Novel two-stage processes for optimal chemical production in microbes

Abstract

1Microbial metabolism can be harnessed to produce a broad range of industrially important chemicals. 2 Often, three key process variables: Titer, Rate and Yield (TRY) are the target of metabolic engineering 3 efforts to improve microbial hosts toward industrial production. Previous research into improving the 4 TRY metrics have examined the efficacy of having distinct growth and production stages to achieve 5 enhanced productivity. However, these studies assumed a switch from a maximum growth to a maximum 6 production phenotype. Hence, the choice of operating points for the growth and production stages of 7 two-stage processes is yet to be explored. The impact of reduced growth rates on substrate uptake adds 8 to the need for intelligent choice of operating points while designing two-stage processes. In this work, we 9 present a computational framework that scans the phenotypic space of microbial metabolism to identify 10 ideal growth and production phenotypic targets, to achieve optimal TRY values. Using this framework, 11 with Escherichia coli as a model organism, we compare two-stage processes that use dynamic pathway 12 regulation, with one-stage processes that use static intervention strategies. Our results indicate that 13 two-stage processes with intermediate growth during the production stage always result in the highest 14 productivity. By analyzing the flux distributions for the production enhancing strategies, we identify 15 key reactions and reaction subsystems that need to be downregulated for a wide range of metabolites 16 in E. coli. We also elucidate the importance of flux perturbations that increase phosphoenolpyruvate 17 and NADPH availability among strategies to design production platforms. Furthermore, reactions in 18 the pentose phosphate pathway emerge as key control nodes that function together to increase the 19 availability of precursors to most products in E. coli. Due to the presence of these common patterns 20 in the flux perturbations, we propose the possibility of a universal production strain that enhances the 21 production of a large number of metabolites. 22 23 Keywords: dynamic pathway engineering, two-stage processes, industrial bioprocesses, phenotypic choices, 24 production platforms, substrate uptake effects 25 1 26The use of microbes for the production of chemicals through metabolic engineering has garnered significant 27 interest in the past few decades. The naturally modular arrangement of metabolic networks makes micro-28 bial strains amenable to be used as chemical production platforms 1 . Metabolic networks have a bow-tie 29 architecture which allows a large number of metabolites to be produced from a few universal precursors 2 . 30This has allowed us to successfully engineer microbes to be biocatalysts for the production of a wide range of 31 commodity chemicals 3,4 , pharmaceuticals 5,6 , biofuels, 7,8 and other natural and non-natural compounds 9 . 32While few such processes have been successful at an industrial scale 10,11 , large strain development costs 3...