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

doi:10.13523/j.cb.2106019

China Biotechnology

2021, Vol. 41

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

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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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

Received: 11 June 2021 Published: 08 November 2021

ZTFLH:

Q789

Corresponding Authors: Meng-hao CAI E-mail: cmh022199@ecust.edu.cn

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	Xin-jie CHEN
	Zhi-lan QIAN
	Qi LIU
	Qing ZHAO
	Yuan-xing ZHANG
	Meng-hao CAI

Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2106019 OR https://manu60.magtech.com.cn/biotech/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

Table 1 Primers used in this study

Table 2 HPLC Gradient elution procedure

Fig.1 Intracellular metabolic pathway of cinnamic acid and ρ-coumaric acid in recombinant Pichia pastoris The heterologous expression of Rhodotorula glutinis phenylalanine ammonia lyase (RgPAL) in Pichia pastoris GS115 can catalyze the transformation of endogenous aromatic amino acids into cinnamic acid (CA) and ρ-coumaric acid (ρ-CA) (green). Then we overexpressed key enzymes (or their mutants) from P. pastoris (purple) and Saccharomyces cerevisiae (orange) in the biosynthetic pathway of aromatic amino acids, respectively, to improve the production capacity of recombinant engineering strain. DAHPS1/2/3: DAHPS synthetase, ARO3^K222L/4^K229L: feedback inhibition insensitive mutant of DAHPS synthetase, CM: chorismate mutase, ARO7^G141S: feedback inhibition insensitive mutant of chorismate mutase, Glycolysis: glycolysis pathway, PPP: pentose phosphate pathway, PEP: phosphoenolpyruvate, E4P: erythritose 4-phosphate, DAHP: 3-deoxy-D-arabino-heptulosonate 7-phosphate, EPSP: 5-enolpyruvylshikimate-3-phosphate, CHA: chorismate acid, L-Trp: L-tryptophan, PPA: prephenate, HPP: phenylpyruvate, PPY: 4-hydroxyphenylpyruvate, L-Phe: L-phenylalanine, L-Tyr: L-tyrosine

Fig.2 HPLC analysis of the standards of cinnamic acid, ρ-coumaric acid and their mixture,as well as the sample of GS115 and P.p/ESAD-Rg strains of Pichia pastoris The detection wavelength was at 278 nm (a) for cinnamic acid (CA) and 310 nm (b) for ρ-coumaric acid (ρ-CA) respectively. (1) CA standard; (2) Mixture of CA and ρ-CA at 278 nm; (3) Culture extract of GS115 for CA; (4) Culture extract of P.p/ESAD-Rg for CA; (5) ρ-CA standard; (6) Mixture of CA and ρ-CA at 310 nm; (7) Culture extract of GS115 for ρ-CA; (8) Culture extract of P.p/ESAD-Rg for ρ-CA

Fig.3 Shake flask fermentation of Pichia pastoris P.p/ESAD-Rg strain in YNE medium with different concentrations of L-phenylalanine (0, 0.5, 1, 1.5 g/L) or L-tyrosine (0, 0.5, 1, 1.5 g/L) as a precursor (a) Growth when L-phenylalanine was the precursor (b) Cinnamic acid (CA) production (c) Growth when L-tyrosine was the precursor (d) ρ-Coumaric acid (ρ-CA) production. Statistical significance of cinnamic acid production and ρ-coumaric acid production at different concentrations of precursors compared with no precursor at two time points is shown (^** P< 0.01, ^* P< 0.05 at 72 h; ⁺⁺ P< 0.01, ⁺ P< 0.05 at 96 h; n.s., not signifcance)

