产蒎烯人工酵母细胞的构建

doi:10.11949/j.issn.0438-1157.20180784

摘要/Abstract

摘要：

蒎烯可衍生为高能量密度燃料，但在酿酒酵母中的全生物合成却未见报道。酿酒酵母由于拥有强大的蛋白表达和翻译后修饰系统以及完整的内膜系统，相比于大肠杆菌等原核生物更适于P450等蛋白的表达，因此将酿酒酵母作为宿主细胞，对于蒎烯或者其他物质实现如“疯狂碳环”的高能量化是至关重要的。本研究在酿酒酵母底盘中表达内源焦磷酸香叶酯合成酶（ERG20）的突变体ERG20^ww和火炬松来源的蒎烯合酶（PtPS）构建了蒎烯的合成路径。通过截短PtPS N端2～51位氨基酸残基（tPtPS），蒎烯产量较初始产量（0.329 mg·L^-1）提高了2.23倍。在过表达异戊二烯焦磷酸异构酶（IDI1）和RNA聚合酶Ш负调控因子（MAF1）的基础上，表达ERG20^ww和tPtPS的融合蛋白，蒎烯产量进一步提高了5.16倍。通过将内源基因ERG20启动子原位替换为弱启动子HXT1，下调ERG20的转录，蒎烯的产量提高了26.0%。最终通过调节发酵过程中的培养基pH使蒎烯产量达11.7 mg·L^-1，较初始产量提高了34.5倍。本研究在酿酒酵母中实现蒎烯的从头合成，并获得已知蒎烯摇瓶水平的最高产量。

关键词: 代谢工程, 合成生物学, 蒎烯, 酿酒酵母

Abstract:

The derivatives of pinene can be employed as high energy density fuels. However, the biosynthesis of pinene from single carbon sources has not been realized in yeast. As Saccharomyces cerevisiae has stronger ability on protein expression and post-translational modification as well as processes a maturity endomembrane system, it is more suitable for expressing complex proteins (e.g. cytochrome P450) by yeast than by prokaryotic hosts such as Escherichia coli. Therefore, it is crucial to engineer S. cerevisiae as the host cell to produce high energy density fuels (like “Crazy Carbon Ring”) based on the derivatization of pinene or other terpenes compounds. Here, in order to achieve pinene synthesis in yeast, endogenous farnesyl diphosphate synthase (ERG20) mutant ERG20^ww and Pinus taeda pinene synthase (PtPS) were expressed in Saccharomyces cerevisiae strain, obtaining an initial pinene titer of 0.329 mg·L^-1. N-terminus truncation (from 2A to 51P) of PtPS (obtaining tPtPS) improve the pinene production by 2.23-fold. The pinene titer was further enhanced by 5.16-fold by expression the fusion of ERG20^ww/tPtPS on the basis of overexpression of isoprene pyrophosphate isomerase (IDI1) and the repressor of RNA polymerase III (MAF1). Replacing the promoter of gene ERG20 by a weaker promoter HXT1 down-regulated the transcription of ERG20. And correspondingly the pinene output was increased by 26.0%. Eventually, adjusting the pH of the fermentat-ion medium further increased the pinene production by 42.2% (to 11.7 mg·L^-1). This titer was 34.5-fold higher than the initial one. This study is the first to achieve de novo synthesis of terpenes in Saccharomyces cerevisiae and to obtain the highest yield of known terpene shake flasks.

Key words: metabolic engineering, synthetic biology, pinene, Saccharomyces cerevisiae

中图分类号:

Q 819

陈天华, 张若思, 姜国珍, 姚明东, 刘宏, 王颖, 肖文海, 元英进. 产蒎烯人工酵母细胞的构建[J]. 化工学报, 2019, 70(1): 179-188.

Tianhua CHEN, Ruosi ZHANG, Guozhen JIANG, Mingdong YAO, Hong LIU, Ying WANG, Wenhai XIAO, Yingjin YUAN. Metabolic engineering of Saccharomyces cerevisiae for pinene production[J]. CIESC Journal, 2019, 70(1): 179-188.

