Model-guided development of an evolutionarily stable yeast chassis.

Filipa Pereira,Britta Meyer,Paulo Maia,Isabel Rocha,Justyna Nocon,Peter Kötter,Paula Jouhten,Kiran R Patil,Miguel Rocha,Eleni Kafkia,Helder Lopes,Dimitrios Konstantinidis

doi:10.15252/msb.202110253

Abstract

First‐principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. Yet, the theory of model‐based chassis design has rarely been put to rigorous experimental test. Here, we report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome‐scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product‐specific TCA cycle disruptions resulted in the secretion of the corresponding acid. Adaptive laboratory evolution further improved production of succinate and fumarate, demonstrating the evolutionary robustness of the engineered cells. In the case of malate, multi‐omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. In all three cases, flux balance analysis integrating transcriptomics, proteomics and metabolomics data confirmed the flux re‐routing predicted by the model. Taken together, our modelling and experimental results have implications for the computer‐aided design of microbial cell factories.

Highlights

First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations
The robustness of the predictions was verified across different versions of yeast genome-scale metabolic models and against different simulation methods (Appendix Tables S5–S6)
Model-guided design of chassis microbial strains has the potential to overcome the cost and time burden associated with cell factory development, but so far has only been explored theoretically (Layton & Trinh, 2014; Trinh et al, 2015; Jouhten et al, 2016)

Summary

Introduction

First-principle metabolic modelling holds potential for designing microbial chassis that are resilient against phenotype reversal due to adaptive mutations. The theory of model-based chassis design has rarely been put to rigorous experimental test. We report the development of Saccharomyces cerevisiae chassis strains for dicarboxylic acid production using genome-scale metabolic modelling. The chassis strains, albeit geared for higher flux towards succinate, fumarate and malate, do not appreciably secrete these metabolites. As predicted by the model, introducing product-specific TCA cycle disruptions resulted in the secretion of the corresponding acid. In the case of malate, multi-omics analysis revealed a flux bypass at peroxisomal malate dehydrogenase that was missing in the yeast metabolic model. Our modelling and experimental results have implications for the computer-aided design of microbial cell factories

Methods

Results

Conclusion