微生物学报

Escherichia coli strains	Description	Sources
DH5α	F^– φ80 lacZ ΔM15 Δ (lacZYA-rgF) U169 recA1 endA1 hsdR17 (r_K^–, m_K⁺) phoA supE44 λ^– thi-1 gyrA96 relA1	ThermoFisher Scientific
JM109	endA1 recA1 gyrA96 thi-1 hsdR17 (r_k⁻, m_k⁺) relA1 supE44 D (lac-proAB) [F'traD36 proAB laqI^qZ ΔM15]	ThermoFisher Scientific
BL21(DE3)	fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS, λ DE3=λ sBamH Io ΔEcoR I-B int: : (lacI: : lacUV5:: T7-gene1) i21 Δnin5	New England Biolabs
BW25113/pIJ790	K-12 derivative; ΔaraBAD ΔrhaBAD, Cam^R	[17]
BL21 Star(DE3)	F⁻ ompT hsdS_B (r_B⁻ m_B) gal dcm rne131 (DE3) pLysS, Cam^R	ThermoFisher Scientific
CA01	BL21(DE3) containing pCTCQ-1	This work
CA02	BL21 Star(DE3) containing pCTCQ-1	This work
CA03	BL21 Star(DE3) containing pCTCQ-2	This work
CA04	BL21 Star(DE3) containing pCTCQ-3	This work
CA05	BL21 Star(DE3) containing pCTCQ-4	This work
CA06	BL21 Star(DE3) containing pCTCQ-5	This work
CA07	BL21 Star(DE3) containing pCTCQ-6	This work
CA08	BL21 Star(DE3) containing pCTCQ-7	This work
CA09	BL21 Star(DE3) containing pCTCQ-8	This work
CA10	BL21 Star(DE3) containing pCTCQ-9	This work
CA11	BL21 Star(DE3) containing pCTCQ-10	This work
CA12	BL21 Star(DE3) containing pCTCQ-15	This work
CA13	BL21 Star(DE3) containing pCTCQ-16	This work
CA14	BL21 Star(DE3) containing pCTCQ-17	This work
DH01	DH5α containing pCTCQ-11	This work
DH02	DH5α containing pCTCQ-12	This work
DH03	DH5α containing pCTCQ-13	This work

Escherichia coli strains	Description	Sources
DH5α	F^– φ80 lacZ ΔM15 Δ (lacZYA-rgF) U169 recA1 endA1 hsdR17 (r_K^–, m_K⁺) phoA supE44 λ^– thi-1 gyrA96 relA1	ThermoFisher Scientific
JM109	endA1 recA1 gyrA96 thi-1 hsdR17 (r_k⁻, m_k⁺) relA1 supE44 D (lac-proAB) [F'traD36 proAB laqI^qZ ΔM15]	ThermoFisher Scientific
BL21(DE3)	fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS, λ DE3=λ sBamH Io ΔEcoR I-B int: : (lacI: : lacUV5:: T7-gene1) i21 Δnin5	New England Biolabs
BW25113/pIJ790	K-12 derivative; ΔaraBAD ΔrhaBAD, Cam^R	[17]
BL21 Star(DE3)	F⁻ ompT hsdS_B (r_B⁻ m_B) gal dcm rne131 (DE3) pLysS, Cam^R	ThermoFisher Scientific
CA01	BL21(DE3) containing pCTCQ-1	This work
CA02	BL21 Star(DE3) containing pCTCQ-1	This work
CA03	BL21 Star(DE3) containing pCTCQ-2	This work
CA04	BL21 Star(DE3) containing pCTCQ-3	This work
CA05	BL21 Star(DE3) containing pCTCQ-4	This work
CA06	BL21 Star(DE3) containing pCTCQ-5	This work
CA07	BL21 Star(DE3) containing pCTCQ-6	This work
CA08	BL21 Star(DE3) containing pCTCQ-7	This work
CA09	BL21 Star(DE3) containing pCTCQ-8	This work
CA10	BL21 Star(DE3) containing pCTCQ-9	This work
CA11	BL21 Star(DE3) containing pCTCQ-10	This work
CA12	BL21 Star(DE3) containing pCTCQ-15	This work
CA13	BL21 Star(DE3) containing pCTCQ-16	This work
CA14	BL21 Star(DE3) containing pCTCQ-17	This work
DH01	DH5α containing pCTCQ-11	This work
DH02	DH5α containing pCTCQ-12	This work
DH03	DH5α containing pCTCQ-13	This work

Plasmids	Relevant characteristics	Sources
Kan^R: Kanamycin resistance; Amp^R: Ampicillin resistance; Cm^R: Chloramphenicol resistance.
pACYCDuet-1	p15A ori, T7, Cm^R	Novagen
pET23a	pf1 ori, T7, Amp^R	Novagen
pACYCDuet-2	A derivative of pACYCDuet-1 containing kanamycin resistance gene (Kan^R) replacing chloramphenicol resistance gene (Cm^R)	This work
pCTCQ-1	pACYCDuet-2 containing FjTAL gene driven by T7 promoter and EchpaBC gene driven by T7 promoter	This work
pCTCQ-2	pCTCQ-1 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-3	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101 promoter	This work
pCTCQ-4	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101* promoter	This work
pCTCQ-5	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101** promoter	This work
pCTCQ-6	pCTCQ-1 with the promoter of EchpaBC gene replaced by glnSm promoter	This work
pCTCQ-7	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101 promoter	This work
pCTCQ-8	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101* promoter	This work
pCTCQ-9	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101** promoter	This work
pCTCQ-10	pCTCQ-8 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-11	pET23a containing padR gene driven by lpp0.2 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-12	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-13	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9* promoter with two PadR binding boxes	This work
pCTCQ-14	pCTCQ-10 with the promoter of EchpaBC gene replaced by P9 promoter	This work
pCTCQ-15	pCTCQ-14 along with padR gene driven by lpp0.2 promoter	This work
pCTCQ-16	pCTCQ-14 along with padR gene driven by oxb20 promoter	This work
pCTCQ-17	pCTCQ-10 added with EchpaBC gene driven by J23101* promoter	This work

Plasmids	Relevant characteristics	Sources
Kan^R: Kanamycin resistance; Amp^R: Ampicillin resistance; Cm^R: Chloramphenicol resistance.
pACYCDuet-1	p15A ori, T7, Cm^R	Novagen
pET23a	pf1 ori, T7, Amp^R	Novagen
pACYCDuet-2	A derivative of pACYCDuet-1 containing kanamycin resistance gene (Kan^R) replacing chloramphenicol resistance gene (Cm^R)	This work
pCTCQ-1	pACYCDuet-2 containing FjTAL gene driven by T7 promoter and EchpaBC gene driven by T7 promoter	This work
pCTCQ-2	pCTCQ-1 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-3	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101 promoter	This work
pCTCQ-4	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101* promoter	This work
pCTCQ-5	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101** promoter	This work
pCTCQ-6	pCTCQ-1 with the promoter of EchpaBC gene replaced by glnSm promoter	This work
pCTCQ-7	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101 promoter	This work
pCTCQ-8	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101* promoter	This work
pCTCQ-9	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101** promoter	This work
pCTCQ-10	pCTCQ-8 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-11	pET23a containing padR gene driven by lpp0.2 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-12	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-13	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9* promoter with two PadR binding boxes	This work
pCTCQ-14	pCTCQ-10 with the promoter of EchpaBC gene replaced by P9 promoter	This work
pCTCQ-15	pCTCQ-14 along with padR gene driven by lpp0.2 promoter	This work
pCTCQ-16	pCTCQ-14 along with padR gene driven by oxb20 promoter	This work
pCTCQ-17	pCTCQ-10 added with EchpaBC gene driven by J23101* promoter	This work

