Parallel experimental evolution reveals a novel repressive control of GalP on xylose fermentation in Escherichia coli

Abstract

Efficient xylose utilization will facilitate microbial conversion of lignocellulosic sugar mixtures into valuable products. In Escherichia coli, xylose catabolism is controlled by carbon catabolite repression (CCR). However, in E. coli such as the succinateproducing strain KJ122 with disrupted CCR, xylose utilization is still inhibited under fermentative conditions. To probe the underlying genetic mechanisms inhibiting xylose utilization, we evolved KJ122 to enhance its xylose fermentation abilities in parallel and characterized the potential convergent genetic changes shared by multiple independently evolved strains. Whole-genome sequencing revealed that convergent mutations occurred in the galactose regulon during adaptive laboratory evolution potentially decreasing the transcriptional level or the activity of GalP, a galactose permease. We showed that deletion of galP increased xylose utilization in both KJ122 and wild-type E. coli, demonstrating a common repressive role of GalP for xylose fermentation. Concomitantly, induced expression of galP from a plasmid repressed xylose fermentation. Transcriptome analysis using RNA sequencing indicates that galP inactivation increases transcription levels of many catabolic genes for secondary sugars including xylose and arabinose. The repressive role of GalP for fermenting secondary sugars in E. coli suggests that utilization of GalP as a substitute glucose transporter is undesirable for conversion of lignocellulosic sugar mixtures.

Directed evolution of Zymomonas mobilis sugar facilitator Glf to overcome glucose inhibition

“…This result indicates that Glf is able to serve as an efficient transporter for both glucose and xylose. Our previous work suggests that utilization of GalP as a substitute glucose transporter is undesirable for conversion of lignocellulosic sugar mixtures because GalP represses xylose fermentation and uses proton gradients (Kurgan, Sievert, et al, 2019 ). Thus, Glf is a more suitable substitute glucose transporter for lignocellulosic bioconversion compared to GalP since Glf is more energy efficient and has no repressive effect on xylose fermentation.…”

“…When performing cosugar fermentations, 66 g l –1 glucose and 34 g l –1 xylose were used, a typical mass ratio representative of lignocellulosic sugar compositions (Saha, 2003 ). 100 mg l –1 ampicillin and 10 µM IPTG were included in all fermentation tests for the cells with plasmids to maintain the plasmids and induce suitable expression for transporter genes as previously described (Kurgan, et al, 2019a , b ). All batch fermentations were inoculated using an initial OD 550nm of 0.05 and fermentation was controlled at 37°C and pH 7.0 with automatic additions of 6 M potassium hydroxide.…”

Glucose Transport in Escherichia coli: From Basics to Transport Engineering

“…Remarkably, resulting PTS − mutants abolishing the CCR or inducer exclusion mechanisms can simultaneously use glucose and xylose mixtures as carbon sources for production metabolite purposes such as succinate and cis, cis-muconic acid, demonstrating that product yield and productivity were significantly improved in these PTS − mutants ( Table 3 ). The overexpression of GalP in the Δ galP derivative strain of E. coli KJ122 showed a negative regulatory control mechanism of the expression of catabolic genes of other sugars such as xylose and arabinose, suggesting that the selection of GalP as the leading glucose transporter in the PTS − derivative was an efficient strategy [ 28 , 82 ].…”

Biosynthesis of value-added bioproducts from hemicellulose of biomass through microbial metabolic engineering

“…It has been found that some intermediates produced from glucose can inhibit the transcription of genes for other sugars' metabolism ( Lu et al, 2016 ; Vinuselvi et al, 2012 ). CCR is a global regulatory mechanism of bacteria, and about 5–10% of genes in bacteria are regulated by CCR ( Kurgan et al, 2019 ).…”