构建酿酒酵母细胞工厂从头合成倍半萜类化合物α-新丁香三环烯和β-石竹烯

doi:10.12211/2096-8280.2021-014

合成生物学 ›› 2021, Vol. 2 ›› Issue (5): 792-803.DOI: 10.12211/2096-8280.2021-014

构建酿酒酵母细胞工厂从头合成倍半萜类化合物α-新丁香三环烯和β-石竹烯

李晓东¹^,², 杨成帅³, 王平平¹, 严兴¹, 周志华¹

^1.中国科学院分子植物科学卓越创新中心，合成生物学重点实验室，上海　200032
^2.中国科学院大学，北京　100049
^3.中国科学院深圳先进技术研究院，深圳合成生物学创新研究院，中国科学院定量工程生物学重点实验室，广东　深圳　518055

收稿日期:2021-01-31 修回日期:2021-03-10 出版日期:2021-11-19 发布日期:2021-11-19
通讯作者: 周志华
作者简介:李晓东（1992—），男，博士研究生。研究方向为天然产物合成生物学。E-mail：lixiaodong@cemps.ac.cn
周志华（1966—），女，研究员，博士，博士生导师。研究方向为天然化合物合成生物学、丝状真菌分子生物学及基因组编辑技术等。E-mail：zhouzhihua@sippe.ac.cn
基金资助:
国家重点研发计划(2019YFA0904300);国家自然科学基金(31921006)

Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae

Xiaodong LI¹^,², Chengshuai YANG³, Pingping WANG¹, Xing YAN¹, Zhihua ZHOU¹

^1.CAS-key Laboratory of Synthetic Biology，Center for Excellence in Molecular Plant Sciences，Chinese Academy of Sciences，Shanghai 200032，China
^2.University of Chinese Academy of Sciences，Beijing 100049，China
^3.CAS Key Laboratory of Quantitative Engineering Biology，Shenzhen Institute of Synthetic Biology，Shenzhen Institutes of Advanced Technology，Chinese Academy of Sciences，Shenzhen 518055，Guangdong，China

Received:2021-01-31 Revised:2021-03-10 Online:2021-11-19 Published:2021-11-19
Contact: Zhihua ZHOU

摘要/Abstract

摘要：

倍半萜类化合物（sesquiterpenoids）α-新丁香三环烯（α-neoclovene）和β-石竹烯（β-caryophyllene）都是人参挥发油中的主要组成成分，不仅具有重要的药用开发价值，而且在高能量密度生物能源的开发中也受到关注。然而，它们在人参和其他植物中含量均十分稀少且难以分离纯化，其开发与应用研究严重滞后。本研究利用天然产物合成生物学的方法，以BY4742为出发菌株构建了高产倍半萜类化合物前体FPP的通用底盘菌株SQTBY03。在此底盘菌株中异源表达了密码子优化的植物内生真菌Hypoxylon sp. EC38来源的多产物倍半萜合酶元件基因ec38-cs和黄花蒿（Artemisia annua）来源的倍半萜合酶元件基因QHS1，分别构建了合成α-新丁香三环烯和β-石竹烯的两个倍半萜细胞工厂NCVBY01和CPLBY01。细胞工厂NCVBY01的主要倍半萜产物为α-新丁香三环烯（>39%），细胞工厂CPLBY01的主要产物为β-石竹烯（>96%）。它们的摇瓶发酵产量分别达到25.8 mg/L和250.4 mg/L。通过在1.3 L发酵罐中进行批次补料发酵，细胞工厂NCVBY01合成α-新丁香三环烯的产量达到了487.1 mg/L，为目前首次在微生物细胞工厂中合成α-新丁香三环烯；CPLBY01合成β-石竹烯的产量达到2949.1 mg/L，为目前报道的最高水平。上述研究成果为α-新丁香三环烯和β-石竹烯的规模化制备提供新的途径。

关键词: 倍半萜类, 酿酒酵母, 细胞工厂, α-新丁香三环烯, β-石竹烯

Abstract:

