Characterizing and Engineering Promoters for Metabolic Engineering of

Overview

Journal Synth Syst Biotechnol

Specialty Biotechnology

Date 2022 Jan 3

PMID 34977394

Citations 12

Authors

Chunxiao Yan

Wei Yu

Xiaoxin Zhai

Lun Yao

Xiaoyu Guo

Jiaoqi Gao

Yongjin J Zhou

Affiliations

Soon will be listed here.

Abstract

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. , one of the methylotrophic yeasts, possesses advantages in broad substrate spectrum, thermal-tolerance, and capacity to achieve high-density fermentation. However, a limited number of available promoters hinders the engineering of for bio-productions. Here, we systematically characterized native promoters in by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P , P , P , P , P , P , P , P , P and P ) were obtained with the activity range of 13%-130% of the common promoter P (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P and P were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P , rhamnose-induced P and P , and a bidirectional promoter (P -P ) that is strongly induced by sucrose, further expanded the promoter toolbox in . Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P by 4.7-10.4 times without obvious leakage expression. Therefore, this study provided a group of constitutive, inducible, and hybrid promoters for metabolic engineering of , and also a feasible strategy for rationally regulating the promoter strength.

Citing Articles

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References

Rajkumar A, Liu G, Bergenholm D, Arsovska D, Kristensen M, Nielsen J . Engineering of synthetic, stress-responsive yeast promoters. Nucleic Acids Res. 2016; 44(17):e136. PMC: 5041464. DOI: 10.1093/nar/gkw553. View

Fernie A, Carrari F, Sweetlove L . Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol. 2004; 7(3):254-61. DOI: 10.1016/j.pbi.2004.03.007. View

Faber K, Haima P, Harder W, Veenhuis M, Ab G . Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet. 1994; 25(4):305-10. DOI: 10.1007/BF00351482. View

Peng B, Plan M, Carpenter A, Nielsen L, Vickers C . Coupling gene regulatory patterns to bioprocess conditions to optimize synthetic metabolic modules for improved sesquiterpene production in yeast. Biotechnol Biofuels. 2017; 10:43. PMC: 5320780. DOI: 10.1186/s13068-017-0728-x. View

Ledeboer A, Edens L, Maat J, Visser C, Bos J, Verrips C . Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res. 1985; 13(9):3063-82. PMC: 341221. DOI: 10.1093/nar/13.9.3063. View

Chen X, Gao C, Guo L, Hu G, Luo Q, Liu J . DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals. Chem Rev. 2017; 118(1):4-72. DOI: 10.1021/acs.chemrev.6b00804. View

Engstrom M, Pfleger B . Transcription control engineering and applications in synthetic biology. Synth Syst Biotechnol. 2018; 2(3):176-191. PMC: 5655343. DOI: 10.1016/j.synbio.2017.09.003. View

Paddon C, Westfall P, Pitera D, Benjamin K, Fisher K, McPhee D . High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013; 496(7446):528-32. DOI: 10.1038/nature12051. View

Manfrao-Netto J, Gomes A, Skorupa Parachin N . Advances in Using as Chassis for Recombinant Protein Production. Front Bioeng Biotechnol. 2019; 7:94. PMC: 6504786. DOI: 10.3389/fbioe.2019.00094. View

10.

Brown S, Clastre M, Courdavault V, OConnor S . De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci U S A. 2015; 112(11):3205-10. PMC: 4371906. DOI: 10.1073/pnas.1423555112. View

11.

Blazeck J, Alper H . Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J. 2012; 8(1):46-58. DOI: 10.1002/biot.201200120. View

12.

Fillet S, Adrio J . Microbial production of fatty alcohols. World J Microbiol Biotechnol. 2016; 32(9):152. DOI: 10.1007/s11274-016-2099-z. View

13.

Menendez J, Valdes I, Cabrera N . The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter. Yeast. 2003; 20(13):1097-108. DOI: 10.1002/yea.1028. View

14.

Nehlin J, Carlberg M, Ronne H . Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J. 1991; 10(11):3373-7. PMC: 453065. DOI: 10.1002/j.1460-2075.1991.tb04901.x. View

15.

Puseenam A, Kocharin K, Tanapongpipat S, Eurwilaichitr L, Ingsriswang S, Roongsawang N . A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica. FEMS Microbiol Lett. 2018; 365(20). DOI: 10.1093/femsle/fny238. View

16.

Blazeck J, Reed B, Garg R, Gerstner R, Pan A, Agarwala V . Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica. Appl Microbiol Biotechnol. 2012; 97(7):3037-52. DOI: 10.1007/s00253-012-4421-5. View

17.

Zhou Y, Buijs N, Zhu Z, Qin J, Siewers V, Nielsen J . Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat Commun. 2016; 7:11709. PMC: 4894961. DOI: 10.1038/ncomms11709. View

18.

Kurylenko O, Ruchala J, Vasylyshyn R, Stasyk O, Dmytruk O, Dmytruk K . Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, . Biotechnol Biofuels. 2018; 11:197. PMC: 6052537. DOI: 10.1186/s13068-018-1203-z. View

19.

Dulermo R, Brunel F, Dulermo T, Ledesma-Amaro R, Vion J, Trassaert M . Using a vector pool containing variable-strength promoters to optimize protein production in Yarrowia lipolytica. Microb Cell Fact. 2017; 16(1):31. PMC: 5316184. DOI: 10.1186/s12934-017-0647-3. View

20.

Xiong X, Chen S . Expanding Toolbox for Genes Expression of to Include Novel Inducible, Repressible, and Hybrid Promoters. ACS Synth Biol. 2020; 9(8):2208-2213. DOI: 10.1021/acssynbio.0c00243. View