Cloning, sequencing, and characterization of the bifunctional xylosidase–arabinosidase from the anaerobic thermophile Thermoanaerobacter ethanolicus

The axy43A gene encoding the intracellular trifunctional xylanolytic enzyme from Paenibacillus curdlanolyticus B-6 was cloned and expressed in Escherichia coli. Recombinant PcAxy43A consisting of a glycoside hydrolase family 43 and a family 6 carbohydrate-binding module exhibited endo-xylanase, ␤-xylosidase, and arabinoxylan arabinofuranohydrolase activities. PcAxy43A hydrolyzed xylohexaose and birch wood xylan to release a series of xylooligosaccharides, indicating that PcAxy43A contained endo-xylanase activity. PcAxy43A exhibited ␤-xylosidase activity toward a chromogenic substrate, p-nitrophenyl-␤-D-xylopyranoside, and xylobiose, while it preferred to hydrolyze long-chain xylooligosaccharides rather than xylobiose. In addition, surprisingly, PcAxy43A showed arabinoxylan arabinofuranohydrolase activity; that is, it released arabinose from both singly and doubly arabinosylated xylose,Moreover, the combination of PcAxy43A and P. curdlanolyticus B-6 endo-xylanase Xyn10C greatly improved the efficiency of xylose and arabinose production from the highly substituted rye arabinoxylan, suggesting that these two enzymes function synergistically to depolymerize arabinoxylan. Therefore, PcAxy43A has the potential for the saccharification of arabinoxylan into simple sugars for many applications. IMPORTANCEIn this study, the glycoside hydrolase 43 (GH43) intracellular multifunctional endo-xylanase, ␤-xylosidase, and arabinoxylan arabinofuranohydrolase (AXH) from P. curdlanolyticus B-6 were characterized. Interestingly, PcAxy43A AXH showed a new property that acted on both the C(O)-2 and C(O)-3 positions of xylose residues doubly substituted with arabinosyl, which usually obstruct the action of xylanolytic enzymes. Furthermore, the studies here show interesting properties for the processing of xylans from cereal grains, particularly rye arabinoxylan, and show a novel relationship between PcAxy43A and endo-xylanase Xyn10C from strain B-6, providing novel metabolic potential for processing arabinoxylans into xylose and arabinose.

Section: Resultssupporting

confidence: 85%

mentioning

confidence: 99%

Novel Trifunctional Xylanolytic Enzyme Axy43A from Paenibacillus curdlanolyticus Strain B-6 Exhibiting Endo-Xylanase, β- d -Xylosidase, and Arabinoxylan Arabinofuranohydrolase Activities

Teeravivattanakit

Baramee

Phitsuwan

et al. 2016

“…6). Genes encoding a bifunctional xylanosidase/arabinosidase (xarB), associated cellobiose transporters (xarG), and ␤-glucosidase (cglT) have previously been cloned and characterized for Thermoanaerobacter ethanolicus JW200 (25). The genomes of 39E and X514 were compared to genomes of eight additional Thermoanaerobacter species to determine if the variability of this region extends across multiple Thermoanaerobacter species and if this variability can be linked to observed physiological phenotypes (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Glucose (25,50,75, 100, and 150 mg/liter) was used as the standard. Samples (200 l) were diluted to the appropriate concentrations and mixed with 200 l 5% phenol and 1 ml of 98% H 2 SO 4 .…”

Section: Whole-genome Sequencing and Analysis (I) Organism Source Anmentioning

confidence: 99%

Correlation of Genomic and Physiological Traits of Thermoanaerobacter Species with Biofuel Yields

Hemme

Fields

et al. 2011

Self Cite

Thermophilic anaerobic noncellulolytic Thermoanaerobacter species are of great biotechnological importance in cellulosic ethanol production due to their ability to produce high ethanol yields by simultaneous fermentation of hexose and pentose. Understanding the genome structure of these species is critical to improving and implementing these bacteria for possible biotechnological use in consolidated bioprocessing schemes (CBP) for cellulosic ethanol production. Here we describe a comparative genome analysis of two ethanologenic bacteria, Thermoanaerobacter sp. X514 and Thermoanaerobacter pseudethanolicus 39E. Compared to 39E, X514 has several unique key characteristics important to cellulosic biotechnology, including additional alcohol dehydrogenases and xylose transporters, modifications to pentose metabolism, and a complete vitamin B 12 biosynthesis pathway. Experimental results from growth, metabolic flux, and microarray gene expression analyses support genome sequencing-based predictions which help to explain the distinct differences in ethanol production between these strains. The availability of whole-genome sequence and comparative genomic analyses will aid in engineering and optimizing Thermoanaerobacter strains for viable CBP strategies.Global energy demands will increase significantly in coming decades (16), and renewable energy sources such as biofuels have been proposed to help reduce dependence upon fossil energy (8,23,27,49). Current efforts focus on biofuel production from renewable lignocellulosic feedstock (e.g., switchgrass), which constitutes ϳ50% of the world's biomass (2,8,23,27,49). Although intensive research and development have been performed on the effective utilization of lignocellulose, problems associated with practical use of this material have not been resolved fully (13). When enzymatic hydrolysis is adopted for cellulosic ethanol production, different levels of process integration can be visualized: (i) separate (or sequential) hydrolysis and fermentation (SHF), where the enzymes (cellulases) are used separately from fermentation tanks; (ii) simultaneous saccharification and fermentation (SSF), which consolidates enzymatic hydrolysis with fermentation of hexose or pentose; (iii) simultaneous saccharification and cofermentation (SSCF), which further combines the fermentation of hexose and pentose together; and (iv) consolidated bioprocessing (CBP), where all required enzymes and ethanol are produced in a single reactor (1,13,24,29,46,51). While these production schemes represent increasing levels of simplification through process consolidation, consolidation of multiple steps often results in a loss of process efficiency. Thus, improving the efficiency of individual steps, such as cellulose hydrolysis and ethanol fermentation, remains an important task for the development of economically feasible cellulosic bioethanol.Recent efforts have focused on metabolic engineering and (more recently) synthetic biology to produce strains or consortia capable of producing biofuels. Efforts to ...

“…Xylodextrin utilization has been demonstrated in a variety of bacteria (10,15,32,40,43,44,47). Bacillus stearothermophilus contains a gene cluster involved in the transport and metabolism of large soluble products from methylglucuronoxylan (40).…”

mentioning

confidence: 99%

Cloning, Characterization, and Functional Expression of the Klebsiella oxytoca Xylodextrin Utilization Operon ( xynTB ) in Escherichia coli

Qian

Yomano

Preston

et al. 2003

Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na ؉ /melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.