Primers	Sequences (5′→3′)	Purpose (construction)
Underlined sequences for restriction enzyme recognition sites, and lower-case letters for overlapping between DNA sequences.
FjTAL-F	CATCACCACAGCCAGGATCCATGAACACCATTAACGAATATCTG	pCTCQ-1
FjTAL-R	AATTGAGCTCTTAGTTGTTAATCAGATGATCTTTCACT
EcHpaBC-F	AATTCATATGAAACCAGAAGATTTCCGCGCC	pCTCQ-1
EcHpaBC-R	AATTCTCGAGTTAAATCGCAGCTTCCATTT
pACYC-glnSm-Up-F	tcagataaaatatttctagaTCTGCTTTATGCCTGATGCG	pCTCQ-2
FjTAL-glnSm-Dn-R	tattcgttaatggtgttcatCGTGGATTCCTCAAAGCGTA
pACYC-J23101-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-3
FjTAL-J23101-Dn-R	cagatattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101*-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-4
FjTAL-J23101*-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101**-Up-F	tcagataaaatatttctagaTTTACACTAGCTCAGTCCTAGG	pCTCQ-5
FjTAL-J23101**-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
glnSm-F	AATTGCGGCCGCTCTGCTTTATGCCTGATGCG	pCTCQ-6
glnSm-R	TTAACATATGCGTGGATTCCTCAAAGCGTA
J23101-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-7
J23101-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTAAAGC
J23101*-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-8
J23101*-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGCTGTAAAGC
J23101**-F	GGCCGCTTTACACTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-9
J23101**-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGTGTAAAGC
LacZ-F	AATTACTAGTTTATTTTTGACACCAGACCAACTGGTAATGGTAG	pCTCQ-11
LacZ-R	TTAAGCATGCCCATGATTACGGATTCACTGG
oxb20-PadR-Up-F	accgccagagataatttactcgagatcaaaCTATTTACAAGAGGGGGGCGTG	pCTCQ-12
PadR-oxb20-Dn-R	gcgtattttaatactctcatttttactcctgtcatGCCGGGTAATACCGGATAGTC
P9-F	AATTGCGGCCGCTAAATTATCTCTGGCGGTGTT	pCTCQ-14
P9-R	TTAACATATGGATACCTTTCTCCTCTTTAATGAATT
PadR-t0-Dn-R	gtccctcttccacctgctgacttaagATTTGTCCTACTCAGGAGAGCGTTC	pCTCQ-15
P9-lpp0.2-PadR-Up-F	acaccgccagagataatttagcggccgcAAAATATTGACAACATAAAAAACTTTGTGTT
oxb20-P9-Up-F	aacaccgccagagataatttagcggccgcCTATTTACAAGAGGGGGGCGTG	pCTCQ-16
t0-EcHpaBC-Up-F	aaatggaagctgcgatttaaaattGCATGCGTCCAGTAATGACCTCAGAA	pCTCQ-17
EcHpaBC-Dn-R	agcggtttctttaccagactcgagTTAAATCGCAGCTTCCATTT

Primers	Sequences (5′→3′)	Purpose (construction)
Underlined sequences for restriction enzyme recognition sites, and lower-case letters for overlapping between DNA sequences.
FjTAL-F	CATCACCACAGCCAGGATCCATGAACACCATTAACGAATATCTG	pCTCQ-1
FjTAL-R	AATTGAGCTCTTAGTTGTTAATCAGATGATCTTTCACT
EcHpaBC-F	AATTCATATGAAACCAGAAGATTTCCGCGCC	pCTCQ-1
EcHpaBC-R	AATTCTCGAGTTAAATCGCAGCTTCCATTT
pACYC-glnSm-Up-F	tcagataaaatatttctagaTCTGCTTTATGCCTGATGCG	pCTCQ-2
FjTAL-glnSm-Dn-R	tattcgttaatggtgttcatCGTGGATTCCTCAAAGCGTA
pACYC-J23101-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-3
FjTAL-J23101-Dn-R	cagatattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101*-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-4
FjTAL-J23101*-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101**-Up-F	tcagataaaatatttctagaTTTACACTAGCTCAGTCCTAGG	pCTCQ-5
FjTAL-J23101**-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
glnSm-F	AATTGCGGCCGCTCTGCTTTATGCCTGATGCG	pCTCQ-6
glnSm-R	TTAACATATGCGTGGATTCCTCAAAGCGTA
J23101-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-7
J23101-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTAAAGC
J23101*-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-8
J23101*-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGCTGTAAAGC
J23101**-F	GGCCGCTTTACACTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-9
J23101**-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGTGTAAAGC
LacZ-F	AATTACTAGTTTATTTTTGACACCAGACCAACTGGTAATGGTAG	pCTCQ-11
LacZ-R	TTAAGCATGCCCATGATTACGGATTCACTGG
oxb20-PadR-Up-F	accgccagagataatttactcgagatcaaaCTATTTACAAGAGGGGGGCGTG	pCTCQ-12
PadR-oxb20-Dn-R	gcgtattttaatactctcatttttactcctgtcatGCCGGGTAATACCGGATAGTC
P9-F	AATTGCGGCCGCTAAATTATCTCTGGCGGTGTT	pCTCQ-14
P9-R	TTAACATATGGATACCTTTCTCCTCTTTAATGAATT
PadR-t0-Dn-R	gtccctcttccacctgctgacttaagATTTGTCCTACTCAGGAGAGCGTTC	pCTCQ-15
P9-lpp0.2-PadR-Up-F	acaccgccagagataatttagcggccgcAAAATATTGACAACATAAAAAACTTTGTGTT
oxb20-P9-Up-F	aacaccgccagagataatttagcggccgcCTATTTACAAGAGGGGGGCGTG	pCTCQ-16
t0-EcHpaBC-Up-F	aaatggaagctgcgatttaaaattGCATGCGTCCAGTAATGACCTCAGAA	pCTCQ-17
EcHpaBC-Dn-R	agcggtttctttaccagactcgagTTAAATCGCAGCTTCCATTT

咖啡酸合成途径重构及其在大肠杆菌中的异源表达

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刘蓉 ¹^,² , 王美燕 ² , 杜红毅 ¹ , 刘硕 ² , 栾孟澳 ² , 唐游 ¹ , 廖凤霞 ³ , 牛国清 ²^,^*

微生物学报 | 研究报告 2024,64(11): 4371-4387

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微生物学报 | 研究报告 2024, 64(11): 4371-4387

咖啡酸合成途径重构及其在大肠杆菌中的异源表达

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刘蓉¹^,², 王美燕², 杜红毅¹, 刘硕², 栾孟澳², 唐游¹, 廖凤霞³, 牛国清²^,^*

作者信息

1 重庆中烟工业有限责任公司烟叶资源科学利用重庆市重点实验室, 重庆 400060

2 西南大学农学与生物科技学院, 重庆 400715

3 重庆太极集团股份有限公司医药研究院, 重庆 401147

Reconstruction and heterologous expression of a biosynthetic pathway for caffeic acid in Escherichia coli

Rong LIU¹^,², Meiyan WANG², Hongyi DU¹, Shuo LIU², Meng'ao LUAN², You TANG¹, Fengxia LIAO³, Guoqing NIU²^,^*

Affiliations

1 Chongqing Key Laboratory of Scientific Utilization of Tobacco Resources, China Tobacco Chongqing Industrial Co., Ltd., Chongqing 400060, China

2 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China

3 Institutes of Biomedical Research, Chongqing Taiji Group Co., Ltd., Chongqing 401147, China

出版时间: 2024-07-24 doi: 10.13343/j.cnki.wsxb.20240354

文章导航

摘要

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【目的】通过咖啡酸合成途径重构，在大肠杆菌中实现咖啡酸的从头合成；通过优化合成基因的表达提升咖啡酸的合成效率，为咖啡酸及其衍生物的高效合成奠定基础。【方法】克隆约氏黄杆菌(Flavobacterium johnsoniaeu)酪氨酸氨解酶编码基因FjTAL和大肠杆菌(Escherichia coli) 4-羟基苯乙酸-3-单加氧酶/核黄素氧化还原酶复合体编码基因EchpaBC，通过基因共表达重构咖啡酸合成途径，导入常用大肠杆菌中进行表达。通过组成型启动子筛选、对香豆酸生物传感器动态调控和关键基因拷贝数增加相结合的方式，构建一系列工程菌株，并利用HPLC分析这些菌株中对香豆酸和咖啡酸的产生情况。随后比较添加不同氮源和底物对咖啡酸产量的影响。【结果】在大肠杆菌中实现了咖啡酸的从头合成；通过启动子工程大幅提升了咖啡酸的合成效率，以葡萄糖为底物时咖啡酸产量从1.40 mg/L提升到96.40 mg/L，以酪氨酸为底物时咖啡酸从1.78 mg/L提升到123.31 mg/L；将驱动EchpaBC表达的组成型启动子替换为对香豆酸生物传感器，咖啡酸产量达到162.73 mg/L；额外增加一个EchpaBC的拷贝数促进对香豆酸的转化，咖啡酸产量提高到185.15 mg/L。【结论】本研究采用组成型启动子改造、生物传感器调控和关键基因拷贝数增加相结合的策略，优化了FjTAL和EchpaBC的表达，成功获得了咖啡酸高产工程菌株，为咖啡酸的高效合成奠定了基础。

