Engineering Geobacillus thermodenitrificans to introduce cellulolytic activity; expression of native and heterologous cellulase genes.

BACKGROUND: Consolidated bioprocessing (CBP) is a cost-effective approach for the conversion of lignocellulosic biomass to biofuels and biochemicals. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and ß-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to harbor desired features for CBP, although it lacks the desired endo and exoglucanases required for the conversion of cellulose. Here, we report the expression of both endoglucanase and exoglucanase encoding genes by G. thermodenitrificans T12, in an initial attempt to express cellulolytic enzymes that complement the enzymatic machinery of this strain. RESULTS: A metagenome screen was performed on 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 (GH5) were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-ß-glucanase from S. cerevisiae. However, we determined GE39 to be a ß-xylosidase having pronounced activity towards p-nitrophenyl-ß-D-xylopyranoside. Structure modelling of GE40 revealed its protein architecture to be similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic activity against 2-hydroxyethylcellulose, carboxymethylcellulose and barley ß-glucan. Additionally, we introduced expression constructs into T12 containing Geobacillus sp. 70PC53 endoglucanase gene celA and both endoglucanase genes (M1 and M2) from Geobacillus sp. WSUCF1. Finally, we introduced expression constructs into T12 containing the C. thermocellum exoglucanases celK and celS genes and the endoglucanase celC gene. CONCLUSIONS: We identified a novel G. thermodenitrificans ß-xylosidase (GE39) and a novel endoglucanase (GE40) using a metagenome screen based on multiple HMM profiles. We successfully expressed both genes in E. coli and functionally expressed the GE40 endoglucanase in G. thermodenitrificans T12. Additionally, the heterologous production of active CelK, a C. thermocellum derived exoglucanase, and CelA, a Geobacillus derived endoglucanase, was demonstrated with strain T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of G. thermodenitrificans T12 as a host for consolidated bioprocessing.

Complete Genome Sequence of Geobacillus thermodenitrificans T12, A Potential Host for Biotechnological Applications.

In attempt to obtain a thermophilic host for the conversion of lignocellulose derived substrates into lactic acid, Geobacillus thermodenitrificans T12 was isolated from a compost heap. It was selected from over 500 isolates as a genetically tractable hemicellulolytic lactic acid producer, requiring little nutrients. The strain is able to ferment glucose and xylose simultaneously and can produce lactic acid from xylan, making it a potential host for biotechnological applications. The genome of strain T12 consists of a 3.64 Mb chromosome and two plasmids of 59 and 56 kb. It has a total of 3.676 genes with an average genomic GC content of 48.7%. The T12 genome encodes a denitrification pathway, allowing for anaerobic respiration. The identity and localization of the responsible genes are similar to those of the denitrification pathways found in strain NG80-2. The hemicellulose utilization (HUS) locus was identified based on sequence homology against G. stearothermophilus T-6. It appeared that T12 has all the genes that are present in strain T-6 except for the arabinan degradation cluster. Instead, the HUS locus of strain T12 contains genes for both an inositol and a pectate degradation pathway. Strain T12 has complete pathways for the synthesis of purine and pyrimidine, all 20 amino acids and several vitamins except D-biotin. The host-defense systems present comprise a Type II and a Type III restriction-modification system, as well as a CRISPR-Cas Type II system. It is concluded that G. thermodenitrificans T12 is a potentially interesting candidate for industrial applications.

Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12.

BACKGROUND: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. RESULTS: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 °C and 80 °C, with an optimum activity at 60 °C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 °C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. CONCLUSIONS: The G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 °C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates.

Isolation of a genetically accessible thermophilic xylan degrading bacterium from compost.

BACKGROUND: Due to the finite nature of global oil resources we are now faced with the challenge of finding renewable resources to produce fuels and chemicals in the future. Lactic acid has great potential as a precursor for the production of bioplastics alternatives to conventional plastics. Efficient lactic acid fermentation from non-food lignocellulosic substrates requires pretreatment and saccharification to generate fermentable sugars. A fermentation process that requires little to no enzyme additions, i.e. consolidated bioprocessing would be preferred and requires lactic acid-producing organisms that have cellulolytic and/or hemicellulolytic activity. RESULTS: To obtain candidate production strains we have enriched and isolated facultative anaerobic (hemi) cellulolytic bacterial strains from compost samples. By selecting for growth on both cellulose and xylan, 94 Geobacillus strains were isolated. Subsequent screening for lactic acid production was carried out from C6 and C5 sugar fermentations and a selection of the best lactic acid producers was made. The denitrifying Geobacillus thermodenitrificans T12 was selected for further research and was rendered genetically accessible. In fermentations on a mixture of glucose and xylose, a total of 20.3 g of lactic acid was produced with a yield of 0.94 g product/g sugar consumed. In addition, strain T12 is capable of direct conversion of beech wood xylan to mainly lactic acid in minimal media. CONCLUSIONS: We have demonstrated that G. thermodenitrificans T12 is genetically accessible and produces lactic acid as its main fermentation product on glucose, xylose and a mixture thereof. Strain T12 was additionally used for the direct conversion of xylan to lactic acid. The genetic accessibility of the T12 strain provides a solid basis for the development of this strain into a host for consolidated bioprocessing of biomass to lactic acid.

Isolation and screening of thermophilic bacilli from compost for electrotransformation and fermentation: characterization of Bacillus smithii ET 138 as a new biocatalyst.

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216(T) and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10(3) colonies per µg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.