Fine-tunable Rebalancing of Escherichia coli for Optimal Production of Butyric acid and n-Butanol

Abstract

Global demand for the development of environmentally favorable microbial processes that producing petroleum-derived chemicals and fuels, is increasing dramatically owing to environmental concerns and uncertainty of oil supply. Certainly, advances in synthetic biology and metabolic engineering has been fueled these strides to design optimal microbial cell factories, capable of converting biomass into various bio-chemicals with maximal yield and productivity. These cellular phenotypes are particularly important for the high-volume (and low-value) bulk chemicals and biofuels such as n-butanol, butyrate.

n-Butanol obviously have spotlighted as an advanced ‘drop-in’ alternative for gasoline owing to its close physicochemical properties, such as high energy density, low corrosiveness, and low vapor pressure, compared to gasoline than ethanol. Moreover, n-butanol has versatility not only as an advanced biofuels but also as a feedstock and solvent for various industrial applications, requiring large global demand.

Butyric acid, a short-chain fatty acid, and its derivatives are also utilized as feedstock for various industrial products, including plastics, fibers, food additives, and pharmaceuticals. Furthermore, direct hydrogenation of butyric acid by copper-based catalysts can produce n-butanol, a chemical in the world spotlight as mentioned above.

Here, I proposed novel synthetic biology-driven engineering approaches to design optimal cell factory for butyric acid and n-butanol in Escherichia coli. First issue to be solved is balancing the intracellular redox state that plays a governing factor for the continuation of both catabolic and anabolic pathways. Beyond stoichiometric-dependent redox rebalancing, the effect of optimizing catalytic amounts of reducing powers depending on cellular demands, which termed carbon flux-associated redox rebalancing, was investigated to improve yield and productivity of bio-chemical synthesis. Secondly, the optimization of metabolic pathways between glycolysis and the engineered product-forming pathway was evaluated using proposed a metabolic valve of glycolytic flux to further increase yield and/or productivity of desired products.

Followings are summarized contents by chapters.

In chapter 1, the recent advances for the production of advanced biofuels in tractable microorganisms, mostly in E. coli, are outlined following specific engineering approaches: i) Engineering of heterologous product-forming pathways ii) Engineering of endogenous pathways iii) Cofactor engineering iv) Protein engineering v) Tolerance engineering vi) Diversification of carbon sources. Furthermore, traditional approaches for the redox rebalancing are categorized in detailed.

In chapter 2, the native redox cofactor regeneration system in E. coli was engineered to construct base strain for the production of butyric acid as model system. By implementing a nonnative butyrate synthetic pathway, rendering butyrate as the only final electron acceptor, the engineered strain (JHL26) produced 4.35 g/L of butyrate from glucose in 24 hours batch fermentation, which corresponds to 83.4% of theoretical maximum yield. Moreover, selective fermentation of butyric acid over acetic acid enabled (Butyrate/Acetate ~ 41 in the engineered JHL26 strain, but 5~7 in the native butyrate host, Clostridia). To the best of my knowledge, the data represented the highest yield and selectivity for butyric acid fermentation. Moving forward, the advanced concept of redox rebalancing, i.e., carbon flux-associated redox rebalancing was demonstrated using n-butanol as model compound. The intracellular redox state was modulated by anaerobically activating the pyruvate dehydrogenase (PDH) complex and was further balanced by fine-tuning expression levels of yeast NAD+-dependent formate dehydrogenase (Fdh1) through UTR engineering. In a batch fermentation, optimized strain (JHL85) showed the highest productivity of n-butanol in E. coli (0.26 g/L/h) at the 69% of theoretical maximum yield. Furthermore, 4.44g/L of n-butanol was produced from galactose for the first time and also demonstrated that different amounts of reducing equivalents were required to efficiently produce the desired products, depending on the carbon flux of substrates. Finally, the production of n-butanol from galactose further enhanced through the implementation of galactose utilization module. The resulting strain (GAL_061) dramatically increased productivity of n-butanol from galactose, about 2.5-fold increase, due to 66.4% improvement in specific galactose uptake rate. By optimizing intracellular redox state depending on the enhanced carbon flux enabled 22% further increase in productivity (0.13 g/L/h). To the best of my knowledge, the data represented the highest productivity of n-butanol from galactose.

In chapter 3, glucose-specific transporter (encoded by ptsG) is proposed as a metabolic valve to control overall glycolytic flux. The specific glucose uptake rate ranged from -28% to +21% compared to wild type E. coli W3110 through UTR engineering of ptsG. The n-butanol system further optimized using the metabolic valve, the yield controlled from 65% to 93% of theoretical maximum. In another case, the productivity of butyric acid 7% increased than the parental strain while the yield was maintained around 83% of theoretical maximum. To the best of my knowledge, the data represented in this study showed the highest yield and productivity of n-butanol and butyric acid.