毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸<sup>*</sup>

doi:10.13523/j.cb.2106019

中国生物工程杂志

2021, Vol. 41

Issue (10): 52-61 DOI: 10.13523/j.cb.2106019

技术与方法

毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*

陈鑫洁¹,钱芷兰¹,刘启¹,赵清²,张元兴¹,蔡孟浩^1,^**(

)

1 华东理工大学生物反应器工程国家重点实验室上海 200237
2 中国科学院上海辰山植物科学研究中心上海市资源植物功能基因组学重点实验室上海 201602

Modification of Aromatic Amino Acid Synthetic Pathway in Pichia pastoris to Produce Cinnamic Acid and ρ-Coumaric Acid

CHEN Xin-jie¹,QIAN Zhi-lan¹,LIU Qi¹,ZHAO Qing²,ZHANG Yuan-xing¹,CAI Meng-hao^1,^**(

)

1 State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China
2 Plant Science Research Center, Shanghai Chenshan Botanical Garden, Shanghai Key Laboratoryof Plant Functional Genomics and Resources, Shanghai 201602, China

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摘要：

目的:改造毕赤酵母使其异源合成类黄酮生物合成途径的重要中间体肉桂酸、对香豆酸,并优化前体芳香族氨基酸生物合成途径以提高毕赤酵母的生产能力。方法:在毕赤酵母GS115中利用乙醇诱导型人工转录系统表达Rhodotorula glutinis来源的苯丙氨酸解氨酶,并在该重组菌株中分别过表达胞内芳香族氨基酸生物合成途径中的关键酶或其突变体以进行优化。结果:异源表达苯丙氨酸解氨酶可使毕赤酵母将自身产生的L-苯丙氨酸、L-酪氨酸转化为肉桂酸(38.8 mg/L)、对香豆酸(34.2 mg/L),而通过过表达相关酶进行优化,最终肉桂酸和对香豆酸的产量分别达到124.1 mg/L和302.0 mg/L。结论:利用新的异源宿主毕赤酵母成功合成了肉桂酸、对香豆酸,并对胞内的芳香族氨基酸生物合成途径进行了优化,表明毕赤酵母具有生产黄酮类化合物的应用潜力,也为其他芳香族氨基酸衍生物或植物化合物在毕赤酵母中的异源合成奠定了基础。

关键词： 毕赤酵母; 芳香族氨基酸; 肉桂酸; 对香豆酸

Abstract:

Objective: The Pichia pastoris strain was engineered to heterologously synthesize cinnamic acid and ρ-coumaric acid,which were important intermediates of flavonoid biosynthetic pathway. The biosynthetic pathway of precursors aromatic amino acids was optimized to improve the production capacity of P. pastoris. Methods: Phenylalanine ammonia lyase from Rhodotorula glutinis was expressed in P. pastoris GS115 by ethanol induced artificial transcription system, and the key enzymes or their mutants in the biosynthetic pathway of intracellular aromatic amino acids were overexpressed in the recombinant strain. Results: Heterologous expression of phenylalanine ammonia lyase could convert L-phenylalanine and L-tyrosine produced by P. pastoris into cinnamic acid (38.8 mg/L) and ρ-coumaric acid (34.2 mg/L). Through overexpression of related enzymes, the yields of cinnamic acid and ρ-coumaric acid reached 124.1 mg/L and 302.0 mg/L, respectively. Conclusion: Cinnamic acid and ρ-coumaric acid were successfully synthesized by P. pastoris, and the biosynthetic pathway of intracellular aromatic amino acids was optimized. It shows that P. pastoris has the application potential to produce flavonoids, and it also lays a foundation for the heterologous synthesis of other aromatic amino acid derivatives or plant compounds in P. pastoris.

Key words: Pichia pastoris Aromatic amino acid Cinnamic acid ρ-Coumaric acid

收稿日期: 2021-06-11 出版日期: 2021-11-08

ZTFLH:

Q789

基金资助: * 国家重点研发计划(2018YFC1706202)

通讯作者: 蔡孟浩 E-mail: cmh022199@ecust.edu.cn

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引用本文:

陈鑫洁,钱芷兰,刘启,赵清,张元兴,蔡孟浩. 毕赤酵母底盘芳香族氨基酸合成途径改造生产肉桂酸及对香豆酸^*[J]. 中国生物工程杂志, 2021, 41(10): 52-61.

CHEN Xin-jie,QIAN Zhi-lan,LIU Qi,ZHAO Qing,ZHANG Yuan-xing,CAI Meng-hao. Modification of Aromatic Amino Acid Synthetic Pathway in Pichia pastoris to Produce Cinnamic Acid and ρ-Coumaric Acid. China Biotechnology, 2021, 41(10): 52-61.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2106019 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I10/52

