在大肠杆菌中从头生物合成3-苯丙醇

doi:10.12211/2096-8280.2021-098

摘要/Abstract

摘要：

3-苯丙醇是一种具有芳香味的高价值香料，在医药、化妆品、食品等领域有广泛用途，是生产多种药品和化学品的重要前体。其目前的生产方法主要依赖于植物提取和化学合成，存在产物得率低、生产周期长和环境不友好等缺点。为解决这些问题，构建微生物细胞工厂利用可再生资源合成3-苯丙醇具有重要的意义。本研究通过将目标化合物与微生物自身代谢网络建立联系，基于底物或中间体与产物的结构类似性以及化合物间的基团转移关系，设计并构建了两条不同的3-苯丙醇的人工生物合成途径。其中，依赖羧酸还原酶的苯丙醇生物合成途径具有较高的生产效率。在大肠杆菌中实现了以甘油为碳源，从头生物合成3-苯丙醇，产量达91 mg/L。通过消除限速步骤，增加莽草酸途径碳通量以及敲除竞争途径等代谢工程策略的实施，将苯丙醇的产量提高到了841 mg/L，较初始菌株产量提高了9.2倍，为苯丙醇的绿色、可持续、大规模生产提供了基础。

关键词: 3-苯丙醇, 代谢工程, 莽草酸途径, 人工途径设计

Abstract:

With the increasing consumption of fossil fuels and growing depravation of environment, development of substitutes for petroleum-derived compounds is becoming more and more important. In the past few years, bio-based production of chemicals, fuels, nutraceuticals and pharmaceuticals from renewable raw materials via metabolic engineering has gained significant attention. As a high-value fragrance with aromatic taste, 3-phenylpropanol has been widely used in the production of food additives, cosmetics and etc. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Because of its high efficiency in promoting bile secretion and mild antispasmodic function, 3-phenylpropanol is widely used in the treatment of cholecystitis, gallstones and biliary surgery syndrome. The current production method mainly relies on plant extraction and chemical synthesis, which, however, are challenging due to the high cost of catalyst, strict reaction condition and low product yield. Recently, engineering microorganisms has become an attractive alternative to efficient production of high-value compounds, such as flavors, fragrances, cosmetics, pharmaceuticals, solvents, biofuels and other chemicals. It is of great significance to construct a microbial cell factory to synthesize 3-phenylpropanol from renewable resources. In this study, we designed and constructed two different artificial 3-phenylpropanol biosynthetic pathways by establishing a connection between the target compound and the microorganism's own metabolic network. Especially, the pathway that relies on carboxylic acid reductase exhibited high efficiency in the production of 3-phenylpropanol. When using glycerol as the sole carbon source, the recombinant strain successfully generated 91 mg/L 3-phenylpropanol in shake flask experiments. By eliminating the rate-limiting steps, increasing the carbon flux towards the shikimate pathway and knocking out the competitive pathways, the titer of 3-phenylpropanol in the shake flask fermentation culture was finally increased to 841 mg/L, representing a 9.2-fold increase compared with the titer generated by the original strain. This work provides a green and sustainable approach for the production of 3-phenylpropanol.

Key words: phenylpropanol, metabolic engineering, the shikimate pathway, artificial pathway design

中图分类号:

Q812

高虎涛, 王佳, 孙新晓, 申晓林, 袁其朋. 在大肠杆菌中从头生物合成3-苯丙醇[J]. 合成生物学, 2021, 2(6): 1046-1060.

Hutao GAO, Jia WANG, Xinxiao SUN, Xiaolin SHEN, Qipeng YUAN. De novo biosynthesis of 3-phenylpropanol in E. coli[J]. Synthetic Biology Journal, 2021, 2(6): 1046-1060.

