Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Chunxiao Yan; Wei Yu; Xiaoxin Zhai; Lun Yao; Xiaoyu Guo; Jiaoqi Gao; Yongjin J Zhou

doi:10.1016/j.synbio.2021.12.005

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha

Synth Syst Biotechnol. 2021 Dec 15;7(1):498-505. doi: 10.1016/j.synbio.2021.12.005. eCollection 2022 Mar.

Authors

Chunxiao Yan^{1

2}, Wei Yu^{2

3

4}, Xiaoxin Zhai^{2

3}, Lun Yao^{2

3}, Xiaoyu Guo¹, Jiaoqi Gao^{2

5

3}, Yongjin J Zhou^{2

5

3}

Affiliations

¹ School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, PR China.
² Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian, 116023, PR China.
³ Dalian Key Laboratory of Energy Biotechnology, Dalian Institute of Chemical Physics, CAS, 457 Zhongshan Road, Dalian, 116023, PR China.
⁴ University of Chinese Academy of Sciences, Beijing, 100049, China.
⁵ CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, PR China.

Abstract

Bio-manufacturing via microbial cell factory requires large promoter library for fine-tuned metabolic engineering. Ogataea polymorpha, 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 O. polymorpha for bio-productions. Here, we systematically characterized native promoters in O. polymorpha by both GFP fluorescence and fatty alcohol biosynthesis. Ten constitutive promoters (P _PDH , P _PYK , P _FBA , P _PGM , P _GLK , P _TRI , P _GPI , P _ADH1 , P _TEF1 and P _GCW14 ) were obtained with the activity range of 13%-130% of the common promoter P _GAP (the promoter of glyceraldehyde-3-phosphate dehydrogenase), among which P _PDH and P _GCW14 were further verified by biosynthesis of fatty alcohol. Furthermore, the inducible promoters, including ethanol-induced P _ICL1 , rhamnose-induced P _LRA3 and P _LRA4 , and a bidirectional promoter (P _Mal -P _Per ) that is strongly induced by sucrose, further expanded the promoter toolbox in O. polymorpha. Finally, a series of hybrid promoters were constructed via engineering upstream activation sequence (UAS), which increased the activity of native promoter P _LRA3 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 O. polymorpha, and also a feasible strategy for rationally regulating the promoter strength.

Keywords: EMP, Embden-Meyerhof-Parnas pathway; Fatty alcohols; GFP, Green Fluorescent Protein; Hybrid promoter; Metabolic engineering; Ogataea polymorpha; Promoter; UAS, Upstream Activation Sequence; Upstream activation sequence.