Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis

Abstract

Shewanella oneidensis is unable to metabolize the sugar xylose as a carbon and energy source. In the present study, an otherwise silent xylose catabolic pathway was activated in S. oneidensis by following an adaptive evolution strategy. Genome-wide scans indicated that the S. oneidensis genome encoded two proteins similar to the xylose oxido-reductase pathway enzymes xylose reductase (SO_0900) and xylulokinase (SO_4230), and purified SO_0900 and SO_4230 displayed xylose reductase and xylulokinase activities, respectively. The S. oneidensis genome was missing, however, an Escherichia coli XylE-like xylose transporter. After 12 monthly transfers in minimal growth medium containing successively higher xylose concentrations, an S. oneidensis mutant (termed strain XM1) was isolated for the acquired ability to grow aerobically on xylose as a carbon and energy source. Wholegenome sequencing indicated that strain XM1 contained a mutation in an unknown membrane protein (SO_1396) resulting in a glutamine-to-histidine conversion at amino acid position 207. Homology modeling demonstrated that the Q207H mutation in SO_1396 was located at the homologous xylose docking site in XylE. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis. Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. IMPORTANCEThe activation of an otherwise silent xylose metabolic system in Shewanella oneidensis is a powerful example of how accidental mutations allow microorganisms to adaptively evolve. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis. Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. Xylose is one of the primary products of lignocellulose degradation and, after glucose, is the second most abundant carbohydrate in nature. The xylose polymer xylan is the primary constituent of hemicellulose, which comprises approximately 17% of the dry weight of hardwoods and up to 31% of plants (1). Xylose catabolism is a key component of sustainable processes that produce useful secondary products from lignocellulosic biomass (2-4). Xylose catabolism is also of commercial interest because xylose conversion to useful secondary chemicals such as bioethanol and biodegradable plastics can reduce losses associated with lignocellulose bioprocessing (5, 6). Industrially important by-products of xylose metabolism include xylitol, which is used as a natural sweetener in the food and confectionary industries (7).Xylose metabolic pathways include the oxido-reductase, isomerase, and Weimberg-Dahms pathways (Fig. 1) (1, 8). Extracellular xylose is transported inside the cell via the ATP-bindi...

Understanding and engineering electrochemically active bacteria for sustainable biotechnology

“…NAG: N-acetylglucosamine; NAG-6P: N-acetylglucosamine-6-phosphate; KDG-6P: 2-keto-3-deoxygluconate 6-phosphate; PEP: phosphoenolpyruvate; Q: quinone; QH 2 : quinol; NagP: N-acetylglucosamine transporter; NagK: N-acetylglucosamine kinase; NagA: N-acetylglucosamine-6-phosphate deacetylase; NagB: glucosamine/fructose-6-phosphate aminotransferase; Fbp: fructose-1,6-bisphosphatase; Fba: fructose-bisphosphate aldolase; TpiA: triosephosphate isomerase; Pgi: glucose-6-phosphate isomerase; Zwf: glucose-6-phosphate 1-dehydrogenase; Edd: phosphogluconate dehydratase; Eda: 2-dehydro-3-deoxyphosphogluconate aldolase/ (4S)-4-hydroxy-2-oxoglutarate aldolase; PpsA: pyruvate, water dikinase; PckA: phosphoenolpyruvate carboxykinase; Pdh: pyruvate dehydrogenase; GltA: citrate synthase; Acn: aconitate hydratase; Icd: isocitrate dehydrogenase; SucAB: 2-oxoglutarate dehydrogenase; SucCD: succinyl-CoA synthetase; Sdh: succinate dehydrogenase/fumarate reductase; Fum: fumarate reductase; Mdh: malate dehydrogenase; AceB: malate synthase; AceA: isocitrate lyase; Pta: phosphate acetyltransferase; AckA: acetate kinase; Acs: acetyl-CoA synthetase; Fdh: formate dehydrogenase; Dld: d-lactate dehydrogenase, quinone dependent; Lld: l-lactate dehydrogenase; LdhA: d-lactate dehydrogenase, NAD dependent; Glf: glucose uniporter; GalP: galactose:H + symporter; Glk: glucokinase; Xks1: xylulokinase; Xyl2: xylitol dehydrogenase; Xyl1: NAD(P)H-dependent xylose reductase and xylulokinase (SO_4230) (Sekar et al 2016), while a gene for xylose transporter is not present. Adaptive evolution is a powerful tool to confer a missing function on a bacterial strain, and a xylose-utilizing mutant was obtained from MR-1 after incubation in media containing a high concentration of xylose (Sekar et al 2016). Whole genome sequencing revealed a single nucleotide mutation in a gene encoding unknown membrane protein (SO_1396), which resulted in the substitution of a glutamate residue to histidine and conferred the activity to bind to and transport xylose (Sekar et al 2016 Pyruvate is a major product of glycolysis and utilized in diverse catabolic and anabolic pathways, including gluconeogenesis, pyruvate fermentation, amino-acid biosynthesis and the TCA cycle.…”

“…Adaptive evolution is a powerful tool to confer a missing function on a bacterial strain, and a xylose-utilizing mutant was obtained from MR-1 after incubation in media containing a high concentration of xylose (Sekar et al 2016). Whole genome sequencing revealed a single nucleotide mutation in a gene encoding unknown membrane protein (SO_1396), which resulted in the substitution of a glutamate residue to histidine and conferred the activity to bind to and transport xylose (Sekar et al 2016 Pyruvate is a major product of glycolysis and utilized in diverse catabolic and anabolic pathways, including gluconeogenesis, pyruvate fermentation, amino-acid biosynthesis and the TCA cycle. Among known enzymes for pyruvate oxidation, MR-1 has a pyruvate dehydrogenase complex (PDH, comprised of AceEF and LpdA, SO_0424 to SO_0426) and pyruvate formate lyase (Pfl, SO_2912).…”

Direct conversion of cellulose and hemicellulose to fermentable sugars by a microbially-driven Fenton reaction

“…Wild-type S. oneidensis is unable to metabolize xylose as sole carbon and energy source (Rodionov et al, 2010). Thus, to determine if the xylan degradation products resulting from the microbially-driven Fenton reaction may be fermented to useful bioproducts, spent culture supernatants of xylan degradation products were fed to genetically engineered S. oneidensis strain XM1 (previously isolated for the acquired ability to uptake and grow aerobically on xylose as carbon and energy source) (Sekar et al, 2016). Strain XM1 was genetically engineered to harbor the polyhydroxybutyrate (PHB) biosynthetic gene cassette phaCAB (Steinbuchel, 2001).…”

Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell

“…Firstly, an adaptive evolution approach was developed to activate an otherwise silent xylose metabolic pathway, i.e. oxidoreductase pathway in the WT S. oneidensis , thus generating a S. oneidensis mutant XM1 that could metabolize xylose as the sole carbon and energy source [ 56 ]. Secondly, microbial consortia including fermenters and exoelectrogens were developed to accomplish xylose-powered MFCs, in which the engineered Escherichia coli played as a fermenter to metabolize xylose for the synthesis of metabolites such as lactate and formate to feed the S. oneidensis as the carbon source and electron donor, thus enabling an indirect utilization of xylose by S. oneidensis for bioelectricity production [ 24 ].…”