图/表 9

表1 本实验所用到的菌株

Tab. 1 Strains used in this study

Strains	Genotype	Source
Trans 5α		Lab Stock
BW25113(F′)		Lab Stock
BW25113(F′) ∆pykA ∆pykF		This study
G01	BW25113(F′) harboring pZE-CCR-4CL1	This study
G02	BW25113(F′) harboring pCS-ER	This study
G03	BW25113(F′) harboring pZE-CCR-4CL1 and pCS-ER	This study
G04	BW25113(F′) harboring pZE-ER	This study
G05	BW25113(F′) harboring pCS-Carsfp	This study
G06	BW25113(F′) harboring pZE-ER and pCS-Carsfp	This study
G07	BW25113(F′) harboring pZE-RgTAL-ER and pCS-Carsfp	This study
G08	BW25113(F′) harboring pZE-tac-RgTAL-ER-Carsfp	This study
G09	BW25113(F′)∆pykA∆pykF harboring pZE-tac-RgTAL-ER-Carsfp	This study
G10	BW25113(F′) harboring pCS-lac-APTA and pZE-tac-RgTAL-ER-Carsfp	This study
G11	BW25113(F′) harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp	This study
G12	BW25113(F′)∆pykA∆pykF harboring pCS-tac-APTA and pZE-tac-RgTAL-ER-Carsfp	This study

表2 本实验所用到的基础质粒

Tab. 2 Plasmids used in this study

Plasmids	Description	Source
pZE12-luc	pLlacO-1; luc; ColE1 ori; Ampr	Lab Stock
pCS27	pLlacO-1, P15A ori, Kanr	Lab Stock
pZE-CCR-4CL1	pZE-luc carrying CCR from Leucaena leucocephala, and 4CL1 from Arabidopsis thaliana	This study
pZE-ER	pZE-luc carrying ER from Clostridium acetobutylicum	This study
pCS-ER	pCS27 carrying ER from C. acetobutylicum	This study
pCS-carsfp	pCS27 carrying Car from Mycobacterium marinum and Sfp from Bacillus subtilis	This study
pZE-RgTAL-ER	pZE-luc carrying TAL from Rhodobacter glutinis and ER from C.acetobutylicum	This study

表3 本实验所用到的引物

Tab. 3 Primers used in this study

Primer	Sequence 5′-3′
CCR-4CL1-1-F-KpnI	gggaaaGGTACCatgcctgctgcggctccagc
CCR-4CL1-1-R-BamHI	gggaaaGGATCCttatttggtcggcagcggcaggtg
CCR-4CL1-2-F-BamHI	gggaaaGGATCCaggagatataccatggcgccacaagaacaagcagt
CCR-4CL1-2-R-XbaI	gggaaaTCTAGAttacaatccatttgctagttttgccctc
ER-KpnI-F	gggaaaGGTACCatgaacaaatacaagaaattatttgaaccaatcaaaattgg
ER-XbaI-F	gggaaaTCTAGAttatatatggtttgcaacttcaaaagcatccc
ER框-SpeI-F	gggaaaACTAGTaattgtgagcggataacaattgacattgtga
ER框-SacI-R	gggaaaGAGCTCacaacagataaaacgaaaggcccagtc
TAL框-SpeI-F	gggaaaACTAGTctcgagaattgtgagcggataacaattga
TAL框-SacI-R	gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc
Car-KpnI-F	gggaaaGGTACCatgtcacctatcacccgcgagg
Car-BamHI-R	gggaaaGGATCCtcacagcaagcccagcagac
sfp-BamHI-F	gggaaaGGATCCaggagatataccatgaagatttacggaatttatatgg
sfp-XbaI-R	GGGAAAtctagattataaaagctcttcgtacgagactattgtgat
AP-NheI-R	gggaaaGCTAGCttatttcttcagttcagccaggcttaacc
TA-NheI-F	gggaaaGCTAGCaggagatataccatgtcctcacgtaaagagcttg
APTA-XbaI-F	gggaaaTCTAGAatgacacaacctctttttctgatcggg
APTA-BamHI-R	gggaaaGGATCCttacccgcgacgcgctttta
pCS-tac-反-BamHI-F	gggaaaGGATCCgtcgccaatcacgcgtgaagagc
pCS-tac-反-XbaI-R	gggaaaCATATGttataaaagctcttcgtacgagacta
tac-Car框-F-SpeI	gggaaaACTAGTctcgagttgacaattaatcatcggctcg
tac-Car框-R-SacI	gggaaaGAGCTCcgacaaacaacagataaaacgaaaggcc

