Synthetic Design and Optimization of Metabolic Pathway for Efficient Conversion of Low-cost Carbon Sources to Value-added Bio-chemicals

Abstract

Biorefinery is a sustainable process capable of converting biomass into various types of energy and chemicals. Enormous accomplishments in the production of fuels, platform chemicals, pharmaceuticals, and polymers were achieved with the recent progress in metabolic engineering to design efficient microbial cell factory. Currently, the biorefinery is regarded as a unique process able to replace petroleum-based process in future. To realize this optimistic future of biorefinery, it should convert various carbon sources from abundant biomass into value-added bio-chemicals.

The current biorefinery process is relying on glucose from starch crops as the main carbon source. Although most industrial microorganisms favor utilizing glucose, its supply in massive quantity is not desirable as it can be utilized as a food. Thus, other abundant carbon sources should be considered for utilization as a feedstock for biorefinery process. For future feedstock, sugars from lignocellulosic or marine biomass have been suggested. In addition, crude glycerol from biodiesel industry and synthetic gases are also considered.

However, most industrial microorganisms are not able to rapidly convert those carbon sources due to deficient or inefficient utilization pathway. Moreover, carbon catabolite repression hinders simultaneous utilization of multiple sugars, resulting in low productivity and yield. Thus, the microorganisms should be engineered for enhanced and simultaneous utilization of various carbon sources to achieve efficient conversion for biochemical production.

Recent progress in synthetic biology enabled us to design microorganisms in a predictable manner. The expression of multiple genes encoding metabolic enzymes can be precisely controlled with predictable genetic devices. With those tools, metabolic pathway can be amplified and optimized for maximum catalytic activity. In this manner, the slow utilization pathway for low-cost carbon sources can be efficiently reconstructed for increased metabolism. In addition, optimization of the entire pathway including sugar utilization and biochemical production is achievable by precise tuning of the expression level of key enzymes.

Here, the strategy for synthetic design and optimization of metabolic pathway is suggested for efficient low-cost carbon utilization to produce value-added bio-chemicals. The first issue is redesigning of microorganisms to efficiently utilize a non-preferred sugar in abundant biomass. Specifically, native galactose utilization pathway in Escherichia coli was synthetically reconstructed for rapid assimilation. This pathway also showed the simultaneous utilization of glucose to maintain high productivity. Secondly, the developed pathway was further associated with n-butanol production pathway and the overall metabolic pathway was optimized with precise control of key enzymes to balance intracellular redox state. Lastly, this design and optimization strategy was also demonstrated in 3-hydroxypropionic acid(3-HP) production from glycerol. The strategies used in this study could be broadly applicable to convert various carbons to value-added chemicals.

Followings are summarized contents by chapters.

In chapter 1, the available carbon sources from abundant biomass, industrial wastes were introduced. In addition, the recent efforts in engineering microorganisms (mainly E. coli and yeast) to efficiently utilize those carbon sources were described.

In chapter 2, the native galactose utilization pathway was redesigned in Escherichia coli to construct platform strain for rapid conversion of seaweed-derived sugars. The removal of native regulation and expression of metabolic enzymes under synthetic expression cassettes enabled 53% increased sugar uptake rate and 44.8% faster growth rate in galactose medium. Moreover, the developed strain showed simultaneous galactose utilization even with the presence of glucose.

In chapter 3, the redesigned galactose utilization pathway was associated with an amplified n-butanol production pathway. The amplification of production pathway alone showed much reduced n-butanol production (1.80 g/L) compared to the result with glucose, however, implementation of synthetic pathway significantly improved capability to produce n-butanol from galactose (248%, 4.47 g/L). Furthermore, the optimization of intracellular redox state by precise control of fdh1 expression additionally increased n-butanol production to 6.25 g/L in 48 hours. This was the best n-butanol production from galactose by engineered E. coli so far.

In chapter 4, the pathway for conversion of glycerol to 3-HP was amplified and optimized for maximized conversion efficiency. In glycerol-dependent 3-HP production, accumulation of 3-hydroxypropionic aldehyde(3-HPA) is known to a crucial hurdle to achieve high productivity and yield. Although the initial strain with maximized expression of biosynthetic enzymes showed poor 3-HP production (1.69 g/L), precise expression control not to accumulate 3-HPA, allowed a dramatic increase (4.50 g/L) from glycerol. After fed-batch fermentation, the engineered strain produced 41.8 g/L 3-HP in 30 hours with the highest yield until date (0.97 g/g glycerol, 0.53 g/g total sugar).