Fig.4 Optimization of aromatic amino acids biosynthetic pathway by overexpression of endogenous enzymes of Pichia pastoris Growth (a), cinnamic acid production (b) and ρ-coumaric acid production (c) in shake flask fermentation of P.p/ESAD-Rg, P.p/IRg-DAHPS1, P.p/IRg-DAHPS2, P.p/IRg-DAHPS3, P.p/IRg-CM, P.p/IRg-PD strains of P. pastoris in YNE medium. Statistical significance of cinnamic acid production and ρ-coumaric acid production of each strain overexpressing endogenous enzymes compared with strain P.p/ESAD-Rg at two time points is shown (^** P< 0.01, ^* P< 0.05 at 72 h; ⁺⁺ P< 0.01, ⁺ P< 0.05 at 96 h; n.s., not signifcance)

Fig.5 Optimization of aromatic amino acids biosynthetic pathway by overexpression mutants of key enzymes of Saccharomyces cerevisiae Growth (a), cinnamic acid production (b) and ρ-coumaric acid production (c) in shake flask fermentation of P.p/ESAD-Rg, P.p/IRg-4^fbr7^fbr, P.p/IRg-D3^fbr4^fbr7^fbr and P.p/IRg-A3^fbr4^fbr7^fbr strains of P. pastoris in YNE medium. Statistical significance of cinnamic acid production and ρ-coumaric acid production of each strain overexpressing mutants of key enzymes compared with strain P.p/ESAD-Rg at two time points is shown (^** P< 0.01, ^* P< 0.05 at 72 h; ⁺⁺ P< 0.01, ⁺ P< 0.05 at 96 h; n.s., not signifcance)


[1]	郭欣慰, 黄丛林, 吴忠义, 等. 植物类黄酮生物合成的分子调控. 北方园艺, 2011(4): 204-207.

[1]	Guo X W, Huang C L, Wu Z Y, et al. Molecular regulation of plant flavonoid biosynthesis pathway. Northern Horticulture, 2011(4): 204-207.

[2]	Wang L, Song J K, Liu A L, et al. Research progress of the antiviral bioactivities of natural flavonoids. Natural Products and Bioprospecting, 2020, 10(5): 271-283. doi: 10.1007/s13659-020-00257-x pmid: 32948973

[3]	Liu Q L, Yu T, Li X W, et al. Rewiring carbon metabolism in yeast for high level production of aromatic chemicals. Nature Communications, 2019, 10(1): 4976. doi: 10.1038/s41467-019-12961-5

[4]	Li J H, Tian C F, Xia Y H, et al. Production of plant-specific flavones baicalein and scutellarein in an engineered E. coli from available phenylalanine and tyrosine. Metabolic Engineering, 2019, 52: 124-133. doi: 10.1016/j.ymben.2018.11.008

[5]	Williams I S, Chib S, Nuthakki V K, et al. Biotransformation of chrysin to baicalein: selective C6-hydroxylation of 5, 7-dihydroxyflavone using whole yeast cells stably expressing human CYP1A1 enzyme. Journal of Agricultural and Food Chemistry, 2017, 65(34): 7440-7446. doi: 10.1021/acs.jafc.7b02690 pmid: 28782952

[6]	Liu X N, Cheng J, Zhu X X, et al. De novo biosynthesis of multiple pinocembrin derivatives in Saccharomyces cerevisiae. ACS Synthetic Biology, 2020, 9(11): 3042-3051. doi: 10.1021/acssynbio.0c00289

[7]	Gasser B, Mattanovich D. A yeast for all seasons-Is Pichia pastoris a suitable chassis organism for future bioproduction. FEMS Microbiology Letters, 2018, 365(17): 181.

