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Coexpression of a β-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

ABSTRACT: Caldicellulosiruptor species are hyperthermophilic, Gram-positive anaerobes and the most thermophilic cellulolytic bacteria so far described. They have been engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. Xylooligomers, such as xylobiose and xylotriose, that result from the breakdown of plant biomass more strongly inhibit cellulase activity than do glucose or cellobiose. High concentrations of xylobiose and xylotriose are present in C. bescii fermentations after 90 h of incubation, and removal or breakdown of these types of xylooligomers is crucial to achieving high conversion of plant biomass to product. In previous studies, the addition of exogenous β-d-xylosidase substantially improved the performance of glucanases and xylanases in vitro. β-d-Xylosidases are, in fact, essential enzymes in commercial preparations for efficient deconstruction of plant biomass. In addition, the combination of xylanase and β-d-xylosidase is known to exhibit synergistic action on xylan degradation. In spite of its ability to grow efficiently on xylan substrates, no extracellular β-d-xylosidase was identified in the C. bescii genome. Here, we report that the coexpression of a thermal stable β-d-xylosidase from Thermotoga maritima and a xylanase from Acidothermus cellulolyticus in a C. bescii strain containing the A. cellulolyticus E1 endoglucanase significantly increased the activity of the exoproteome as well as growth on xylan substrates. The combination of these enzymes also resulted in increased growth on crystalline cellulose in the presence of exogenous xylan. IMPORTANCE Caldicellulosiruptor species are bacteria that grow at extremely high temperature, more than 75°C, and are the most thermophilic bacteria so far described that are capable of growth on plant biomass. This native ability allows the use of unpretreated biomass as a growth substrate, eliminating the prohibitive cost of preprocessing/pretreatment of the biomass. They only grow under strictly anaerobic conditions, and the combination of high temperature and the lack of oxygen reduces the cost of fermentation and contamination by other microbes. They have been genetically engineered to convert switchgrass to ethanol without pretreatment and represent a promising platform for the production of fuels, chemicals, and materials from plant biomass. In this study, we introduced genes from other cellulolytic bacteria and identified a combination of enzymes that improves growth on plant biomass. An important feature of this study is that it measures growth, validating predictions made from adding enzyme mixtures to biomass.

SUBMITTER: Kim SK

PROVIDER: S-EPMC8231722 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The ability to deconstruct plant biomass without conventional pretreatment has made members of the genus Caldicellulosiruptor the target of investigation for the consolidated processing of lignocellulosic biomass to biofuels and bioproducts. These Gram-positive bacteria are hyperthermophilic anaerobes and the most thermophilic cellulolytic organisms so far described. They use both C5 and C6 sugars simultaneously and have the ability to grow well on xylan, a major component of plant cell walls. This is an important advantage for their use to efficiently convert biomass at yields sufficient for an industrial process. For commodity chemicals, yield from substrate is perhaps the most important economic factor. In an attempt to improve even further the ability of C. bescii to use xylan, we introduced two xylanases from Acidothermus cellulolyticus. Acel_0180 includes tandem carbohydrate-binding modules (CBM2 and CBM3) located at the C-terminus, one of which, CBM2, is not present in C. bescii. Also, the sequences of Xyn10A and Acel_0180 have very little homology with the GH10 domains present in C. bescii. For these reasons, we selected these xylanases as potential candidates for synergistic interaction with those in the C. bescii exoproteome. RESULTS:Heterologous expression of two xylanases from Acidothermus cellulolyticus in Caldicellulosiruptor bescii resulted in a modest, but significant increase in the activity of the exoproteome of C. bescii on xylan substrates. Even though the increase in extracellular activity was modest, the ability of C. bescii to grow on these substrates was dramatically improved suggesting that the xylan substrate/microbe interaction substantially increased deconstruction over the secreted free enzymes alone. CONCLUSIONS:We anticipate that the ability to efficiently use xylan, a major component of plant cell walls for conversion of plant biomass to products of interest, will allow the conversion of renewable, sustainable, and inexpensive plant feedstocks to products at high yields.

Project description:Background:Caldicellulosiruptor bescii, a promising biocatalyst being developed for use in consolidated bioprocessing of lignocellulosic materials to ethanol, grows poorly and has reduced conversion at elevated medium osmolarities. Increasing tolerance to elevated fermentation osmolarities is desired to enable performance necessary of a consolidated bioprocessing (CBP) biocatalyst. Results:Two strains of C. bescii showing growth phenotypes in elevated osmolarity conditions were identified. The first strain, ORCB001, carried a deletion of the FapR fatty acid biosynthesis and malonyl-CoA metabolism repressor and had a severe growth defect when grown in high-osmolarity conditions-introduced as the addition of either ethanol, NaCl, glycerol, or glucose to growth media. The second strain, ORCB002, displayed a growth rate over three times higher than its genetic parent when grown in high-osmolarity medium. Unexpectedly, a genetic complement ORCB002 exhibited improved growth, failing to revert the observed phenotype, and suggesting that mutations other than the deleted transcription factor (the fruR/cra gene) are responsible for the growth phenotype observed in ORCB002. Genome resequencing identified several other genomic alterations (three deleted regions, three substitution mutations, one silent mutation, and one frameshift mutation), which may be responsible for the observed increase in osmolarity tolerance in the fruR/cra-deficient strain, including a substitution mutation in dnaK, a gene previously implicated in osmoresistance in bacteria. Differential expression analysis and transcription factor binding site inference indicates that FapR negatively regulates malonyl-CoA and fatty acid biosynthesis, as it does in many other bacteria. FruR/Cra regulates neighboring fructose metabolism genes, as well as other genes in global manner. Conclusions:Two systems able to effect tolerance to elevated osmolarities in C. bescii are identified. The first is fatty acid biosynthesis. The other is likely the result of one or more unintended, secondary mutations present in another transcription factor deletion strain. Though the locus/loci and mechanism(s) responsible remain unknown, candidate mutations are identified, including a mutation in the dnaK chaperone coding sequence. These results illustrate both the promise of targeted regulatory manipulation for osmotolerance (in the case of fapR) and the challenges (in the case of fruR/cra).

Dataset Information

Coexpression of a β-d-Xylosidase from Thermotoga maritima and a Family 10 Xylanase from Acidothermus cellulolyticus Significantly Improves the Xylan Degradation Activity of the Caldicellulosiruptor bescii Exoproteome.

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