图/表 10

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Fuels	Density/(g·ml^-1)	Heating value/(MJ·L^-1)
pinene dimers	0.938	39.5
JP-10	0.94	39.6
RJ-5	1.08	44.9

Fuels	Density/(g·ml^-1)	Heating value/(MJ·L^-1)
pinene dimers	0.938	39.5
JP-10	0.94	39.6
RJ-5	1.08	44.9

Plasmids/strains	Description	Source
plasmids
pRS425K	shuttle plasmid for E. coli and S. cerevisiae, Kan^r, LEU2	this laboratory
pRS424	shuttle plasmid for E. coli and S. cerevisiae, Amp, TRP3	this laboratory
PRS425K_LDJ04	pRS425K harboring cassette GPM1t_GAL7p_GPDt	this laboratory
PRS425K_LDJ09	pRS425K harboring cassette GPDt_GAL1p_FBA1t	this laboratory
PRS425K_LDJ10	pRS425K harboring cassette FBA1t_GAL7p_PGK1t	this laboratory
PRS425K_LDJ14	pRS425K harboring cassette PGK1t_GAL3p_CYC1t	this laboratory
PRS424_LDJ04	pRS424 harboring cassette GPM1t_GAL7p_GPDt	this study
ZRS1	pRS424 harboring cassette GPM1t_GAL7p_PtPS_GPDt	this study
ZRS2	pRS425K harboring cassette GPDt_GAL1p_ERG20^ww _FBA1t	this study
ZRS3	pRS424 harboring cassette GPM1t_GAL7p_tPtPS_GPDt	this study
ZRS4	pRS424 harboring cassette GPM1t_GAL7p_ERG20^ww -tPtPS_GPDt	this study
ZRS5	pRS425K harboring cassette FBA1t_GAL7p_IDI1_PGK1t	this study
ZRS6	pRS425K harboring cassette PGK1t_GAL3p_MAF1_CYC1t	this study
ZRS7	pRS425K harboring cassette GPDt_GAL1p_ERG20^ww _FBA1t_GAL7p _IDI1_PGK1t_GAL3p_MAF1_CYC1t	this study
*S. cerevisiae* strains
YJGZ1	MAT a; URA3-52, TRP1-289, LEU2-3,112, HIS3Δ1, MAL2-8C, SUC2, GAL80 :: IDI1_GAL1,10p_tHMGR	this laboratory
YJGZ1_HXT1	YJGZ1, ERG20p_ERG20 :: HXT1p_ERG20	this study
SyBE_Sc02090001	YJGZ1, ZRS1, ZRS2	this study
SyBE_Sc02090002	YJGZ1, ZRS3, ZRS2	this study
SyBE_Sc02090003	YJGZ1, ZRS4, ZRS2	this study
SyBE_Sc02090004	YJGZ1, ZRS3, ZRS7	this study
SyBE_Sc02090005	YJGZ1, ZRS4, ZRS7	this study
SyBE_Sc02090006	YJGZ1_HXT1, ZRS4, ZRS7	this study