[8]	Winkel-Shirley B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiology, 2001, 126(2): 485-493. pmid: 11402179

[9]	Braus G H. Aromatic amino acid biosynthesis in the yeast Saccharomyces cerevisiae: a model system for the regulation of a eukaryotic biosynthetic pathway. Microbiological Reviews, 1991, 55(3): 349-370. doi: 10.1128/mr.55.3.349-370.1991 pmid: 1943992

[10]	Liu X Z, Niu H, Li Q, et al. Metabolic engineering for the production of l-phenylalanine in Escherichia coli. 3 Biotech, 2019, 9(3): 85. doi: 10.1007/s13205-019-1619-6

[11]	Liu Y F, Xu Y R, Ding D Q, et al. Genetic engineering of Escherichia coli to improve L-phenylalanine production. BMC Biotechnology, 2018, 18(1): 5. doi: 10.1186/s12896-018-0418-1

[12]	Reifenrath M, Boles E. Engineering of hydroxymandelate synthases and the aromatic amino acid pathway enables de novo biosynthesis of mandelic and 4-hydroxymandelic acid with Saccharomyces cerevisiae. Metabolic Engineering, 2018, 45: 246-254. doi: S1096-7176(17)30407-X pmid: 29330068

[13]	Hassing E J, de Groot P A, Marquenie V R, et al. Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae. Metabolic Engineering, 2019, 56: 165-180. doi: 10.1016/j.ymben.2019.09.011

[14]	Liu Y Q, Bai C X, Liu Q, et al. Engineered ethanol-driven biosynthetic system for improving production of acetyl-CoA derived drugs in Crabtree-negative yeast. Metabolic Engineering, 2019, 54: 275-284. doi: 10.1016/j.ymben.2019.05.001

[15]	Chen Y C, Shen S C, Chow J M, et al. Flavone inhibition of tumor growth via apoptosis in vitro and in vivo. International Journal of Oncology, 2004, 25(3): 661-670.

[16]	Way T D, Kao M C, Lin J K. Apigenin induces apoptosis through proteasomal degradation of HER2/neu in HER2/neu-overexpressing breast cancer cells via the phosphatidylinositol 3-kinase/Akt-dependent pathway. Journal of Biological Chemistry, 2004, 279(6): 4479-4489. doi: 10.1074/jbc.M305529200

[17]	Song J W, Long J Y, Xie L, et al. Applications, phytochemistry, pharmacological effects, pharmacokinetics, toxicity of Scutellaria baicalensis Georgi. and its probably potential therapeutic effects on COVID-19: a review. Chinese Medicine, 2020, 15: 102. doi: 10.1186/s13020-020-00384-0

[18]	Wu J J, Du G C, Zhou J W, et al. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy. Metabolic Engineering, 2013, 16: 48-55. doi: 10.1016/j.ymben.2012.11.009

[19]	Zhao Q, Cui M Y, Levsh O, et al. Two CYP82D enzymes function as flavone hydroxylases in the biosynthesis of root-specific 4'-deoxyflavones in Scutellaria baicalensis. Molecular Plant, 2018, 11(1): 135-148. doi: 10.1016/j.molp.2017.08.009

[20]	Hwang E I, Kaneko M, Ohnishi Y, et al. Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster. Applied and Environmental Microbiology, 2003, 69(5): 2699-2706. doi: 10.1128/AEM.69.5.2699-2706.2003 pmid: 12732539

[21]	Ji D N, Li J H, Xu F L, et al. Improve the biosynthesis of baicalein and scutellarein via manufacturing self-assembly enzyme reactor in vivo. ACS Synth Biol, 2021, 10(5): 1087-1094. doi: 10.1021/acssynbio.0c00606

[22]	Yan Y J, Kohli A, Koffas M A G. Biosynthesis of natural flavanones in Saccharomyces cerevisiae. Applied and Environmental Microbiology, 2005, 71(9): 5610-5613. doi: 10.1128/AEM.71.9.5610-5613.2005

[23]	McCandliss R J, Poling M D, Herrmann K M. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. Journal of Biological Chemistry, 1978, 253(12): 4259-4265. pmid: 26682

[24]	Liu S P, Xiao M R, Zhang L, et al. Production of l-phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110. Process Biochemistry, 2013, 48(3): 413-419. doi: 10.1016/j.procbio.2013.02.016

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