图1 3-苯丙醇合成途径的设计RgTALRgTAL—来自Rhodobacter glutinis的苯丙氨酸氨裂合酶；4CL1—来自Arabidopsis thaliana香豆酸辅酶A连接酶；CCR—来自Leucaena leucocephala的肉桂酰辅酶A还原酶；ER—来自Clostridium acetobutylicum的烯酸还原酶；Car—来自Mycobacterium marinum的羧酸还原酶；sfp—来自Bacillus subtilis的磷酸泛肽基转移酶

Fig. 1 Design of synthetic routes of 3-phenylpropanolRgTAL—phenylalanine ammonia lyase from Rhodobacter glutinis; 4CL1—from Arabidopsis thaliana coumarate-CoA ligase; CCR— cinnamoyl-CoA reductase from Leucaena leucocephala; ER—enoic acid reduction from Clostridium acetobutylicum Enzyme; Car—carboxylic acid reductase from Marine Mycobacterium; sfp—phosphoubiquitin transferase from Bacillus subtilis

图2 验证辅酶A依赖的还原途径1

Fig. 2 Validation of coenzyme A-dependent reduction pathway 1

图3 验证羧酸还原酶依赖的途径2

Fig. 3 Validation of carboxylic acid reductase-dependent pathway 2

图4 在菌株G07和菌株G08中从头合成3-苯丙醇

Fig. 4 De novo production of 3-phenylpropanol in strains G07 and G08

图5 pykA/F敲除对3-苯丙醇产量的影响DHAP—磷酸二羟丙酮；PYR—丙酮酸；PEP—磷酸烯醇式丙酮酸；E4P—D-赤藓糖-4-磷酸；DAHP—3-脱氧-D-阿拉伯糖庚酸七磷酸酯；TktA—转酮酶；PykA/F—丙酮酸激酶；PpsA—磷酸烯醇式丙酮酸合酶；AroF/AroGfbr/AroH—3-脱氧-D -阿拉伯糖庚酸七磷酸酯合酶

Fig. 5 Production of 3-phenylpropanol in pykA/F knockout strainDHAP—dihydroxyacetone phosphate; PYR—pyruvate; PEP—phosphoenolpyruvate; E4P—D-erythrose-4-phosphate; DAHP—3-deoxy-D-arabinoheptanoate heptaphosphate; TktA—Transketolase; PykA/F—pyruvate kinase; PpsA—phosphoenolpyruvate synthase; AroF/AroGfbr/AroH—3-deoxy-D-arabinoheptanoate heptaphosphate synthase

图6 增强莽草酸途径对3-苯丙醇产量的影响DHQ—3-脱氢奎尼酸；DHS—3-脱氢莽草酸；SHK—莽草酸；CHOR—分支酸；AroB—脱氢奎尼酸合成酶；AroD—脱氢奎尼酸脱水酶；AroE—莽草酸脱氢酶；AroK/AroL/AroA/AroC—脱氢莽草酸脱水酶；PheA—预苯酸脱水酶

Fig. 6 Strengthening of shikimate pathway on production of 3-phenylpropanolDHQ—3-dehydroquinic acid; DHS—3-dehydroshikimate; SHK—shikimic acid; CHOR—chorismate; AroB—dehydroquinic acid synthase; AroD—dehydroquinic acid dehydratase, AroE—Shikimate dehydrogenase; AroK/AroL/AroA/AroC—dehydroshikimate dehydratase; PheA—prephenate dehydrogenase

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