关键词

咖啡酸 / 大肠杆菌 / 启动子工程 / 生物传感器 / 动态调控

Abstract

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[Objective] To realize the de novo biosynthesis of caffeic acid from glucose by reconstruction of its biosynthetic pathway in Escherichia coli. Fine-tuning gene expression allows us to improve caffeic acid production, which paves a way for the high production of caffeic acid and its derivatives in E. coli. [Methods] The biosynthetic pathway of caffeic acid was reconstructed based on FjTAL and EchpaBC, which encoded the tyrosine ammonia lyase in Flavobacterium johnsoniaeu and the 4-hydroxyphenylacetate 3-hydroxylase complex in E. coli, respectively. The reconstructed pathway was then introduced into commonly used E. coli strains. We improved the expression levels of FjTAL and EchpaBC by screening constitutive promoters, utilizing an intermediate-based biosensor, and increasing the copy number of the key gene. Thus, a total of fourteen recombinant strains were obtained, and the production of caffeic acid and the intermediate p-coumaric acid in these strains was quantified by HPLC. Moreover, the effects of different nitrogen sources and substrate concentrations on the production of caffeic acid were investigated. [Results] We realized de novo biosynthesis of caffeic acid from glucose in E. coli. The use of constitutive promoters other than the commonly used T7 promoter contributed to the yield increase of caffeic acid. When glucose was used as the substrate, the yield of caffeic acid was increased from 1.40 mg/L to 96.40 mg/L. When tyrosine was used as the substrate, the yield of caffeic acid was increased from 1.78 mg/L to 123.31 mg/L. Furthermore, the yield of caffeic acid reached 162.73 mg/L when a p-coumaric acid biosensor instead of a constitutive promoter was used to drive the expression of EchpaBC. Moreover, the yield of caffeic acid was improved to 185.15 mg/L in the case of introducing an extra copy of EchpaBC. [Conclusion] We constructed the strains with high production of caffeic acid by promoter engineering, using an intermediate-base biosensor, and increasing copy number of the key gene. Our study laid a solid foundation for the high production of caffeic acid.

Key words

caffeic acid / Escherichia coli / promoter engineering / biosensor / dynamic regulation

引用本文

刘蓉, 王美燕, 杜红毅, 刘硕, 栾孟澳, 唐游, 廖凤霞, 牛国清. 咖啡酸合成途径重构及其在大肠杆菌中的异源表达. 微生物学报, 2024 , 64 (11) : 4371 -4387 . DOI: 10.13343/j.cnki.wsxb.20240354

Rong LIU, Meiyan WANG, Hongyi DU, Shuo LIU, Meng'ao LUAN, You TANG, Fengxia LIAO, Guoqing NIU. Reconstruction and heterologous expression of a biosynthetic pathway for caffeic acid in Escherichia coli[J]. Acta Microbiologica Sinica, 2024 , 64 (11) : 4371 -4387 . DOI: 10.13343/j.cnki.wsxb.20240354

正文

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咖啡酸(3, 4-二羟基肉桂酸)是一种天然酚类化合物，具有强抗氧化活性，市场需求在食品、化妆品和化工等领域均巨大。同时，咖啡酸具有抗病毒、抑菌、抗感染和抗肿瘤等多种生物活性^[1-3]，已应用于白细胞减少症和心脑血管疾病的治疗。此外，咖啡酸是合成多种高价值酚酸类化合物的关键前体，如迷迭香酸、咖啡酸苯乙酯、咖啡酸苯乙胺和白皮杉醇等^[4-6]。目前，咖啡酸主要通过植物提取、化学合成和微生物发酵3种方法制备。然而植物中咖啡酸的含量偏低，生长周期长，提取工艺复杂，导致生产成本较高。化学合成法存在有毒试剂使用和副产物残留等不足。相较而言，微生物合成具有以下优势：(1) 常用工业微生物菌株，如大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)和酿酒酵母(Saccharomyces cerevisiae)等，能以葡萄糖为底物产生酪氨酸，可为咖啡酸合成提供直接前体；(2) 微生物合成兼具绿色环保、周期短和成本低等特性。因此，微生物合成已成为咖啡酸生产最具竞争力的方式。

微生物利用葡萄糖通过莽草酸途径形成酪氨酸，后者经酪氨酸氨解酶(tyrosine ammonia lyase, TAL)和4-羟基苯乙酸3-羟基化酶(4-hydroxyphenylacetate 3-hydroxylase, HpaB)/核黄素氧化还原酶[NAD(P)H-flavin oxidoreductase, HpaC]复合体(HpaBC)依次催化合成咖啡酸^[7-8]。为了提高咖啡酸的产量，国内外学者已在外源酶筛选、辅因子再生和发酵条件优化等方面开展了一系列研究工作^[8-10]，如Zhou等比较了不同来源的HpaBC酶，并从中筛选了来自肺炎克雷伯氏菌(Klebsiella pneumoniae)的KpHpaBC进行咖啡酸催化反应，减少了酪氨酸到副产物的转化^[11]。Wang等通过敲除合成酪氨酸的竞争途径、增强辅因子水平和引入咖啡酸外排基因等手段提高了咖啡酸产量^[12]。此外，启动子工程和生物传感器介导的动态调控是实现天然产物微生物合成的重要手段^[13-15]，如Pan等将l-缬氨酸合成关键基因的原始启动子替换为不同的组成型启动子，获得的工程菌株显著提升了l-缬氨酸的产量^[14]。Guo等通过基于酪氨酸生物传感器的群体筛选策略，提高了酪氨酸高产菌株在群体中的相对比例，将苯酚的产量提高了3.9倍^[15]。Zhou等在大肠杆菌中构建基于对香豆酸和柚皮素生物传感器的动态调控网络，并对柚皮素合成相关的多个基因进行协调控制，显著提高了柚皮素的产量^[16]。

本研究选择了约氏黄杆菌(Flavobacterium johnsoniae)的酪氨酸解氨酶FjTAL和大肠杆菌(Escherichia coli)的4-羟基苯乙酸-3-单加氧酶/核黄素氧化还原酶复合体EcHpaBC重构咖啡酸合成通路，在大肠杆菌中进行咖啡酸的从头合成。在此基础上，综合应用启动子工程、生物传感器介导的动态调控和增加关键基因拷贝数的策略，对咖啡酸异源合成途径进行优化，提升了咖啡酸产量，这些研究成果有助于咖啡酸及其高值衍生物的高效合成和绿色生产。

1 材料与方法

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1.1 材料

1.1.1 菌株、质粒和引物

本研究所用菌株见表1，所涉及质粒见表2。本研究所用引物均由生工生物工程(上海)股份有限公司合成，引物序列如表3所示。

1.1.2 培养基

大肠杆菌JM109和DH5α主要用于质粒构建，BL21 Star(DE3)作为咖啡酸合成宿主细胞。

LB培养基(g/L)：胰蛋白胨10.0，酵母粉5.0，氯化钠10.0。LB培养基用于大肠杆菌常规培养。

M9培养基(g/L)：M9 Broth 11.30，葡萄糖5.00，氯化钙0.014 7，硫酸镁0.246，硫酸亚铁0.003，盐酸硫胺素0.01，CaCO₃ 5.00。M9培养基用于咖啡酸合成重组菌株发酵培养。