Primers	Sequence (5'→3')
sRgPAL_kz_F	CAACTAATTATTCGAACTCGAGTTCGAAATGGCTCCTTCTGT
sRgPAL_kz_R	AACTCAATGATGATGATGATGATGGTCGACAGGTACCATGGCCATCATC
ICL1-KZ-F	GAGACCTTCGTTTGTGCGGATCCAGATCTTCATCTAACACTTTGTATAGC
ICL1-KZ-R	GCTATGGTGTGTGGGGGATCCTCTCACTTAATCTTCTGTACTCTGAAGAG
DAHPS1-F	AATCAATTGAACAACTATTTCGAAATGACCTCCACACCAGTTCAAGAAGAATACG
DAHPS1-R	AACTCAATGATGATGATGATGATGGTCGACAGCAGCGTTCTTTAATGCTCTT
DAHPS2-F	AATCAATTGAACAACTATTTCGAAATGACAGTACAAGAGGTCGACC
DAHPS2-R	AACTCAATGATGATGATGATGATGGTCGACTGTAATGAGTTTCAATGAATTACTTATTTTTACCTCT
DAHPS3-F	AATCAATTGAACAACTATTTCGAAATGTTCATTCAAAACGATCATGTCG
DAHPS3-R	AACTCAATGATGATGATGATGATGGTCGACATTCTTGAGATTACGACGTTCAATGAC
CM-F	AATCAATTGAACAACTATTTCGAAATGGAGTTCAAGAAACCCGC
CM-R	AACTCAATGATGATGATGATGATGGTCGACCCACAAGCTGTTGGATAATAGTCT
PD-F	AATCAATTGAACAACTATTTCGAAATGACTAATATAGCATATTTGGGGCCCCAGGGAACGTATTC
PD-R	AACTCAATGATGATGATGATGATGGTCGACGTCCCAGTACTTTTTGGACCG
1_ARO3_KZ_F	AATCAATTGAACAACTATTTCGAAATGTTCATTAAAAACGATCACGCC
2_ARO3_KZ_R	AACTCAATGATGATGATGATGATGGTCGACTTTTTTCAAGGCCTTTCTTCTGTTTCT
1_ARO4_KZ_F	AATCAATTGAACAACTATTTCGAAATGAGTGAATCTCCAATGTTCGC
2_ARO4_KZ_R	AACTCAATGATGATGATGATGATGGTCGACTTTCTTGTTAACTTCTCTTCTTTGTCTGAC
1_ARO7_KZ_F	AATCAATTGAACAACTATTTCGAAATGGATTTCACAAAACCAGAAACTG
2_ARO7_KZ_R	AACTCAATGATGATGATGATGATGGTCGACCTCTTCCAACCTTCTTAGCAAGTAT
4ARO3~K222L-KZ-F		TTCTGTCACATTGCCAGGTGTCACTGCTATCGTGGGCAC
GAPZa-KZ-R		TGGCCTTTTGCTCACATGTTGGTCTCCAGCTTGCAAATTAAAGC
GAPZa-KZ-F		AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG
3ARO3~K222L-KZ-R		CACCTGGCAATGTGACAGAAAGGAAGTAATGTTCATGTGCAGC
4ARO4~K229L-KZ-F		ACTTTGCATGGTGTTGCTGCTATCACCACTACTAAGGGTAACGAACA
3ARO4~K229L-KZ-R		GCAGCAACACCATGCAAAGTAACACCCATGAAATGGTGAGAAT
4ARO7~G141S-KZ-F		AACTTCTCTTCTGTTGCCACTAGAGATATAGAATGTTTGCAAAGCTTGAG
3ARO7~G141S-KZ-R		GTGGCAACAGAAGAGAAGTTATTCTTATCATCACCATCTCTTTTCGAAAT
DAHPS3~K222L-KZ-F		CATTACCAGGAGTTGTGGCTATTGTCGGTACCGAAGGAAATG
DAHPS3~K222L-KZ-R		AGCCACAACTCCTGGTAATGTGACAGACAGGAAATGGTGAGG
BamH IupGAP-KZ-F		GTACGCTGCAGGTCGACGGATCAGGTCATGCATGAGATCAGATCT
AOX1TT-KZ-R		GCACAAACGAAGGTCTCACTTAA
TTupGAP-KZ-F		TTAAGTGAGACCTTCGTTTGTGCGGATTTTGGTCATGCATGAGATC
doTTBgl II-KZ-R		AAGGCAAGCTAAACAGATCTGGCGCGCCAGAAACATTTTGAAGCTATGGTGTG
TTBamH IupGAP-KZ-F		ACCTTCGTTTGTGCGGATCGGTCATGCATGAGATCAGATCT
AOXITT(BamH I)-KZ-R		TTGAAGCTATGGTGTGTGGGGGATCCGCACAAACGAAGGTCTCACTT
TTBamH IupGAP-KZ-F2		ACCTTCGTTTGTGCGGATCCCGAAAACTCACGTTAAGGGATTTT
AOXITT(BamH I)-KZ-R2		TTGAAGCTATGGTGTGTGGGGGATCCTCTGGAAGAGTAAAAAAGGAGTAGAAACAT
CM-R		AACTCAATGATGATGATGATGATGGTCGACCCACAAGCTGTTGGATAATAGTCT
PD-F		AATCAATTGAACAACTATTTCGAAATGACTAATATAGCATATTTGGGGCCCCAGGGAACGTATTC
PD-R		AACTCAATGATGATGATGATGATGGTCGACGTCCCAGTACTTTTTGGACCG
1_ARO3_KZ_F		AATCAATTGAACAACTATTTCGAAATGTTCATTAAAAACGATCACGCC
2_ARO3_KZ_R		AACTCAATGATGATGATGATGATGGTCGACTTTTTTCAAGGCCTTTCTTCTGTTTCT

表1 本研究所用引物

表2 HPLC检测梯度洗脱程序

图1 重组毕赤酵母合成肉桂酸及对香豆酸的胞内代谢途径

图2 毕赤酵母菌株GS115及P.p/ESAD-Rg摇瓶诱导96h发酵液样品的HPLC检测

图3 毕赤酵母菌株P.p/ESAD-Rg在不同浓度的芳香族氨基酸作为前体时的摇瓶发酵结果

图4 过表达毕赤酵母来源的关键酶对芳香族氨基酸合成途径的优化效果

图5 过表达酿酒酵母来源的关键酶的突变体对芳香族氨基酸合成途径的优化效果

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