Plasmids/strains	Description	Source
plasmids
pRS425K	shuttle plasmid for E. coli and S. cerevisiae, Kan^r, LEU2	this laboratory
pRS424	shuttle plasmid for E. coli and S. cerevisiae, Amp, TRP3	this laboratory
PRS425K_LDJ04	pRS425K harboring cassette GPM1t_GAL7p_GPDt	this laboratory
PRS425K_LDJ09	pRS425K harboring cassette GPDt_GAL1p_FBA1t	this laboratory
PRS425K_LDJ10	pRS425K harboring cassette FBA1t_GAL7p_PGK1t	this laboratory
PRS425K_LDJ14	pRS425K harboring cassette PGK1t_GAL3p_CYC1t	this laboratory
PRS424_LDJ04	pRS424 harboring cassette GPM1t_GAL7p_GPDt	this study
ZRS1	pRS424 harboring cassette GPM1t_GAL7p_PtPS_GPDt	this study
ZRS2	pRS425K harboring cassette GPDt_GAL1p_ERG20^ww _FBA1t	this study
ZRS3	pRS424 harboring cassette GPM1t_GAL7p_tPtPS_GPDt	this study
ZRS4	pRS424 harboring cassette GPM1t_GAL7p_ERG20^ww -tPtPS_GPDt	this study
ZRS5	pRS425K harboring cassette FBA1t_GAL7p_IDI1_PGK1t	this study
ZRS6	pRS425K harboring cassette PGK1t_GAL3p_MAF1_CYC1t	this study
ZRS7	pRS425K harboring cassette GPDt_GAL1p_ERG20^ww _FBA1t_GAL7p _IDI1_PGK1t_GAL3p_MAF1_CYC1t	this study
*S. cerevisiae* strains
YJGZ1	MAT a; URA3-52, TRP1-289, LEU2-3,112, HIS3Δ1, MAL2-8C, SUC2, GAL80 :: IDI1_GAL1,10p_tHMGR	this laboratory
YJGZ1_HXT1	YJGZ1, ERG20p_ERG20 :: HXT1p_ERG20	this study
SyBE_Sc02090001	YJGZ1, ZRS1, ZRS2	this study
SyBE_Sc02090002	YJGZ1, ZRS3, ZRS2	this study
SyBE_Sc02090003	YJGZ1, ZRS4, ZRS2	this study
SyBE_Sc02090004	YJGZ1, ZRS3, ZRS7	this study
SyBE_Sc02090005	YJGZ1, ZRS4, ZRS7	this study
SyBE_Sc02090006	YJGZ1_HXT1, ZRS4, ZRS7	this study

Primer name	Sequence(5'-3')
MAF1-F	GGTCTCCAATGAAAGTATGTTATCACTCTAAAACTG
MAF1-R	GGTCTCCTTTACTGTAGGGATTCTTCTTGATCTG
IDI1-BsaI-F	GGTCTCCAATGACTGCCGACAACAATAG
IDI1-BsaI-R	GGTCTCCTTTATAGCATTCTATGAATTTGCCTG
tPtPS-BsaI-F	GGTCTCCAATGAGAGGTGGTAAATCTATTGCACC
tPtPS-BsaI-R	GGTCTCCTTTATAATGGAACAGTTTCAACAACTGTTCTAG
ERG20^ww-GS-F	GGTCTCCATAAGAATGCGGCCGCGGTCTCCAATGGCTTCAGAAAAAGAAATTAGG
GS-ERG20^ww-R	GCTACCGCTACCGCTGCCGCTACCTTTGCTTCTCTTGTAAACTTTGTTCAAG
GS-tPtPS-F	GGTAGCGGCAGCGGTAGCGGTAGCATGGCTAGAAGAATTGCTGGTCATCATTC
tPtPS-GS-R	GGTCTCCATAAGAATGCGGCCGCGGTCTCCTTTATAATGGAACAGTTTCAACAACTGTTC
P_HXT1-F	GTGATATCAGATCCACTAGTGGCCTATGCGTGCAGGTCTCATCTGGAATATAATTCC
P_HXT1-R	CTCTCTCCTAATTTCTTTTTCTGAAGCCATGATTTTACGTATATCAACTAGTTGAC
URA3-Cre-loxp-F	ATTCTTTTTTCAATAGTCGAAGTCAGCTTCAGCTGAAGCTTCGTACGCTGCAGGTC
URA-Cre-loxp-R	AGATGAGACCTGCAGCATAGGCCACTAGTGGATCTGATATCACCTAATAACTTCG
Actin-F	GCCTTGGACTTCGAACAAGA
Actin-R	CCAAACCCAAAACAGAAGGA
ERG20-F	GCTATACCACGAATATGAAG
ERG20-R	GAACGCAGTTAAGACATC