为探究不同氮源对重组菌株发酵合成咖啡酸产量的影响，本研究制备了含有不同氮源的3种M9培养基，即在M9培养基中分别添加0.00−2.00 g/L酵母提取物(yeast extract, YE)、10.00 g/L胰蛋白胨(tryptone, TP)和10.00 g/L玉米浆(corn steep liquor, CSL)，相应的培养基分别命名为M9YE、M9TP和M9CSL。

必要时在培养基中加入抗生素，氨苄青霉素和卡那霉素的使用浓度为100.00 μg/mL，氯霉素使用浓度为25.00 μg/mL。

1.1.3 主要试剂和仪器

色谱级甲醇，北京迪科马科技有限公司；卡那霉素、氨苄青霉素和氯霉素和质粒快速小提试剂盒，生工生物工程(上海)股份有限公司；TIANamp Bacteria DNA Kit，天根生化科技(北京)有限公司；高保真GXL DNA聚合酶(PrimeSTAR^® GXL DNA Polymerase)，T4 DNA连接酶和限制性内切酶，TaKaRa公司；同源重组酶(ClonExpress^® II One Step Cloning Kit)，南京诺唯赞生物科技股份有限公司。

PCR仪和电穿孔系统，Bio-Rad公司；振荡培养箱，上海知楚仪器有限公司；恒温培养箱，上海博迅医疗生物仪器股份有限公司；溶剂蒸发工作站，赛默飞世尔科技(中国)有限公司；高效液相色谱仪，岛津企业管理(中国)有限公司。

1.2 咖啡酸合成相关质粒和菌株的构建

为了能够在含氯霉素抗性的BL21 Star(DE3)中使用，首先利用λ-Red介导的重组(λ-Red-mediated recombination)方法将pACYCDuet-1的氯霉素抗性编码基因替换为卡那霉素抗性编码基因，得到重组质粒pACYCDuet-2^[17]。来自约氏黄杆菌的酪氨酸氨解酶编码基因FjTAL (WP_012023194.1)^[12]，由苏州金唯智生物科技有限公司经密码子优化后合成，EchpaBC扩增自BL21(DE3)基因组DNA，该基因组DNA采用TIANamp Bacteria DNA Kit提取，具体步骤详见试剂盒说明书。为构建pCTCQ-1，以合成的携带FjTAL基因质粒为模板，利用引物FjTAL-F和FjTAL-R进行PCR扩增合成FjTAL片段，其PCR扩增的反应体系(50.0 μL)：PrimeSTAR^® GXL DNA Polymerase (1.25 U/µL) 1.0 µL，5×PrimeSTAR GXL Buffer 10.0 µL，dNTP Mixture (2.5 mmol/L) 4.0 µL，上、下游引物(10 µmol/L)各2.0 µL，含FjTAL的质粒模板1.0 µL，ddH₂O 30.0 µL。PCR反应条件：98 ℃预变性5 min；98 ℃变性30 s，60 ℃退火30 s，68 ℃延伸1.6 min，共30个循环；68 ℃终延伸5 min。后续PCR扩增的反应体系和反应条件与该PCR扩增相似，将不赘述。将pACYCDuet-2目标载体和所得FjTAL片段通过BamH I和Sac I酶切连接后获得中间载体。随后，通过引物EcHpaBC-F和EcHpaBC-R对BL21(DE3)基因组DNA进行扩增，获得EchpaBC片段，经Nde I和Xho I酶切连接到上述中间载体相应位置，构建得到重组质粒pCTCQ-1。本研究筛选了4种大肠杆菌常用组成型启动子(glnSm、J23101、J23101*和J23101**)，分别替换驱动FjTAL和EchpaBC表达的T7启动子^[18-19]，得到相应的重组质粒pCTCQ-2−pCTCQ-10，相关重组质粒见表2。最后，将构建所得重组质粒转化至所需感受态细胞中，获得对应的重组菌株见表1。相关载体的构建和详细转化操作参考分子克隆实验指南^[20]。

为了构建以对香豆酸为响应信号、利用lacZ作为报告基因的生物传感器，合成了pET23a- lpp0.2-padR-P9质粒^[21]。使用LacZ-F和LacZ-R引物从BL21(DE3)基因组DNA上扩增lacZ片段，通过Spe I和Sph I酶切连接至上述合成质粒中，获得pCTCQ-11重组质粒。随后，通过同源重组将pCTCQ-11中驱动padR表达的lpp0.2组成型弱启动子更换为oxb20组成型强启动子^[22]，得到重组质粒pCTCQ-12。将pCTCQ-12重组质粒中的P9启动子改造为包含2个PadR特异性结合位点的P9*启动子，得到了重组质粒pCTCQ-13。将上述3个重组质粒分别导入DH5α中，获得相应的工程菌株DH01−DH03 (表1)。此外，以pCTCQ-11为模板，利用引物P9-F和P9-R进行PCR扩增获得P9启动子片段，经Nde I和Not I酶切后，连入pCTCQ-10相应位置，得到重组质粒pCTCQ-14。采用PadR-t0-Dn-R和P9-lpp0.2- PadR-Up-F引物从pCTCQ-11上扩增了lpp0.2-padR片段，通过同源重组方法将其插入到pCTCQ-14中，获得重组质粒pCTCQ-15；利用相同策略构建重组质粒pCTCQ-16。以pCTCQ-10为模板，利用t0-EcHpaBC-Up-F和EcHpaBC-Dn-R引物扩增得到J2310*-EchpaBC片段，通过同源重组方法连接至pCTCQ-10相应位置，得到重组质粒pCTCQ-17。PCR扩增反应体系及程序参考PrimeSTAR^® GXL DNA Polymerase操作说明书，同源重组反应体系和流程见ClonExpress^® II One Step Cloning Kit使用说明书。

1.3 β-半乳糖苷酶活性测定

参照文献[23]报道的β-半乳糖苷酶活性测定方法进行样品前处理与分析，具体操作如下：分别挑取菌株DH01−DH03单菌落于3 mL LB液体培养基中，37 ℃、220 r/min摇床培养10 h，待菌液OD₆₀₀=1.0后，按1:100接种到3 mL M9液体培养基中培养1 h，然后加入不同浓度的对香豆酸，继续培养2 h后进行OD₆₀₀测试。随后，取一定量菌液12 000 r/min离心1 min收集菌体，加入1 mL Z buffer (60 mmol/L Na₂HPO₄，40 mmol/L NaH₂PO₄，10 mmol/L KCl，1 mmol/L MgSO₄，pH 7.0)重悬。加入100 μL氯仿和50 μL SDS (sodium dodecyl sulfate，0.1%)，剧烈涡旋裂解细胞，并在28 ℃下加入200 μL ONPG (2-nitrophenyl β-galactopyranoside，4 mg/mL)，记录起始时间为t₁，待溶液变黄后立即加入500 μL Na₂CO₃ (1 mol/L)终止反应，记录终止时间t₂。

最后，以12 000 r/min离心10 min，取上清液进行OD₄₂₀测定，记录数值后计算β-半乳糖苷酶活性，如公式(1)所示^[23]。

(1)

1.4 重组菌株的发酵培养

挑取单菌落于3.00 mL LB液体培养基，在37 ℃、220 r/min培养10 h，待菌液OD₆₀₀=1.0后，按1:100接种到10.00 mL LB液体培养基中继续培养4 h。随后，5 000 r/min离心5 min收集菌体，用M9液体培养基重悬菌体沉淀，转接到100 mL锥形瓶中(含30.00 mL M9液体培养基，OD₆₀₀终浓度值约0.1)，在37 ℃、220 r/min发酵培养48 h。

