Pinene is a natural monoterpene with a wide range of applications. Especially, its dimers have high energy value, thus it has great application prospects in the fuel field. Previous studies had shown that the activity of pinene synthase was the rate-limiting step for efficient synthesis of pinene in Saccharomyces cerevisiae. In this study, we hoped to obtain pinene synthase mutants with high catalytic efficiency in S. cerevisiae, which was proposed to provide a reference for efficient synthesis of pinene in yeast. Saturation mutants of the Q456 site of pinene synthase Pt30 were expressed in S. cerevisiae with enhanced mevalonate (MVA) pathway. And the ability of the recombinant strain to produce pinene was analyzed by two-phase shake flask fermentation and gas chromatographic detection. The results showed that among these mutants, Pt30 (Q456K) and Pt30 (Q456I) increased the pinene titer from non-detectable to 0.017 and 0.041 mg/L, respectively. In conclusion, the mutants Pt30 (Q456K) and Pt30 (Q456I) promoted pinene synthesis in S. cerevisiae. This indicated that Q456 was essential for Pt30 and provided a reference for rational modifications of other pinene synthases.
REN Quanlu
,
LU Xinyao
,
ZONG Hong
,
ZHUGE Bin
. Key enzyme mutations promote pinene production in Saccharomyces cerevisiae[J]. Food and Fermentation Industries, 2021
, 47(22)
: 30
-34
.
DOI: 10.13995/j.cnki.11-1802/ts.027337
[1] BEHR A, JOHNEN L.Myrcene as a natural base chemical in sustainable chemistry:A critical review[J].ChemSusChem, 2009, 2(12):1 072-1 095.
[2] HARVEY B G, WRIGHT M E, QUINTANA R L.High-density renewable fuels based on the selective dimerization of pinenes[J].Energy & Fuels, 2010, 24(1):267-273.
[3] YANG J M, NIE Q J, REN M, et al.Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene[J].Biotechnology for Biofuels, 2013, 6(1):60.
[4] VICKERS C E, WILLIAMS T C, PENG B Y, et al.Recent advances in synthetic biology for engineering isoprenoid production in yeast[J].Current Opinion in Chemical Biology, 2017, 40:47-56.
[5] CHEN Y, WANG Y, LIU M, et al.Primary and secondary metabolic effects of a key gene deletion (ΔYPL062W) in metabolically engineered terpenoid-producing Saccharomyces cerevisiae[J].Applied and Environmental Microbiology, 2019, 85(7).DOI:10.1128/aem.01990-18.
[6] IGNEA C, CVETKOVIC I, LOUPASSAKI S, et al.Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids[J].Microbial Cell Factories, 2011, 10(1):1-18.
[7] LIU J D, ZHANG W P, DU G C, et al.Overproduction of geraniol by enhanced precursor supply in Saccharomyces cerevisiae[J].Journal of Biotechnology, 2013, 168(4):446-451.
[8] IGNEA C, PONTINI M, MAFFEI M E, et al.Engineering monoterpene production in yeast using a synthetic dominant negative geranyl diphosphate synthase[J].ACS Synthetic Biology, 2014, 3(5):298-306.
[9] 陈天华, 张若思, 姜国珍, 等.产蒎烯人工酵母细胞的构建[J].化工学报, 2019, 70(1):179-188.
CHEN T H, ZHANG R S, JIANG G Z, et al.Metabolic engineering of Saccharomyces cerevisiae for pinene production[J].CIESC Journal, 2019, 70(1):179-188.
[10] SARRIA S, WONG B, MARTÍN H G, et al.Microbial synthesis of pinene[J].ACS Synthetic Biology, 2014, 3(7):466-475.
[11] TASHIRO M, KIYOTA H, KAWAI-NOMA S, et al.Bacterial production of pinene by a laboratory evolved pinene synthase[J].ACS Synthetic Biology, 2016, 5(9):1 011-1 020.
[12] YEE D A, DENICOLA A B, BILLINGSLEY J M, et al.Engineered mitochondrial production of monoterpenes in Saccharomyces cerevisiae[J].Metabolic Engineering, 2019, 55:76-84.
[13] LIU T, ZHANG C B, LU W.Heterologous production of levopimaric acid in Saccharomyces cerevisiae[J].Microbial Cell Factories, 2018, 17(1):1-10.
[14] CATALDO V F, ARENAS N, SALGADO V, et al.Heterologous production of the epoxycarotenoid violaxanthin in Saccharomyces cerevisiae[J].Metabolic Engineering, 2020, 59:53-63.
[15] JIANG G Z, YAO M D, WANG Y, et al.Manipulation of GES and ERG20 for geraniol overproduction in Saccharomyces cerevisiae[J].Metabolic Engineering, 2017, 41:57-66.
[16] CHENG S, LIU X, JIANG G Z, et al.Orthogonal engineering of biosynthetic pathway for efficient production of limonene in Saccharomyces cerevisiae[J].ACS Synthetic Biology, 2019, 8(5):968-975.
[17] LINDAHL A L, OLSSON M E, MERCKE P, et al.Production of the artemisinin precursor amorpha-4,11-diene by engineered Saccharomyces cerevisiae[J].Biotechnoligy Letter, 2006, 28(8):571-580.
[18] PHILLIPS M A, WILDUNG M R, WILLIAMS D C, et al.cDNA isolation, functional expression, and characterization of (+)-α-pinene synthase and (-)-α-pinene synthase from loblolly pine (Pinus taeda):Stereocontrol in pinene biosynthesis[J].Archives of Biochemistry and Biophysics, 2003, 411(2):267-276.
[19] JONES R P, GADD G M.Ionic nutrition of yeast-physiological mechanisms involved and implications for biotechnology[J].Enzyme and Microbial Technology, 1990, 12(6):402-418.
[20] WU Q, PENG Z L, ZHANG Y, et al.COACH-D:Improved protein-ligand binding sites prediction with refined ligand binding poses through molecular docking[J].Nucleic Acids Research, 2018, 46(W1):W438-W442.
[21] YANG J Y, ROY A, ZHANG Y.Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment[J].Bioinformatics, 2013, 29(20):2 588-2 595.
