Construction of a Metabolite Valve in Saccharomyces Cerevisiae Increases Pathway Yields

Background: Microbial fermentation has the potential to produce various chemicals from simple and inexpensive starting materials that are otherwise derived from non-renewable or limited resources. Typical methods to improve product yields include overexpressing pathway enzymes and deleting competing pathways. However, these methods are constrained by the inter-connectedness of metabolic pathways and the finite nature of cellular resources. Overexpression of pathway enzymes comes in the expense of endogenous ones due to consumption of common precursors. On the other hand, deletion of competing pathways may necessitate media supplementation and impair cellular function. Methods: To overcome this, we constructed of a metabolite valve that has the potential to improve pathway yields without affecting cellular function. A metabolite valve enables (1) down-regulation of competing pathway enzyme(s) and (2) dynamic control of these enzyme levels. As a proof-of-concept, we looked at directing metabolic flux away from glycolysis into a model pathway, gluconate. In a Saccharomyces cerevisiae strain where Glucokinase 1 and Hexokinase 2 have been deleted, a metabolite valve was constructed at the only remaining hexokinase, Hexokinase 1. The promoter of Hexokinase 1 was replaced with the inducible tetracycline transactivator protein (tTA) system, and glucose dehydrogenase from B. subtilis was expressed for the production of gluconate. Results: Regulation of Hexokinase 1 expression at the transcription level resulted in a maximum 10-fold decrease in enzyme activity upon induction with doxycycline. This reduction in enzyme activity resulted in an increase in available glucose for the gluconate pathway. Consequently, a 300-fold increase in yield of gluconate from glucose was observed, from 0.1 mol/mol % to 18 mol/mol % upon induction with doxycycline. Moreover, a 30% increase in biomass yield was observed; repressing Hexokinase 1 with the valve resulted in increased carbon efficiency and less ethanol production. Conclusions: This proof-of-concept presents an innovative strategy to increase product yields by controlling competing native metabolic pathways. With the success of this proof-of-concept, we would like to implement a metabolite valve to improve the yield of glucaric acid, a “top 10 value added chemical” listed by the USDA. Here, multiple valves can be constructed at competing enzymes such as PFK1, PFK2, and PIS1. In addition to control at the transcription level with the existing tTA system, post-translational methods such as controlled protein degradation and the CRISPR Interference system will be explored.