1.5 重组菌株发酵产物的HPLC分析

取1.00 mL发酵液加入等体积乙酸乙酯涡旋振荡1 min，12 000 r/min离心10 min后取上清液，重复一次，合并两次乙酸乙酯萃取液。利用溶剂蒸发工作站蒸发完溶剂后，加入0.30 mL色谱纯甲醇溶解，溶液样品过0.22 μm滤膜后备用。用色谱纯甲醇制备对香豆酸和咖啡酸标准溶液，取一定量的对香豆酸和咖啡酸标准溶液和上述预处理样品一起进行HPLC检测。HPLC测定条件：色谱柱为ZORBAX SB-C18反相色谱柱(250 mm×4.6 mm，粒径为5 μm)，流动相A为0.1%甲酸的去离子水，流动相B为色谱纯甲醇；流速为0.80 mL/min；柱温为30 ℃；进样量为10 μL；咖啡酸和对香豆酸检测波长分别为324 nm和308 nm。本研究采用梯度洗脱的方法，具体流程：0−8 min，5%−50%甲醇；8−10 min，50%−100%甲醇；10−12 min，100%−5%甲醇；12−24 min，5%甲醇。以上述标准品绘制标准曲线，根据标准曲线计算重组菌株发酵产物中咖啡酸和对香豆酸的含量。

2 结果与分析

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2.1 咖啡酸合成途径重构及其在大肠杆菌中的表达

在大肠杆菌中，诱导型启动子T7常用于外源基因的表达^[7]。因此，本研究首先选择带有T7启动子的pACYCDuet-1质粒用于咖啡酸合成途径重构。考虑到BL21 Star(DE3)具有氯霉素抗性，pACYCDuet-1无法在该菌株中表征，利用λ-Red-mediated recombination方法将该质粒的抗性基因替换为卡那霉素抗性基因，得到重组质粒pACYCDuet-2。随后，分别将源自约氏黄杆菌的酪氨酸解氨酶编码基因FjTAL和大肠杆菌的4-羟基苯乙酸-3-单加氧酶/核黄素氧化还原酶复合体编码基因EchpaBC克隆到pACYCDuet-2的相应位置，得到重组质粒pCTCQ-1 (图1A)。将该质粒分别导入大肠杆菌BL21(DE3)和BL21 Star(DE3)中，得到相应的重组菌株CA01和CA02。所得菌株经发酵后进行HPLC分析，结果显示：在M9培养基中添加酪氨酸底物时，在2种重组菌株发酵液中均检测到咖啡酸，值得注意的是，CA02的咖啡酸色谱峰远高于CA01 (图1B)。同时，在M9培养基中添加葡萄糖底物进行发酵，仅在CA02发酵液中检测到咖啡酸，而CA01中并未检测到咖啡酸(图1B)。上述结果表明，本研究构建的合成途径仅在BL21 Star(DE3)中实现由葡萄糖从头合成咖啡酸，该菌株是咖啡酸异源合成的最适表达宿主。

在此基础上，对CA02中的咖啡酸进行了产量测定。在M9培养基中添加葡萄糖时，CA02的咖啡酸产量较低，约为1.40 mg/L (图2)。为了探究咖啡酸产量偏低的原因，分别添加了FjTAL和EcHpaBC的直接作用底物酪氨酸和对香豆酸，结果显示，以酪氨酸为底物时，咖啡酸产量为1.78 mg/L，其含量与葡萄糖为底物时相当，对香豆酸无积累；添加对香豆酸底物时，咖啡酸产量达到67.60 mg/L，对香豆酸含量约1.22 mg/L (图2)。上述结果表明，FjTAL表达水平对咖啡酸的产量影响较大，是咖啡酸合成的主要限制因素。

2.2 基于启动子工程的咖啡酸高产菌株构建

基于T7启动子重组菌株的咖啡酸从头合成能力较低，本研究采用启动子工程优化FjTAL和EchpaBC的表达水平。首先，筛选了glnSm、J23101、J23101*和J23101**共4个不同强度的组成型启动子，其中glnSm启动子是由谷氨酰胺-tRNA合成酶(glutaminyl-tRNA synthetase)编码基因glnS的启动子改造所得^[18]，J23101、J23101*和J23101**是3个人工合成的不同强度组成型启动子^[19]。首先利用这4个启动子分别驱动FjTAL的表达，得到相应的重组菌株CA03−CA06。这些菌株在M9培养基中发酵后，通过HPLC分析发现所有重组菌株的咖啡酸产量均显著提升，其中glnSm驱动FjTAL表达的CA03中咖啡酸产量最高，以葡萄糖或酪氨酸为底物时，产量分别达到83.31 mg/L和98.46 mg/L (图3A)。采用类似的策略，将这4个启动子分别用于驱动EchpaBC的表达，得到重组菌株CA07−CA10。HPLC结果显示，含有J23101*启动子的CA09中咖啡酸增产最高，在添加葡萄糖或酪氨酸底物时，咖啡酸产量分别为8.87 mg/L和21.63 mg/L (图3B)。与CA02相比，启动子改造后工程菌株的咖啡酸产量均有所提升。结果表明，启动子改造优化FjTAL和EchpaBC的表达水平可以有效提高咖啡酸的产量。在该研究基础上，将FjTAL和EchpaBC的最佳启动子进行组合，得到重组质粒pCTCQ-10，将其导入BL21 Star(DE3)后获得重组菌株CA11。该菌株在添加5.00 g/L葡萄糖或100.00 mg/L酪氨酸的M9培养基中进行发酵，所得咖啡酸产量分别为74.18 mg/L和103.26 mg/L (图3C)。该菌株咖啡酸产量与CA03相当，远高于其他启动子改造所得菌株CA04−CA10。因此，选择CA11进行咖啡酸高产菌株的后续试验。

2.3 咖啡酸高产菌株发酵培养基优化

前期研究发现发酵培养基的成分，尤其是氮源对菌株生长和目标产物合成具有重要影响，含有有机氮源酵母提取物(YE)的M9YE培养基常被用作咖啡酸的发酵培养基^[9]。因此，先比较了CA11在添加了不同酵母提取物的M9YE培养基中发酵所得对香豆酸与咖啡酸含量，发现酵母提取物添加量从0.00 g/L增加到2.00 g/L时，咖啡酸产量从71.18 mg/L逐渐降低到30.71 mg/L (图4A)，说明酵母提取物在一定程度上会抑制咖啡酸的合成。为此，本研究尝试了另外2个常用氮源胰蛋白胨(TP)和玉米浆(CSL)，结果发现CA11在M9TP和M9CSL培养基中发酵所得咖啡酸产量分别为10.31 mg/L和5.99 mg/L，远低于M9培养基中目标产物的产量(图4B)。随后，比较了M9培养基中添加不同浓度的葡萄糖或酪氨酸对咖啡酸产量的影响。当葡萄糖含量从5.00 g/L增加到20.00 g/L时，对香豆酸和咖啡酸的产量均随之增加，对香豆酸积累量由2.81 mg/L提高到4.41 mg/L，咖啡酸产量由76.32 mg/L提高到了92.89 mg/L；当葡萄糖含量由20.00 g/L增加到40.00 g/L时，咖啡酸产量略有增加，最高可达96.40 mg/L，而对香豆酸的积累量并无明显增加(图4C)。酪氨酸底物从100.00 mg/L增加到200.00 mg/L时，咖啡酸产量由102.35 mg/L增加到110.18 mg/L，同时检测到大量的对香豆酸积累，最高可达83.36 mg/L (图4D)；当酪氨酸底物由200.00 mg/L增加到400.00 mg/L时，咖啡酸产量出现先增加(123.31 mg/L)后降低的趋势(119.13 mg/L)，对香豆酸转化也呈现下降趋势，其含量分别为60.48 mg/L和68.34 mg/L。综上所述，M9培养基是咖啡酸合成的最佳发酵培养基，最适葡萄糖和酪氨酸添加浓度分别为20.00 g/L和200.00 mg/L。

