Deciphering the Molecular Underpinnings of the Cryptic Cellobiose Metabolism in Escherichia coli

Abstract

Consolidated bioprocessing (CBP) is an efficient process that combines saccharification and fermentation of lignocellulosic biomass into a single microbial host. The choice of ideal microbes for CBP is challenging as it demands efficient functioning of several complex traits including expression of sets of saccharifying enzymes, metabolism of wide range of substrates, tolerance to various inhibitors, and high yield of desired products. One approach to develop an ideal host for CBP is to mimic the native cellulolytic microbes. In accordance with this, most native cellulolytic microbes metabolize cellulose in the form of cellobiose to obtain energetic benefits for growth on cellulose and to avoid feed-back inhibition of cellulase by glucose or cellobiose. In this study, we constructed cellobiose-metabolizing Escherichia coli (named as strain OSS, Original Synthetic Strain or ESS, Evolved Synthetic Strain which differ in their ability to ferment cellobiose) by exploiting its native cryptic chb and asc operons with an aim for using it as a platform host for Consolidated bioprocessing (CBP) or Simultaneous Saccharification and Fermentation (SSF) process. In addition to paving way for efficient consolidated bioprocessing, in depth analysis of strain ESS revealed several interesting molecular mysteries and regulations related to the cryptic operons of E. coli. Noteworthy is the significance of the ascB gene of asc operon which was previously considered less significant for growth on cellobiose. Here, we show through a combination of conventional genetics, adaptive evolution and targeted genome engineering that the ascB gene could serve as one of the most efficient β-glucosidases or even a stand-alone β-glucosidase for cellobiose metabolism in E. coli. In addition, we show that this gene ascB is being controlled by another putative promoter within the operon apart from the cryptic promoter of the asc operon thus opening new directions on the evolution and regulation of these operons. A combination of recombinant DNA technology and high-throughput screening process revealed that a combination of these cryptic operons could help in extending the substrate range of E. coli to metabolize several glucosidases including cellobiose, salicin, arbutin, gentiobiose, raffinose, and amygdalin. It is not just sufficient to enhance the cellobiose metabolic rate in order to make a strain more proficient for consolidated bioprocessing; it is also necessary that cellobiose is metabolized as efficiently as glucose. For instance, glucose is metabolized in a respiro-fermentative mode resulting in the secretion of large amount of acetate due to overflow metabolism whereas cellobiose is metabolized in a respirative manner secreting acetate only during the stationary phase. Such differences urge the need to understand and rewire the central carbon metabolism for an efficient cellobiose metabolism. Here we show that rewiring the flux through the TCA cycle could help in enhancing the cellobiose metabolism in E. coli. We demonstrate here that the transcription factor, YebK helps in enhancing the cellobiose metabolic activity through modulations in the central carbon metabolism. YebK recognizes the two major central carbon intermediates, 2-keto-3-deoxy-phosphogluconate (KDPG) and alpha-ketoglutarate (AKG), and co-ordinates the regulations of the TCA cycle of E. coli. Such regulation is particularly dominant during the down-shift of cells from rich nutrient source to a minimal nutrient source. Finally, we also demonstrate the application of the strain ESS, engineered with efficient cellobiose metabolism for co-metabolism of multiple carbon sources or in consolidated bioprocessing for growth with cellulose as a sole carbon source. Thus, the strain ESS would serve as a potential host for consolidated bio-processing or in simultaneous saccharification and fermentation process. The strain ESS could also serve as a molecular bag to decipher challenging queries related to the evolution of the cryptic genes of E. coli. Finally, through this study we also show that the putative transcription factor binding sites or transcription start sites (TSS) reported within the coding/intergenic regions identified through ChIP-sequencing or deep RNA sequencing technologies could serve as a potential target for metabolic engineering and strain optimization.