Abstract
BackgroundWe previously developed an E. coli strain that overproduces medium-chain methyl ketones for potential use as diesel fuel blending agents or as flavors and fragrances. To date, the strain’s performance has been optimized during growth with glucose. However, lignocellulosic biomass hydrolysates also contain a substantial portion of hemicellulose-derived xylose, which is typically the second most abundant sugar after glucose. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose.ResultsIn this study, genetic manipulations were performed to alleviate carbon catabolite repression in our most efficient methyl ketone-producing strain. A strain engineered for constitutive expression of xylF and xylA (involved in xylose transport and metabolism) showed synchronized glucose and xylose consumption rates. However, this newly acquired capability came at the expense of methyl ketone titer, which decreased fivefold. Further efforts were made to improve methyl ketone production in this strain, and we found that two strategies were effective at enhancing methyl ketone titer: (1) chromosomal deletion of pgi (glucose-6-phosphate isomerase) to increase intracellular NADPH supply and (2) downregulation of CRP (cAMP receptor protein) expression by replacement of the native RBS with an RBS chosen based upon mutant library screening results. Combining these strategies resulted in the most favorable overall phenotypes for simultaneous glucose–xylose consumption without compromising methyl ketone titer at both 1 and 2% total sugar concentrations in shake flasks.ConclusionsThis work demonstrated a strategy for engineering simultaneous utilization of C6 and C5 sugars in E. coli without sacrificing production of fatty acid-derived compounds.
Highlights
We previously developed an E. coli strain that overproduces medium-chain methyl ketones for poten‐ tial use as diesel fuel blending agents or as flavors and fragrances
The glucose-specific EII complex of the PTS system consists of the permease, EIIBCGlc, and EIIAGlc, which has a primary role in modulating carbohydrate metabolism in E. coli
E IIAGlc is dephosphorylated, which prevents either the import of non-glucose sugars or their subsequent metabolism, and as a consequence, bacterial cells are devoid of the inducer for the corresponding operons; this is known as inducer exclusion [8]
Summary
We previously developed an E. coli strain that overproduces medium-chain methyl ketones for poten‐ tial use as diesel fuel blending agents or as flavors and fragrances. Commercialization of the methyl ketone-producing technology would benefit from the increased efficiency resulting from simultaneous, rather than the native sequential (diauxic), utilization of glucose and xylose. Wang et al Microb Cell Fact (2018) 17:12 technology would benefit from the increased efficiency resulting from simultaneous utilization of glucose and xylose [6]. A challenge in cultivating E. coli in growth medium containing both glucose and xylose is diauxic (phased, non-simultaneous) growth, whereby glucose must be depleted before other sugars, such as xylose, can be metabolized [7]. The expression of genes that are involved in the catabolism of sugars other than glucose generally requires the cAMP–CRP complex and, is repressed in the presence of glucose. The arabinose transcriptional regulator (AraC) suppresses the xylose-catabolism genes xylAB and xylFGH by inhibiting the xylose transcriptional activator (XylR), which constitutes the second layer of CCR [9]
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