2.4 对香豆酸生物传感器的构建与优化

如上所述，通过发酵培养基优化，显著提升了咖啡酸的合成能力，但同时也检测到大量对香豆酸的积累。在早期研究中，研究者利用枯草芽孢杆菌(Bacillus subtilis)的转录抑制因子PadR及其响应元件P_padC构建了对香豆酸生物传感器，并成功应用于对香豆酸向原儿茶酚的高效转化^[24-25]。为此，本研究合成了对香豆酸生物传感器，该生物传感器由lpp0.2启动子驱动的padR和基于P_padC改造的P9启动子两部分组成，其中lpp0.2启动子是由大肠杆菌脂蛋白编码基因lpp的启动子改造所得^[21]。将报告基因lacZ置于P9启动子之后，得到重组质粒pCTCQ-11，将其转入DH5α中得到重组菌株DH01。随后，对DH01的β-半乳糖苷酶活性进行了检测，结果显示DH01具有较强的渗漏表达(图5B)。为降低该系统的渗漏表达，本研究将pCTCQ-11中的lpp0.2启动子替换为由大肠杆菌中recA启动子改造所得oxb20组成型强启动子^[22]，得到重组质粒pCTCQ-12；将pCTCQ-12中的P9启动子替换为包含2个PadR特异性结合位点的P9*启动子，得到pCTCQ-13。将重组质粒pCTCQ-12和pCTCQ-13分别转入DH5α中，获得重组菌株DH02和DH03 (图5A)。对DH01−DH03这3个菌株的β-半乳糖苷酶活性进行了比较分析发现：随着对香豆酸浓度的增加(0.00–200.00 mg/L)，3个菌株的半乳糖苷酶活性也随之增加，其中菌株DH01的β-半乳糖苷酶活性信号强度从1 140.91增加到5 241.66，DH02由228.15增加到1 537.41，DH03从717.21增加到2 244.96；当对香豆酸浓度为300.00 mg/L及以上时，3个菌株的β-半乳糖苷酶的活性明显降低，说明它们都可以响应对香豆酸，但高浓度的对香豆酸具有细胞毒性(图5B)。因此，对这3个菌株的细胞生长密度进行了测定，发现所有菌株的生长随着对香豆酸浓度的增加而受到明显的抑制(图5C)。综上所述，DH01在3个菌株中半乳糖苷酶活性最高，DH02本底渗漏最少，而DH03整体性能最差。

2.5 生物传感器介导的动态调控提高咖啡酸的产量

通过同源重组的方法，分别用对香豆酸响应元件lpp0.2-padR-P9和oxb20-padR-P9替换pCTCQ-10中驱动EchpaBC表达的J23101*，得到重组质粒pCTCQ-15和pCTCQ-16，将其导入BL21 Star(DE3)中获得菌株CA12和CA13。随后，将这2种菌株分别在添加葡萄糖或酪氨酸的M9培养基中进行发酵。HPLC分析发现，CA12中咖啡酸产量分别为72.16 mg/L和162.73 mg/L，对香豆酸产量仅为1.84 mg/L和3.62 mg/L (图6A)；CA13中咖啡酸产量分别为1.78 mg/L和9.89 mg/L，对香豆酸产量为28.89 mg/L和120.88 mg/L (图6B)。上述两种不同生物传感器调控咖啡酸合成的菌株中，以lpp0.2启动子驱动padR表达的CA12能更有效地促进对香豆酸向咖啡酸的高效转化。此外，CA11中酪氨酸底物转化效率为0.55 g/g，而CA12的酪氨酸底物转化效率为0.81 g/g，相较于CA11提高了47.27%。由此可见，生物传感器介导的动态调控有助于调节胞内中间体代谢流平衡，提升咖啡酸的合成效率。

2.6 增加EchpaBC的拷贝数提高咖啡酸产量

为了进一步促进对香豆酸的转化，本研究还尝试了额外增加EchpaBC拷贝数的策略。在pCTCQ-10中增加一个由J23101*启动子驱动表达的EchpaBC，得到重组质粒pCTCQ-17 (图7A)，并将其导入BL21 Star(DE3)中获得菌株CA14。HPCL检测结果显示(图7B)：以20.00 g/L葡萄糖为底物时，CA14发酵产物中咖啡酸产量与CA11相比无明显变化；而以200.00 mg/L酪氨酸为底物时，CA14咖啡酸的产量大幅提升，同时对香豆酸的积累量明显降低。CA14中咖啡酸含量最高可达185.15 mg/L，酪氨酸底物转化效率为0.93 g/g，相较于CA11提高了69.09%。由此可见，增加EchpaBC的拷贝数能够显著提高对香豆酸的转化效率。

3 讨论与结论

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为了在大肠杆菌中重构咖啡酸的合成通路，选择了约氏黄杆菌来源FjTAL和大肠杆菌内源性EchpaBC，分别用T7启动子驱动其表达得到重组菌株CA02，实现了由葡萄糖从头合成咖啡酸，但其产量仅为1.40 mg/L。进一步分析发现，添加酪氨酸时，对香豆酸(0.06 mg/L)和咖啡酸(1.78 mg/L)产量均较低，而添加对香豆酸时咖啡酸产量为67.60 mg/L，其咖啡酸转化效率约61.59%，说明FjTAL的催化效率是导致合成咖啡酸偏低的主要限制因素。为进一步提高咖啡酸产量，我们有必要继续筛选不同来源的TAL和HpaBC，以增强这2种酶的催化活性。另外，可以通过多轮定点饱和突变、组合突变及随机突变技术对现有的FjTAL和EcHpaBC进行改造，筛选出高催化活性和底物选择性的突变酶，合成相应的基因用于咖啡酸的高效合成^[26-28]。

选取了4个大肠杆菌常用的组成型启动子glnSm、J23101、J23101*和J23101**分别驱动FjTAL和EchpaBC的表达，结果显示组成型启动子不同程度地提高了咖啡酸的产量。另外，发现启动子glnSm最适于驱动FjTAL的表达，而J23101*最适于驱动EchpaBC的表达，将这2个启动子组合后，所得菌株CA11的咖啡酸从头合成产量达到96.40 mg/L。这些结果表明，启动子工程是提高咖啡酸产量的有效手段，可以扩大启动子的筛选范围，进一步提高咖啡酸的产量。此外，HPLC检测发现CA11发酵过程中有较高的对香豆酸积累，因而尝试了利用对香豆酸生物传感器驱动EchpaBC的表达，其在一定程度上促进了对香豆酸向咖啡酸的转化，与此同时，采用了额外增加EchpaBC拷贝数的策略，对香豆酸积累量显著降低，咖啡酸产量大幅提升。由此可见，增加关键基因的拷贝数有助于提升咖啡酸的产量，而本研究所用的pACYCDuet-1在大肠杆菌中的拷贝数约10−15，选择不同拷贝数的质粒用于FjTAL和EchpaBC的表达，有望进一步提高咖啡酸的合成效率。

如前所述，对香豆酸和咖啡酸具有一定的细胞毒性^[12]，高产的有毒目标产物会对其重组菌株的生长和目标产物合成造成不利影响。为了解决这一难题，本研究构建了感应对香豆酸的生物传感器，用于动态调控EchhpaBC的表达，显著减少了有毒中间体对香豆酸的积累，从而在一定程度上提高了CA12的耐受性，并进一步提升了咖啡酸产量。此外，在CA12菌株基础上，增加一个由对香豆酸生物传感器调控表达的咖啡酸外排基因，增强对香豆酸转化的同时减少咖啡酸在胞内的积累^[12]，有望进一步提高该重组菌株的耐受性。我们也可选取鲁棒性较强的宿主如恶臭假单胞菌替换大肠杆菌宿主来消除这一不利影响^[24]。适应性进化是一种提高宿主耐受性的有效方法^[29-30]，后续研究中，将以高浓度的对香豆酸为选择压力，进行多次传代。在传代过程中，利用对香豆酸生物传感器监测连续繁殖群体的适应性变化，从而筛选出对香豆酸耐受性强的宿主细胞。随后，采用相同的策略进行咖啡酸耐受性的进化实验，获得对香豆酸和咖啡酸耐受性均增强的菌株，应用于咖啡酸的高效合成。

基金

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重庆中烟工业有限责任公司科技项目(HX20220204)

参考文献

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文献

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ZHOU XJ, ZHANG XX, WANG D, LUO RS, QIN Z, LIN FZ, XIA X, LIU XM, HU G.Efficient biosynthesis of salidroside via artificial in vivo enhanced UDP-glucose system using cheap sucrose as substrate[J].ACS Omega,2024,9(20):22386-22397.

https://doi.org/10.1021/acsomega.4c02060

[30]

WANG M, WANG HM, GAO C, WEI WQ, LIU J, CHEN XL, HU GP, SONG W, WU J, ZHANG F, LIU LM.Efficient production of protocatechuic acid using systems engineering of Escherichia coli[J].Metabolic Engineering,2024,82: 134-146.