Sesquiterpenoids α-neoclovene and β-caryophyllene are major components in volatile oils from Panax ginseng, which have been demonstrated to play important roles in antibacteria, antitumor and cardiovascular protection. Moreover, they have attracted attentions for potential use as biofuels with high-energy-density. However, the industrial production of α-neoclovene and β-caryophyllene as well as other sesquiterpenoids are mainly relied on extraction from plant materials, which is too costly for applications at a large scale. Currently, this challenge could be addressed by advances in synthetic biology for natural product biosynthesis. Through heterologously assembling and integrating of their biosynthetic pathways into microbial chassis cells, targeted natural compounds from plants could be produced by microbial fermentation in a sustainable, low-cost and large-scale way. In this study, by comparing the production potential of sesquiterpenes between different Saccharomyces cerevisiae strainsand followed by enhancing the endogenous mevalonate pathway, a yeast sesquiterpene chassis strain (SQTBY03) with an increase of 458 times in farnesyl pyrophosphate production was constructed. Then by inserting the codon-optimized sesquiterpene synthase gene ec38-cs from the endophytic fungi Hypoxylon sp. EC38 and the codon-optimized caryophyllene synthase gene QHS1 from Artemisia annua into SQTBY03, respectively, we built yeast cell factories NCVBY01 and CPLBY01 for de novo production of α-neoclovene and β-caryophyllene at their titers of 25.8 mg/L and 250.4 mg/L, respectively, in shake flasks. Furthermore, fed-batch fermentation using NCVBY01 and CPLBY01 resulted in the de novo production of 487.1 mg/L α-neoclovene and 2949.1 mg/L β-caryophyllene from glucose. It is also possible to further chemically catalyze β-caryophyllene to produce α-neoclovene. Our work provides strategies for the sustainable production of α-neoclovene and β-caryophyllene from glucose through microbial fermentation, which would benefit their applications as medicine and other functional products. In addition, our yeast chassis for the sesquiterpene production could offer a platform for the sustainable production of other valuable sesquiterpenoids via synthetic biology approach.

Key words: sesquiterpenoids, Saccharomyces cerevisiae, cell factory, α-neoclovene, β-caryophyllene

中图分类号:

Q939.9

李晓东, 杨成帅, 王平平, 严兴, 周志华. 构建酿酒酵母细胞工厂从头合成倍半萜类化合物α-新丁香三环烯和β-石竹烯[J]. 合成生物学, 2021, 2(5): 792-803.

Xiaodong LI, Chengshuai YANG, Pingping WANG, Xing YAN, Zhihua ZHOU. Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae[J]. Synthetic Biology Journal, 2021, 2(5): 792-803.

图/表 10

参考文献 30

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Name	Genotype	Source
BY4742	MATa, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0	EUROSCARF
CEN.PK2-1	MATa, wra3-52, trp1-289, leu2-3_112, hisΔ1	EUROSCARF
SQTBY01	BY4742(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t)	this study
SQTCEN01	CEN.PK2-1(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t)	this study
BY4742Δ	BY4742(ΔGAL80)	this study
SQTBY02	BY4742Δ(deltaDNA:: GAL1p-ERG10-TDH2t, GAL10p-ERG13-HXT7t, GAL1p-tHMGR1-tHMGR1t, GAL10p-ERG12-PYK1t, GAL1p-ERG8-PDC1t)	this study
SQTBY03	SQTBY02(rDNA:: GAL1p-tHMGR1-CYC1t, GAL10p-ERG19-ADH1t, GAL1p-IDI1-ENO2t, GAL10p-ERG20-PGIt)	this study
NCVBY01	SQTBY03(X-4:: TEF1p-synec38-cs-PRM9t)	this study
CPLBY01	SQTBY03(X-4:: TEF1p-synQHS1-PRM9t)	this study

