTAAGAAGAAAACAGTTTCGG

图2 构建LCA2G-LLQ4R共培养系统在M9Y培养基中从头合成甘草素(a) LLQ5单培养系统从头合成甘草素的动态曲线; (b) LCA2G-LLQ4R共培养系统的设计; (c) LCA2G-LLQ4R共培养系统接种比例优化

Fig.2 Engineering of LCA2G-LLQ4R coculture for de novo biosynthesis of liquiritigenin in M9Y medium

图3 构建LCA2G-LLQ4R偏利双菌共培养系统在M9培养基中从头合成甘草素(a) LCA2G-LLQ4R偏利共生共培养系统的设计; (b) LCA2G-LLQ4R共培养系统接种比例优化(发酵72 h); (c) 接种比例为2∶1时LCA2G-LLQ4R共培养系统中对香豆酸、柚皮素和甘草素产量的动力学曲线; (d) 接种比例为2∶1时LCA2G-LLQ4R共培养系统中生物量及LCA2G群体比例的动力学曲线

Fig.3 Commensalistic engineering of LCA2G-LLQ4R coculture for de novo biosynthesis of liquiritigenin in M9 medium

图4 构建LCA2G-LDZ10偏利双菌共培养系统在M9培养基中从头合成大豆苷元(a) LCA2G-LDZ10共培养系统的设计; (b) LCA2G-LDZ10共培养系统接种比例优化

Fig.4 Commensalistic engineering of LCA2G-LDZ10 coculture for de novo biosynthesis of daidzein in M9 medium

图5 构建LDZ12-LLQ4R偏利双菌共培养系统在M9培养基中从头合成大豆苷元(a) LDZ12-LLQ4R共培养系统的设计; (b) LDZ12-LLQ4R共培养系统接种比例优化

Fig.5 Commensalistic engineering of LDZ12-LLQ4R coculture for de novo biosynthesis of daidzein in M9 medium

图6 构建LCA2G-LLQ4R-LDZ11偏利三菌共培养系统在M9培养基中从头合成大豆苷元(a) LCA2G-LLQ4R-LDZ11共培养系统的设计; (b) LCA2G-LLQ4R-LDZ11共培养系统接种比例优化

Fig.6 Commensalistic engineering of LCA2G-LLQ4R-LDZ11 coculture for de novo biosynthesis of daidzein in M9 medium

图7 LCA2G-LLQ4R-LDZ11偏利三菌共培养系统在M9培养基中合成大豆苷元的动态曲线(a) 接种比例2∶1∶2时LCA2G-LLQ4R-LDZ11共培养系统在M9培养基中合成的对香豆酸、柚皮素、甘草素和大豆苷元产量的动力学曲线; (b) 接种比例2∶1∶2时LCA2G-LLQ4R-LDZ11共培养系统在M9培养基中的生物量和群体比例的动力学曲线; (c) LDZ8单培养系统在M9Y培养基中合成大豆苷元

Fig.7 The time profile of commensalistic LCA2G-LLQ4R-LDZ11 coculture for biosynthesis of daidzein in M9 medium

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