(632g) Efficient Co-Utilization of Biomass-Derived Sugar Mixtures By Engineered Escherichia coli for Enhanced Production of Aromatic Biochemicals

A significant challenge in biomanufacturing continues to be the efficient co-utilization of mixed sugar feedstocks, which commonly result from biomass hydrolysis. As such, continued efforts to engineer microbes for efficient conversion of substrate mixtures remains critical. Glucose and xylose represent some of the most abundant carbon sources in nature, and are commonly yielded from lignocellulosic waste biomass. Unfortunately, as a result of carbon catabolite repression microbes such as Escherichia coli first catabolize only glucose, after which xylose may then be consumed. However, in addition to reducing productivity, for certain applications, this inherent constraint misses a potential opportunity to realize synergistic benefits associated with the preferential synthesis of unique precursors from glucose vs. xylose, and vice versa. For instance, through glycolysis, glucose catabolism promotes the availability of phosphoenolpyruvate (PEP) whereas xylose catabolism via the pentose phosphate pathway promotes erythrose-4-phosphate (E4P). Since PEP and E4P are the two main precursors to the shikimic acid pathway, we hypothesized that, if efficiently co-utilized, glucose-xylose mixtures might represent and advantaged feedstock for the microbial production of aromatic chemicals. To investigate this prospect and improve our understanding of which precursor may be a limiting, flux balance analysis was performed, through which it was determined that increasing E4P availability would most significantly enhance total carbon-flux through the shikimate pathway. Then, to provide proof-of-concept, a phenylalanine over-producing strain of E. coli was further engineered by introducing a recently discovered mutant copy of the xylose-specific activator XylR (i.e, XylR*). Additional mutations aimed at increasing PEP availability and tuning the glucose consumption rates were also introduced. As a result of these modifications, 100% of the sugars initially provided in a model mixture of 2:1 glucose:xylose (2% (w/v) total sugars) were consumed within 96 h, resulting the production of up to 3.5 g/L L-phenylalanine at a yield of 156 mg/g total sugar in shake flask cultures. Finally, to investigate the utility of this approach with respect to real biomass-derived sugars, the same strain was grown using corn stover hydrolysate (also 2:1 glucose:xylose, here using 1% (w/v) total sugars). In this case, the strain carrying XylR* produced up to 1.2 g/L L-phenylalanine in 25 h (73 mg/g total sugar); representing a ~20% increase over the parent strain. Notably, with the XylR* mutation, maximum rates of xylose consumption were increased by 3-fold (from 0.04 vs. 0.12 g/L-h) without impeding the maximum rate of total sugar consumption (0.44 vs. 0.40g/L-h ). Overall, this study demonstrates a new strategy for improving glucose-xylose co-utilization in aerobic E. coli cultures, and provides evidence to suggest that, when used to target the correct product, substrate mixtures can provide synergistic benefits over single substrates. In particular, we expect that these findings will be readily translated to improve the production of other aromatic chemicals derived from the shikimate pathway.