Name	Genotype	Source
BY4742	MATa, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0	EUROSCARF
CEN.PK2-1	MATa, wra3-52, trp1-289, leu2-3_112, hisΔ1	EUROSCARF
SQTBY01	BY4742(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t)	this study
SQTCEN01	CEN.PK2-1(rDNA:: TEF1p-ERG8-PRM9t, TEF2p-tHMGR1-ENO2t, PGK1p-ERG10-CYC1t)	this study
BY4742Δ	BY4742(ΔGAL80)	this study
SQTBY02	BY4742Δ(deltaDNA:: GAL1p-ERG10-TDH2t, GAL10p-ERG13-HXT7t, GAL1p-tHMGR1-tHMGR1t, GAL10p-ERG12-PYK1t, GAL1p-ERG8-PDC1t)	this study
SQTBY03	SQTBY02(rDNA:: GAL1p-tHMGR1-CYC1t, GAL10p-ERG19-ADH1t, GAL1p-IDI1-ENO2t, GAL10p-ERG20-PGIt)	this study
NCVBY01	SQTBY03(X-4:: TEF1p-synec38-cs-PRM9t)	this study
CPLBY01	SQTBY03(X-4:: TEF1p-synQHS1-PRM9t)	this study

Gene	Description	Accession No.
GAL80	Coding gene of transcription regulator	NM_001182409
ERG10	Coding gene of acetyl-CoA C-acetyltransferase	NM_001183842
ERG13	Coding gene of hydroxymethylglutaryl-CoA synthase	NM_001182489
tHMGR1	Coding gene of 3-hydroxy-3-methyl-glutaryl-CoA reductase	NM_001182434
ERG12	Coding gene of mevalonate kinase	NM_001182715
ERG8	Coding gene of phosphomevalonate kinase	NM_001182727
ERG19	Coding gene of diphosphomevalonate decarboxylase	NM_001183220
IDI1	Coding gene of isopentenyl-diphosphate delta-isomerase	NM_001183931
ERG20	Coding gene of (2E,6E)-farnesyl diphosphate synthase	NM_001181600
ec38-cs	Coding gene of caryophyllene synthase Hypoxylon sp. EC38	OTA59675
QHS1	Coding gene of β-caryophyllene synthase from Artemisia annua	AF472361

Gene	Description	Accession No.
GAL80	Coding gene of transcription regulator	NM_001182409
ERG10	Coding gene of acetyl-CoA C-acetyltransferase	NM_001183842
ERG13	Coding gene of hydroxymethylglutaryl-CoA synthase	NM_001182489
tHMGR1	Coding gene of 3-hydroxy-3-methyl-glutaryl-CoA reductase	NM_001182434
ERG12	Coding gene of mevalonate kinase	NM_001182715
ERG8	Coding gene of phosphomevalonate kinase	NM_001182727
ERG19	Coding gene of diphosphomevalonate decarboxylase	NM_001183220
IDI1	Coding gene of isopentenyl-diphosphate delta-isomerase	NM_001183931
ERG20	Coding gene of (2E,6E)-farnesyl diphosphate synthase	NM_001181600
ec38-cs	Coding gene of caryophyllene synthase Hypoxylon sp. EC38	OTA59675
QHS1	Coding gene of β-caryophyllene synthase from Artemisia annua	AF472361

Strains	FOH/（mg/L）	Squalene/（mg/L）	Lanosterol/（mg/L）
BY4742	0.177±0.011	0.5±0.07	3.1±0.57
SQTBY01	4.556±0.245	51.1±4.55	46.0±9.24
CEN.PK2-1	0.044±0.002	0.3±0.05	3.5±0.16
SQTCEN01	0.504±0.093	32.0±6.23	26.8±1.66

构建酿酒酵母细胞工厂从头合成倍半萜类化合物α-新丁香三环烯和β-石竹烯

Production of sesquiterpenoids α-neoclovene and β-caryophyllene by engineered Saccharomyces cerevisiae

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Retention time/min	Products	Mass spectra match factors/%	Area/%
11.538	(-)-cyperene	92	21.62
12.359	β-caryophyllene	95	8.52
12.752	humulene	97	2.53
12.932	AC1LBUZB	96	5.02
13.067	germacrene D	96	3.54
13.395	β-caryophyllene Fcc	86	18.87
16.865	α-neoclovene	89	39.90

Strain	Products	Yield/(mg/L)
NCVBY01	α-neoclovene	487.1
	(-)-cyperene	247.6
	β-caryophyllene Fcc	221.2
	β-caryophyllene	109.2
CPLBY01	β-caryophyllene	2949.1
CPLBY01	α-humulene	109.8

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