利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究 <sup>*</sup>

doi:10.13523/j.cb.2008123

中国生物工程杂志

2021, Vol. 41

Issue (1): 12-19 DOI: 10.13523/j.cb.2008123

研究报告

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究 ^*

吴弘轩¹,杨金花¹,沈培杰¹,李清晨¹,黄建忠¹,祁峰^1,^2,^**(

)

1 福建师范大学生命科学学院工业微生物发酵技术国家地方联合工程研究中心福州 350117
2 福建师范大学细胞逆境响应与代谢调控福建省高校重点实验室福州 350108

Study on the Production of Indole-3-acetic Acid Using E. coli Cell Factory

WU Hong-xuan¹,YANG Jin-hua¹,SHEN Pei-jie¹,LI Qing-chen¹,HUANG Jian-zhong¹,QI Feng^1,^2,^**(

)

1 School of Life Science, National and Local Joint Engineering Research Center of Industrial Microbial Fermentation Technology,Fujian Normal University, Fuzhou 350117, China
2 Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation,College of Life Sciences, Fujian Normal University, Fuzhou 350108, China

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摘要：

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA。方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide, IAM)途径和色胺(tryptamine, TRP)途径。结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆菌TPA-4以2g/L的色氨酸为底物,可以产生0.803g/L的IAA;敲除控制色氨酸合成副产物吲哚的tnaA基因后,菌株MPA-3的IAA产量达到1.43g/L,提高了78%。第二条TRP途径合成IAA涉及三个酶:左旋色氨酸脱羧酶(TDC),二胺氧化酶(AOC1)和吲哚-3-乙醛脱氢酶(IAD1)。包含这条途径的重组大肠杆菌TPTA-2以2g/L的色氨酸为底物能够合成13.0mg/L的IAA。在菌株MPTA-3中,最终产生了21.0mg/L的IAA,产量增加了61.5%。结论:首次通过IAM途径和TRP途径利用重组大肠杆菌全细胞催化生产IAA,其中IAM途径的IAA产量较高,有较高的工业化应用前景。

关键词： 色氨酸; 吲哚-3-乙酸; 吲哚-3-乙酰胺; 色胺; 全细胞催化; 大肠杆菌

Abstract:

Objective: Indole-3-acetic acid (IAA) was produced from tryptophan in the metabolically engineered E. coli MG1655 using whole-cell catalysis. Methods: Two novel IAA biosynthetic pathways, the indole-3-acetamide (IAM) pathway and the tryptamine (TRP) pathway, were constructed in E. coli MG1655. Results: The IAM pathway involves two enzymes, tryptophan-2- monooxygenase (IAAM) and amidase (AMI1). 2g/L tryptophan as a substrate was used by the constructed recombinant E. coli strain TPA-4. TPA-4 can produce 0.803g/L of IAA; however, in the strain MPA-3 that was knocked out the gene tnaA which divert flux from tryptophan synthesis, the yield of IAA reached 1.43g/L, an increase of 78% compared with the control. The second TRP pathway biosynthesis of IAA involves three enzymes: L-tryptophan decarboxylase (TDC), diamine oxidase (AOC1) and indole-3-acetaldehyde dehydrogenase (IAD1). The recombinant E.coli TPTA-2 that included the TRP pathway can only synthesize 13.0mg/L IAA with 2g/L tryptophan as substrate. In the strain MPTA-3 with disruption of tnaA gene, 21.0mg/L of IAA was finally produced, and the yield increased by 61.5%. Conclusion: It is the first report to realize production of IAA using the metabolically engineered E. coli through the IAM pathway and TRP pathway via whole-cell catalysis. IAA production from the IAM pathway is relatively higher, and it probably has an industrial application prospect.

Key words: Tryptophan Indole-3-acetic acid Indole-3-acetamide Tryptamine Whole cell catalysis E. coli

收稿日期: 2020-08-14 出版日期: 2021-02-09

ZTFLH:

Q819

基金资助: * 国家自然科学基金(21406130)

通讯作者: 祁峰 E-mail: f.qi@fjnu.edu.cn

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引用本文:

吴弘轩, 杨金花, 沈培杰, 李清晨, 黄建忠, 祁峰. 利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究 ^*[J]. 中国生物工程杂志, 2021, 41(1): 12-19.

