Dataset Information

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha.

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.

SUBMITTER: Yan C

PROVIDER: S-EPMC8685918 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha.

Yan Chunxiao C Yu Wei W Zhai Xiaoxin X Yao Lun L Guo Xiaoyu X Gao Jiaoqi J Zhou Yongjin J YJ

Synthetic and systems biotechnology 20211215 1

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 ...[more]

PMID: 34977394

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Project description:MotivationThe computer-aided design of metabolic intervention strategies has become a key component of an integrated metabolic engineering approach and a broad range of methods and algorithms has been developed for this task. Many of these algorithms enforce coupling of growth with product synthesis and may return thousands of possible intervention strategies from which the most suitable strategy must then be selected.ResultsThis work focuses on how to evaluate and rank, in a meaningful way, a given pool of computed metabolic engineering strategies for growth-coupled product synthesis. Apart from straightforward criteria, such as a preferably small number of necessary interventions, a reasonable growth rate and a high product yield, we present several new criteria useful to pick the most suitable intervention strategy. Among others, we investigate the robustness of the intervention strategies by searching for metabolites that may disrupt growth coupling when accumulated or secreted and by checking whether the interventions interrupt pathways at their origin (preferable) or at downstream steps. We also assess thermodynamic properties of the pathway(s) favored by the intervention strategy. Furthermore, strategies that have a significant overlap with alternative solutions are ranked higher because they provide flexibility in implementation. We also introduce the notion of equivalence classes for grouping intervention strategies with identical solution spaces. Our ranking procedure involves in total ten criteria and we demonstrate its applicability by assessing knockout-based intervention strategies computed in a genome-scale model of E.coli for the growth-coupled synthesis of l-methionine and of the heterologous product 1,4-butanediol.Availability and implementationThe MATLAB scripts that were used to characterize and rank the example intervention strategies are available at http://www2.mpi-magdeburg.mpg.de/projects/cna/etcdownloads.html.Supplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha.

Publications

Characterizing and engineering promoters for metabolic engineering of <i>Ogataea polymorpha</i>.

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