https://doi.org/10.1016/j.ymben.2024.02.003

2024年第64卷第11期

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文章信息

doi: 10.13343/j.cnki.wsxb.20240354

接收时间：2024-06-11
首发时间：2026-03-21
出版时间：2024-07-24

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出版历史

收稿日期：2024-06-11
录用日期：2024-07-22

基金

Technology Project of China Tobacco Chongqing Industrial Co., Ltd.(HX20220204)

重庆中烟工业有限责任公司科技项目(HX20220204)

作者信息

1 重庆中烟工业有限责任公司烟叶资源科学利用重庆市重点实验室, 重庆 400060

2 西南大学农学与生物科技学院, 重庆 400715

3 重庆太极集团股份有限公司医药研究院, 重庆 401147

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https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20240354

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Escherichia coli strains	Description	Sources
DH5α	F^– φ80 lacZ ΔM15 Δ (lacZYA-rgF) U169 recA1 endA1 hsdR17 (r_K^–, m_K⁺) phoA supE44 λ^– thi-1 gyrA96 relA1	ThermoFisher Scientific
JM109	endA1 recA1 gyrA96 thi-1 hsdR17 (r_k⁻, m_k⁺) relA1 supE44 D (lac-proAB) [F'traD36 proAB laqI^qZ ΔM15]	ThermoFisher Scientific
BL21(DE3)	fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS, λ DE3=λ sBamH Io ΔEcoR I-B int: : (lacI: : lacUV5:: T7-gene1) i21 Δnin5	New England Biolabs
BW25113/pIJ790	K-12 derivative; ΔaraBAD ΔrhaBAD, Cam^R	[17]
BL21 Star(DE3)	F⁻ ompT hsdS_B (r_B⁻ m_B) gal dcm rne131 (DE3) pLysS, Cam^R	ThermoFisher Scientific
CA01	BL21(DE3) containing pCTCQ-1	This work
CA02	BL21 Star(DE3) containing pCTCQ-1	This work
CA03	BL21 Star(DE3) containing pCTCQ-2	This work
CA04	BL21 Star(DE3) containing pCTCQ-3	This work
CA05	BL21 Star(DE3) containing pCTCQ-4	This work
CA06	BL21 Star(DE3) containing pCTCQ-5	This work
CA07	BL21 Star(DE3) containing pCTCQ-6	This work
CA08	BL21 Star(DE3) containing pCTCQ-7	This work
CA09	BL21 Star(DE3) containing pCTCQ-8	This work
CA10	BL21 Star(DE3) containing pCTCQ-9	This work
CA11	BL21 Star(DE3) containing pCTCQ-10	This work
CA12	BL21 Star(DE3) containing pCTCQ-15	This work
CA13	BL21 Star(DE3) containing pCTCQ-16	This work
CA14	BL21 Star(DE3) containing pCTCQ-17	This work
DH01	DH5α containing pCTCQ-11	This work
DH02	DH5α containing pCTCQ-12	This work
DH03	DH5α containing pCTCQ-13	This work

Escherichia coli strains

Description

Sources

DH5α

F^– φ80 lacZ ΔM15 Δ (lacZYA-rgF) U169 recA1 endA1 hsdR17 (r_K^–, m_K⁺) phoA supE44 λ^– thi-1 gyrA96 relA1

ThermoFisher Scientific

JM109

endA1 recA1 gyrA96 thi-1 hsdR17 (r_k⁻, m_k⁺) relA1 supE44 D (lac-proAB) [F'traD36 proAB laqI^qZ ΔM15]

ThermoFisher Scientific

BL21(DE3)

fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS, λ DE3=λ sBamH Io ΔEcoR I-B int: : (lacI: : lacUV5:: T7-gene1) i21 Δnin5

New England Biolabs

BW25113/pIJ790

K-12 derivative; ΔaraBAD ΔrhaBAD, Cam^R

[17]

BL21 Star(DE3)

F⁻ ompT hsdS_B (r_B⁻ m_B) gal dcm rne131 (DE3) pLysS, Cam^R

ThermoFisher Scientific

CA01

BL21(DE3) containing pCTCQ-1

This work

CA02

BL21 Star(DE3) containing pCTCQ-1

This work

CA03

BL21 Star(DE3) containing pCTCQ-2

This work

CA04

BL21 Star(DE3) containing pCTCQ-3

This work

CA05

BL21 Star(DE3) containing pCTCQ-4

This work

CA06

BL21 Star(DE3) containing pCTCQ-5

This work

CA07

BL21 Star(DE3) containing pCTCQ-6

This work

CA08

BL21 Star(DE3) containing pCTCQ-7

This work

CA09

BL21 Star(DE3) containing pCTCQ-8

This work

CA10

BL21 Star(DE3) containing pCTCQ-9

This work

CA11

BL21 Star(DE3) containing pCTCQ-10

This work

CA12

BL21 Star(DE3) containing pCTCQ-15

This work

CA13

BL21 Star(DE3) containing pCTCQ-16

This work

CA14

BL21 Star(DE3) containing pCTCQ-17

This work

DH01

DH5α containing pCTCQ-11

This work

DH02

DH5α containing pCTCQ-12

This work

DH03

DH5α containing pCTCQ-13

This work

Plasmids	Relevant characteristics	Sources
Kan^R: Kanamycin resistance; Amp^R: Ampicillin resistance; Cm^R: Chloramphenicol resistance.
pACYCDuet-1	p15A ori, T7, Cm^R	Novagen
pET23a	pf1 ori, T7, Amp^R	Novagen
pACYCDuet-2	A derivative of pACYCDuet-1 containing kanamycin resistance gene (Kan^R) replacing chloramphenicol resistance gene (Cm^R)	This work
pCTCQ-1	pACYCDuet-2 containing FjTAL gene driven by T7 promoter and EchpaBC gene driven by T7 promoter	This work
pCTCQ-2	pCTCQ-1 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-3	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101 promoter	This work
pCTCQ-4	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101* promoter	This work
pCTCQ-5	pCTCQ-1 with the promoter of FjTAL gene replaced by J23101** promoter	This work
pCTCQ-6	pCTCQ-1 with the promoter of EchpaBC gene replaced by glnSm promoter	This work
pCTCQ-7	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101 promoter	This work
pCTCQ-8	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101* promoter	This work
pCTCQ-9	pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101** promoter	This work
pCTCQ-10	pCTCQ-8 with the promoter of FjTAL gene replaced by glnSm promoter	This work
pCTCQ-11	pET23a containing padR gene driven by lpp0.2 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-12	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9 promoter	This work
pCTCQ-13	pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9* promoter with two PadR binding boxes	This work
pCTCQ-14	pCTCQ-10 with the promoter of EchpaBC gene replaced by P9 promoter	This work
pCTCQ-15	pCTCQ-14 along with padR gene driven by lpp0.2 promoter	This work
pCTCQ-16	pCTCQ-14 along with padR gene driven by oxb20 promoter	This work
pCTCQ-17	pCTCQ-10 added with EchpaBC gene driven by J23101* promoter	This work

Plasmids

Relevant characteristics

Sources

Kan^R: Kanamycin resistance; Amp^R: Ampicillin resistance; Cm^R: Chloramphenicol resistance.

pACYCDuet-1

p15A ori, T7, Cm^R

Novagen

pET23a

pf1 ori, T7, Amp^R

Novagen

pACYCDuet-2

A derivative of pACYCDuet-1 containing kanamycin resistance gene (Kan^R) replacing chloramphenicol resistance gene (Cm^R)

This work

pCTCQ-1

pACYCDuet-2 containing FjTAL gene driven by T7 promoter and EchpaBC gene driven by T7 promoter

