Commensalistic Escherichia coli coculture for biosynthesis of daidzein

doi:10.11949/0438-1157.20220461

Abstract

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

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

CLC Number:

Q 819

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

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.

Figures/Tables 9

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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

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

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

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

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