Emulsion-based evolution of Escherichia coli for higher growth yield on D-xylose identifies central role of cyclic AMP

Abstract

D-xylose is an abundant sugar found in plant biomass and can be used as a renewable feedstock for the microbial production of diverse biofuels and bioproducts. However, D-xylose metabolism is slow in many industrial microorganisms, at least as compared to glucose metabolism. Not surprisingly, a number of approaches have been developed for improving D-xylose metabolism in diverse microorganisms. In this work, we applied a previously developed evolution strategy based on media-in-oil emulsions for improving the growth yield of Escherichia coli NCM3722 on D-xylose. After 30 rounds of evolutions, we isolated multiple mutants with increased growth yield on D-xylose. In addition, we also observed similar increases in the growth rate. Three mutants were selected for whole-genome sequencing. Two mutants had an amber stop mutation in adenylate cyclase, which truncates nearly 60% of the enzyme. However, the ability of this mutant to grow on xylose indicated that truncated enzyme, lacking the C-terminal regulatory domain, is still active. The other mutant had a point mutation in the cyclic AMP receptor protein (CRP), near the high affinity binding site for cyclic AMP. Both mutations, when introduced into wild type E. coli, were able to increase the growth yields at levels similar to the isolated mutants. In addition to D-xylose, these mutant strains and their genetic mimics also exhibited higher growth rates and yields on glucose, lactose, and L-arabinose. These results suggest that the improved growth rates and yields are due to changes in the production and sensing of intracellular cyclic AMP concentrations and also suggest native concentrations are suboptimal with respect to the growth rate and yield under the growth conditions tested. Collectively, these results may prove useful for engineering strains of E. coli for high-density fermentations or protein production.