Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrial Escherichia coli strains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94% of a glucose–xylose mixture (50 g⋅L−1 each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild type, suggesting its potential wide application in industrial E. coli biocatalysts.
Polyamines are important metabolites in plant development and abiotic and biotic stress responses. Copper-containing amine oxidases (CuAOs) are involved in the regulation of polyamine levels in the cell. CuAOs oxidize primary amines to their respective aldehydes and hydrogen peroxide. In plants, aldehydes are intermediates in various biosynthetic pathways of alkaloids. CuAOs are thought to oxidize polyamines at only one of the primary amino groups, a process frequently resulting in monocyclic structures. These oxidases have been postulated to be involved in pyrrolizidine alkaloid (PA) biosynthesis. Here, we describe the identification and characterization of homospermidine oxidase (HSO), a CuAO of Heliotropium indicum (Indian heliotrope), involved in PA biosynthesis. Virus-induced gene silencing of HSO in H. indicum leads to significantly reduced PA levels. By in vitro enzyme assays after transient in planta expression, we show that this enzyme prefers homospermidine over other amines. NMR and MS analyses of the reaction products demonstrate that HSO oxidizes both primary amino groups of homospermidine to form a bicyclic structure, 1-formylpyrrolizidine. Using tracer feeding, we have further revealed that 1-formylpyrrolizidine is an intermediate in the biosynthesis of PAs. Our study therefore establishes that HSO, a canonical CuAO, catalyzes the second step of PA biosynthesis and provides evidence for an undescribed and unusual mechanism involving two discrete steps of oxidation that might also be involved in the biosynthesis of complex structures in other alkaloidal pathways.
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.
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