Understanding differential gene expression of cells in the non-growing state may inform enzyme targets useful for cofactor regeneration

Abstract

Non-growing cells offer a platform for enhanced production of target product due to the avoidance of channeling precious metabolic energy into cell growth. In addition, cofactors were also not used in anabolic reactions, thereby, saving the resource for biotransformation. More importantly, non-growing cells enable an easier downstream product separation. However, enzyme stability and half-life are issues preventing the attainment of greater volumetric productivity of product. Finally, substrate and product inhibition have been reported to reduce the productivity of non-growing cells biotransformation. Different approaches have been utilized for cofactor regeneration in non-growing cells. These include: (i) feeding cells with different carbon substrate, (ii) expression of a co-enzyme that regenerates needed cofactors from a substrate (i.e., co-enzyme approach), and (iii) use of an enzyme that while performing the biotransformation also regenerates the cofactor (i.e., co-substrate approach). Amongst the approaches, feeding of cells with different substrates and the expression of enzymes that regenerates cofactors are more commonly used. Co-enzymes typically used in cofactor regeneration are glucose-6-phosphate dehydrogenase and formate dehydrogenase. Open questions in the literature on non-growing cell biotransformation pertains to the understanding of the global gene expression patterns of non-growing cells relative to growing cells. Specifically, relatively little is known about the gene expression patterns of non-growing cells belonging to major biotechnological chassis such as Escherichia coli and Saccharomyces cerevisiae. Without information on the types of genes differentially expressed in the nongrowing state as well as the cellular stress experienced, a trial and error approach would have to be used in selecting specific enzymes for over-expression in regenerating cofactors. Such trial and error approaches are likely to yield solutions that are non-optimal for the efficient recycling of cofactors during biotransformation. Thus, RNA-seq and mass spectrometry proteomics were suggested as possible methodologies for uncovering the differential gene expression patterns of non-growing cells relative to growing cells. The list of pathways and genes differentially expressed in non-growing cells will provide targets for over-expression or deletion of genes with the objective of generating strains of E. coli suitable for efficient cofactor regeneration. Specifically, while over-expression of enzymes for enhancing cofactors regeneration is commonly used, there is a large untapped set of genes and pathways that consume cofactors. If found to be non-essential to the maintenance of non-growing cells for biotransformation, these genes and pathways could be deleted and manipulated, respectively. Ultimately, the goal is the generation of strains of E. coli optimized for biotransformation with enhanced cofactor regeneration capacity. Such E. coli strains would be optimized for regeneration of NADH, NADPH and FADH2 individually, and could be coupled with the expression of any gene expression system that require cofactors for biotransformation.

Keywords: cofactor regeneration, Escherichia coli, enzyme targets, differential gene expression, non-growing cells, biotechnology, molecular biology, cell biology, biochemistry, synthetic biology