Multi-scale approaches to overcome endogenous limits of natural metabolism for efficient biosynthesis

Abstract

Abundance of enzymes and metabolic pathways in nature made a potential to effectively produce the huge number of chemicals with the aid of metabolic engineering. However, in spite of considerable achievements by various strategies of metabolic engineering, there is a still limit line in actual production performance caused by endogenous limitations which the production hosts possess. These limitations are placed in difference scales: by inefficiency of metabolic pathways and enzymes. However, because the shortage of efficiency in the pathways and enzymes are defined as the viewpoints of chemical production, cell metabolism been optimized for survival of cells could show the discrepancy between their endogenous design and ideal design for chemical production in several metabolic scales. In spite of these limitations, the tremendous plenty of metabolic pathways composed by enzymatic reactions and a characteristic of enzyme known as promiscuity can be a potential to overcome these endogenous limitations. Introduction of alternative enzymatic reactions to replace the reaction with limited efficiency could give a chance to improve the efficiency of cellular metabolism. Also, enzyme promiscuity can be a driving force to develop the new enzyme with improved performance. And these new enzymes can be constructed by several approaches of enzyme engineering. The followings are summarized contents in this study. First, metabolic pathways in central carbon metabolism were engineered to keep conserving more carbon flux toward the itaconate with minimizing the loss of redox energy and carbon frame. The carbon loss steps in the glycolysis were blocked at phosphoenolpyruvate node to stop the progress of carbon flux toward the decarboxylation of pyruvate. And for smooth entrance of carbon flux toward the TCA cycle, the co-utilization of glucose and acetate was induced with redirecting the glucose-derived flux to pass through anaplerotic CO2 fixation step. Finally, additional flux optimization between TCA cycle and itaconate production by transcription factor- mediating downregulation of icd gene led to considerable improvement of itaconate yield which was higher than the theoretical maximum carbon yield. Second, it is known that aconitase is inefficient for supply of cis-aconitate for the itaconate production and natural eukaryotic producer of itaconate applies physical barrier to secure the cis-aconitate with the help of membranous organelle. However, in case of bacteria which generally have no membranous structure, they cannot use the previous strategy. Instead of that, the new cis-aconitate synthesizing enzyme was developed by application of evolutionary approach with the support of biosensor-aided high throughput screening. The new enzyme was engineered based on 2-methylcitrate dehydratase (PrpD) catalyzing 2-methylcitrate structurally similar with citrate. The newly developed enzyme showed the changed ligand specificity toward the citrate instead of the original substrate. The introduction of effective mutant led to improvement of itaconate production by kinetically separating carbon flux from TCA cycle depending on the enzymatic reaction of PrpD mutant. And additional flux optimization made the itaconate production considerably improved with higher yield.