Developing novel strategies to regulate metabolic resource distribution for enhancing microbial bioproduction
Creators: name: Zhuang, Lei
Publisher: name: No Publisher Supplied
Year: 2024

description: Microbial biosynthesis has been well recognized as a sustainable and environmentally friendly approach for production of various products and has attracted significant attention due to its applicability in different industries. Advances in metabolic engineering and synthetic biology have accelerated the development of novel microbial biosynthesis methods to convert renewable feedstocks into high-value products. However, one of fundamental challenges in microbial biosynthesis, the competition for resources between cell growth and bioproduction, is still largely unresolved. This dissertation focuses on developing innovative strategies to balance the allocation of cellular metabolic resources between cell growth and bioproduction to effectively enhance the biosynthesis of desired products. First, a novel strategy of antimicrobial peptide (AMP)-mediated global metabolism regulation was developed by expressing an AMP in E. coli to inhibit protein synthesis process and thus repress global metabolism. Compared with the traditional method of over-expression of the biosynthetic pathway genes or the method of direct addition of an exogenous antibiotic, this strategy demonstrated significant advantages in enhancing bioproduction of target products. Further improvements were achieved by optimizing the conditions for using AMP. The successful application of AMP-mediated global metabolism regulation across different bioproduction systems highlighted its effectiveness and broad applicability in microbial biosynthesis. Systematic characterization of the engineered E. coli, including the cell growth, bioproduction, protein synthesis, and energy, indicated that this strategy effectively reallocated metabolic resources among key destinations to enhance the desired bioproduction. Metabolomics analysis further revealed that the use of AMP delayed carbon source uptake and significantly increased metabolite pool size, which benefitted the bioproduction with higher pathway enzyme expression than the background metabolism. This strategy was further integrated with co-culture engineering technology to enable the over-production of a pathway intermediate for an E. coli-E. coli co-culture as well as more rational allocation of resources between the co-culture members, which resulted in significant improvement of the production of the final product. This dissertation also explores another novel strategy to address resource allocation in a co-culture by incorporating a metabolite biosensor into the co-culture to dynamically regulate the population composition to meet the need of biosynthesis optimization. Specifically, a biosensor-regulator system responsive to a biosynthetic pathway intermediate was employed in the downstream co-culture strain. Through dynamic regulation of the downstream strain’s growth based on the pathway intermediate accumulation, this system rationally assigned resources to the co-culture strains to balance the pathway intermediate supply and consumption and significantly enhanced the overall bioproduction performance of the target product indigo. The successful application of the engineering strategies developed in this dissertation, including the AMP-mediated global regulation, the biosensor-regulator system, and their integration with co-culture engineering in various bioproduction systems, provided innovative perspectives to not only address the longstanding challenge in microbial biosynthesis but also facilitate the advances in metabolic engineering and synthetic biology.