This work

pCTCQ-2

pCTCQ-1 with the promoter of FjTAL gene replaced by glnSm promoter

This work

pCTCQ-3

pCTCQ-1 with the promoter of FjTAL gene replaced by J23101 promoter

This work

pCTCQ-4

pCTCQ-1 with the promoter of FjTAL gene replaced by J23101* promoter

This work

pCTCQ-5

pCTCQ-1 with the promoter of FjTAL gene replaced by J23101** promoter

This work

pCTCQ-6

pCTCQ-1 with the promoter of EchpaBC gene replaced by glnSm promoter

This work

pCTCQ-7

pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101 promoter

This work

pCTCQ-8

pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101* promoter

This work

pCTCQ-9

pCTCQ-1 with the promoter of EchpaBC gene replaced by J23101** promoter

This work

pCTCQ-10

pCTCQ-8 with the promoter of FjTAL gene replaced by glnSm promoter

This work

pCTCQ-11

pET23a containing padR gene driven by lpp0.2 promoter and lacZ gene driven by P9 promoter

This work

pCTCQ-12

pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9 promoter

This work

pCTCQ-13

pET23a containing padR gene driven by oxb20 promoter and lacZ gene driven by P9* promoter with two PadR binding boxes

This work

pCTCQ-14

pCTCQ-10 with the promoter of EchpaBC gene replaced by P9 promoter

This work

pCTCQ-15

pCTCQ-14 along with padR gene driven by lpp0.2 promoter

This work

pCTCQ-16

pCTCQ-14 along with padR gene driven by oxb20 promoter

This work

pCTCQ-17

pCTCQ-10 added with EchpaBC gene driven by J23101* promoter

This work

Primers	Sequences (5′→3′)	Purpose (construction)
Underlined sequences for restriction enzyme recognition sites, and lower-case letters for overlapping between DNA sequences.
FjTAL-F	CATCACCACAGCCAGGATCCATGAACACCATTAACGAATATCTG	pCTCQ-1
FjTAL-R	AATTGAGCTCTTAGTTGTTAATCAGATGATCTTTCACT
EcHpaBC-F	AATTCATATGAAACCAGAAGATTTCCGCGCC	pCTCQ-1
EcHpaBC-R	AATTCTCGAGTTAAATCGCAGCTTCCATTT
pACYC-glnSm-Up-F	tcagataaaatatttctagaTCTGCTTTATGCCTGATGCG	pCTCQ-2
FjTAL-glnSm-Dn-R	tattcgttaatggtgttcatCGTGGATTCCTCAAAGCGTA
pACYC-J23101-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-3
FjTAL-J23101-Dn-R	cagatattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101*-Up-F	tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA	pCTCQ-4
FjTAL-J23101*-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
pACYC-J23101**-Up-F	tcagataaaatatttctagaTTTACACTAGCTCAGTCCTAGG	pCTCQ-5
FjTAL-J23101**-Dn-R	tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG
glnSm-F	AATTGCGGCCGCTCTGCTTTATGCCTGATGCG	pCTCQ-6
glnSm-R	TTAACATATGCGTGGATTCCTCAAAGCGTA
J23101-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-7
J23101-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTAAAGC
J23101*-F	GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-8
J23101*-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGCTGTAAAGC
J23101**-F	GGCCGCTTTACACTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA	pCTCQ-9
J23101**-R	TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGTGTAAAGC
LacZ-F	AATTACTAGTTTATTTTTGACACCAGACCAACTGGTAATGGTAG	pCTCQ-11
LacZ-R	TTAAGCATGCCCATGATTACGGATTCACTGG
oxb20-PadR-Up-F	accgccagagataatttactcgagatcaaaCTATTTACAAGAGGGGGGCGTG	pCTCQ-12
PadR-oxb20-Dn-R	gcgtattttaatactctcatttttactcctgtcatGCCGGGTAATACCGGATAGTC
P9-F	AATTGCGGCCGCTAAATTATCTCTGGCGGTGTT	pCTCQ-14
P9-R	TTAACATATGGATACCTTTCTCCTCTTTAATGAATT
PadR-t0-Dn-R	gtccctcttccacctgctgacttaagATTTGTCCTACTCAGGAGAGCGTTC	pCTCQ-15
P9-lpp0.2-PadR-Up-F	acaccgccagagataatttagcggccgcAAAATATTGACAACATAAAAAACTTTGTGTT
oxb20-P9-Up-F	aacaccgccagagataatttagcggccgcCTATTTACAAGAGGGGGGCGTG	pCTCQ-16
t0-EcHpaBC-Up-F	aaatggaagctgcgatttaaaattGCATGCGTCCAGTAATGACCTCAGAA	pCTCQ-17
EcHpaBC-Dn-R	agcggtttctttaccagactcgagTTAAATCGCAGCTTCCATTT

Primers

Sequences (5′→3′)

Purpose (construction)

Underlined sequences for restriction enzyme recognition sites, and lower-case letters for overlapping between DNA sequences.

FjTAL-F

CATCACCACAGCCAGGATCCATGAACACCATTAACGAATATCTG

pCTCQ-1

FjTAL-R

AATTGAGCTCTTAGTTGTTAATCAGATGATCTTTCACT

EcHpaBC-F

AATTCATATGAAACCAGAAGATTTCCGCGCC

pCTCQ-1

EcHpaBC-R

AATTCTCGAGTTAAATCGCAGCTTCCATTT

pACYC-glnSm-Up-F

tcagataaaatatttctagaTCTGCTTTATGCCTGATGCG

pCTCQ-2

FjTAL-glnSm-Dn-R

tattcgttaatggtgttcatCGTGGATTCCTCAAAGCGTA

pACYC-J23101-Up-F

tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA

pCTCQ-3

FjTAL-J23101-Dn-R

cagatattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG

pACYC-J23101*-Up-F

tcagataaaatatttctagaTTTACAGCTAGCTCAGTCCTA

pCTCQ-4

FjTAL-J23101*-Dn-R

tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG

pACYC-J23101**-Up-F

tcagataaaatatttctagaTTTACACTAGCTCAGTCCTAGG

pCTCQ-5

FjTAL-J23101**-Dn-R

tattcgttaatggtgttcatGGTATATCTCCTTCTCTAGTCTCTAG

glnSm-F

AATTGCGGCCGCTCTGCTTTATGCCTGATGCG

pCTCQ-6

glnSm-R

TTAACATATGCGTGGATTCCTCAAAGCGTA

J23101-F

GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA

pCTCQ-7

J23101-R

TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATTATACCTAGGACTGAGCTAGCTGTAAAGC

J23101*-F

GGCCGCTTTACAGCTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA

pCTCQ-8

J23101*-R

TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGCTGTAAAGC

J23101**-F

GGCCGCTTTACACTAGCTCAGTCCTAGGTATATGCTAGCTACTAGAGACTAGAGAAGGAGATATACCCA

pCTCQ-9

J23101**-R

TATGGGTATATCTCCTTCTCTAGTCTCTAGTAGCTAGCATATACCTAGGACTGAGCTAGTGTAAAGC

LacZ-F

AATTACTAGTTTATTTTTGACACCAGACCAACTGGTAATGGTAG

pCTCQ-11

LacZ-R

TTAAGCATGCCCATGATTACGGATTCACTGG

oxb20-PadR-Up-F

accgccagagataatttactcgagatcaaaCTATTTACAAGAGGGGGGCGTG

pCTCQ-12

PadR-oxb20-Dn-R

gcgtattttaatactctcatttttactcctgtcatGCCGGGTAATACCGGATAGTC

P9-F

AATTGCGGCCGCTAAATTATCTCTGGCGGTGTT

pCTCQ-14

P9-R

TTAACATATGGATACCTTTCTCCTCTTTAATGAATT

PadR-t0-Dn-R

gtccctcttccacctgctgacttaagATTTGTCCTACTCAGGAGAGCGTTC

pCTCQ-15

P9-lpp0.2-PadR-Up-F

acaccgccagagataatttagcggccgcAAAATATTGACAACATAAAAAACTTTGTGTT

oxb20-P9-Up-F

aacaccgccagagataatttagcggccgcCTATTTACAAGAGGGGGGCGTG

pCTCQ-16

t0-EcHpaBC-Up-F

aaatggaagctgcgatttaaaattGCATGCGTCCAGTAATGACCTCAGAA

pCTCQ-17

EcHpaBC-Dn-R

agcggtttctttaccagactcgagTTAAATCGCAGCTTCCATTT