WU Hong-xuan, YANG Jin-hua, SHEN Pei-jie, LI Qing-chen, HUANG Jian-zhong, QI Feng. Study on the Production of Indole-3-acetic Acid Using E. coli Cell Factory. China Biotechnology, 2021, 41(1): 12-19.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2008123 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I1/12

图1 吲哚-3-乙酸的合成途径

表1 菌株和质粒

Primers	Sequence(5'-3')
iaaM-F	ggaaacagaccatggaattcaaggagatgtacgatcatttcaaca
iaaM-R	gatccccgggtaccgagctcttaataacgataacttgcat
V-iaaM-F	atgcaagttatcgttattaagagctcggtacccgg
V-iaaM-R	aaatgatcgtacatctccttgaattccatggtctgtttcctgt
amiE-F	aaggatcctctagagtcgacaaggagatgcgtcacggcgatatttc
amiE-R	ccgccaaaacagccaagcttttatcaggcctccttctccagtc
V-amiE-F	tggagaaggaggcctgataaaagcttggctgttttggcgg
V-amiE-R	gaaatatcgccgtgacgcatctccttgtcgactctagaggatcctt
ami1-F	aaggatcctctagagtcgacaaggagatggcaaccaataatgattt
ami1-R	ccgccaaaacagccaagcttttaaatgaatgctgccaga
V-ami1-F	gtctggcagcattcatttaaaagcttggctgttttggc
V-ami1-R	ttattggttgccatctccttgtcgactctagaggatcctt
tac-amiE-F	aaggatcctctagagtcgaccacagctaacaccacgtcgt
tac-amiE-R	tcgccgtgacgcatctccttggttaattcctcctgttacg
V-tac-amiE-F	cgtaacaggaggaattaaccaaggagatgcgtcacggcga
V-tac-amiE-R	acgacgtggtgttagctgtggtcgactctagaggatcctt
tac-ami1-F	aaggatcctctagagtcgaccacagctaacaccacgt
tac-ami1-R	ttattggttgccatctccttggttaattcctcctgttacg
V-tac-ami1-F	cgtaacaggaggaattaaccaaggagatggcaaccaataa
V-tac-ami1-R	acgacgtggtgttagctgtggtcgactctagaggatcctt
tdC-F	gatccgaagcagcggcaaaaaggaggatggatatcgaagcatttcg
tdC-R	ttgcatgcctgcaggtcgacttactgcacgtctttactaa
V-tdC-F	ttagtaaagacgtgcagtaagtcgacctgcaggcatgcaa
V-tdC-R	gcttcgatatccatcctcctttttgccgctgcttcggatc
aoc1-F	acctgcaggcatgcaagcttaaggagatgctgccgcatccg
aoc1-R	tctcatccgccaaaacagccttaaatatgggcattacgac
V-aoc1-F	gtcgtaatgcccatatttaaggctgttttggcggatgaga
V-aoc1-R	ggatgcggcagcatctccttaagcttgcatgcctgcaggt
iad1-F	ccatatttaaggctgttttgaaggagatgccgaccctgaatctg
iad1-R	gaaaatcttctctcatccgcttaaatcggtgccggct
V-iad1-F	gccagccggcaccgatttaagcggatgagagaagatttt
V-iad1-R	ttcagggtcggcatctccttcaaaacagccttaaatatgg
tac-aoc1-F	acctgcaggcatgcaagcttcacagctaacaccacgt
tac-aoc1-R	ggatgcggcagcatctccttggttaattcctcctgttacg
V- tac-aoc1-F	cgtaacaggaggaattaaccaaggagatgctgccgcatcc
V- tac-aoc1-R	acgacgtggtgttagctgtgaagcttgcatgcctgcaggt
tac-iad1-F	ccatatttaaggctgttttgcacagctaacaccacgtcgt
tac-iad1-R	ttcagggtcggcatctccttggttaattcctcctgttacg
V- tac-iad1-F	cgtaacaggaggaattaaccaaggagatgccgaccctgaa
V- tac-iad1-R	acgacgtggtgttagctgtgcaaaacagccttaaatatgg
tdc-F	gatccgaagcagcggcaaaaaggaggatgggcagcattgatagtac
tdc-R	ttgcatgcctgcaggtcgacttatgcttctttcagcagat
V- tdc-F	atctgctgaaagaagcataagtcgacctgcaggcatgcaa
V- tdc-R	tcaatgctgcccatcctcctttttgccgctgcttcggatc
Y-F	ataatgttttttgcgccgac
Y-R	atctgtatcaggctgaaaat

表2 引物序列

图2 通过吲哚-3-乙酰胺途径生成IAA

图3 通过TRP途径生成IAA

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