Transkriptionelle Regulation des Escherichia coli-Stoffwechsels und künstlicher Stoffwechselwege
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Philipps-Universität Marburg
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Abstract
A common problem in metabolic engineering projects is to find enzyme levels that enhance productivity and efficiency of synthetic metabolic pathways. This problem is especially important when overexpressing heterologous enzymes that drain metabolites from central metabolism. The additional requirement for precursors and energy causes a growth burden or even leads to cell death (chapter 1). Several strategies have been employed to solve this problem and two out of them are addressed in this thesis: i) decoupled overproduction with a two-phase process and ii) growth coupled production with a one-phase process. A two-phase process decouples growth of the host from the production phase, while the one-phase strategy couples production to growth of the host. In this thesis, these two strategies are investigated using overproduction of three chemicals as case studies: glycerol, carotenoids and arginine. To test the two-phase processes, we inserted glycerol genes into the E. coli genome and used CRISPR interference (CRISPRi) to down-regulate the expression of RNA polymerase (chapter 2). When the CRISPRi system is induced at a relatively high biomass, the glycerol production can be enhanced. This result implies that producing glycerol requires to maintain certain growth instead of draining all metabolites only for overproduction. Therefore, a one-phase process is more feasible for glycerol overproduction.
In chapter 3, to use the concept of one-phase processes and combine it with an additional layer of regulation, we designed an artificial feedback circuit using a transcription factor (TF), Cra. To this end, the consensus binding sequence of Cra was inserted directly after the pBAD promoter sequence expressing glycerol genes. This design allowed native transcriptional regulation by Cra to regulate the expression of overproduction pathways. Proteomic and metabolomic data revealed that the Cra-regulation system can overcome growth burden by slowing down enzyme expression and thereby avoid the complete utilization of pathway precursors even before they are replenished. This delayed time enabled an adaptive metabolic response, a gradual increment of product synthesis (i.e. glycerol and carotenoids). The observed adaptive behavior leads to an increase in the expression of glucose transporters and glycolytic enzymes thereby improving host fitness and productivity in glycerol and carotenoid overproduction pathways. Cra-regulation was engineered in multiple types of promoters including pBAD promoter, pTetR and constitutive promoters (chapter 4) which shows that this regulation enables universal dynamical control.
In chapter 5, we genomically integrated GFP into SS9 intergenic region and used it as a reporter system to measure burden and fitness defects. In this system, the GFP signal decreases when overproduction pathways drain too many cellular resources and thereby the GFP content per cell reflects cellular fitness. Furthermore, it was sensitive enough to detect the metabolic burden even in the absence of a growth phenotype.
In chapter 6, we engineered the arginine pathway with the CRISPRi system to down-regulate the TF of the pathway, ArgR. Our results show that productivity similar to an argR removed overproducing strain can be achieved with a further enhanced growth rate. The result again indicates that the TF-feedback regulated system is capable of altering enzyme expressions for balanced resource utilization.
In conclusion, incorporating TF-based regulation in any designed circuit, can balance host metabolism and the production of several high-value chemicals without retarding the host growth. This is an advantage in comparison to the conventional process that involves complex enzyme screening to obtain optimized enzyme levels. This thesis therefore introduces a new dynamically controlled feedback loop strategy. With its ability to maintain a balance between host metabolism and product formation without causing host cellular burden, this strategy not only can serve as a competitive and facile solution to improve the productivity of a bacterial strain but also can be further expanded in large-scale applications.
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Created: 2020Issued: 2021-12-02Updated: 2021-12-02
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
Transcriptional regulation Dynamic control Metabolic pathways Biotechnology Synthetic Biology Metabolism Microbiology Metabolic engineering H
DFG-subjects
Transkriptionelle Regulierung Dynamische Steuerung Stoffwechselwege Biotechnologie Synthetische Biologie Stoffwechsel Mikrobiologie Kontrolle
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570
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Wang, Chun-Ying: Transkriptionelle Regulation des Escherichia coli-Stoffwechsels und künstlicher Stoffwechselwege. : Philipps-Universität Marburg 2021-12-02. DOI: https://doi.org/10.17192/z2020.0485.
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Except where otherwised noted, this item's license is described as Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 - CC BY NC ND