Dataset Information

Genomic and transcriptomic analysis of the thermophilic lignocellulose-degrading fungus Thielavia terrestris LPH172.

ABSTRACT:

Background

Biomass-degrading enzymes with improved activity and stability can increase substrate saccharification and make biorefineries economically feasible. Filamentous fungi are a rich source of carbohydrate-active enzymes (CAZymes) for biomass degradation. The newly isolated LPH172 strain of the thermophilic Ascomycete Thielavia terrestris has been shown to possess high xylanase and cellulase activities and tolerate low pH and high temperatures. Here, we aimed to illuminate the lignocellulose-degrading machinery and novel carbohydrate-active enzymes in LPH172 in detail.

Results

We sequenced and analyzed the 36.6-Mb genome and transcriptome of LPH172 during growth on glucose, cellulose, rice straw, and beechwood xylan. 10,128 predicted genes were found in total, which included 411 CAZy domains. Compared to other fungi, auxiliary activity (AA) domains were particularly enriched. A higher GC content was found in coding sequences compared to the overall genome, as well as a high GC3 content, which is hypothesized to contribute to thermophilicity. Primarily auxiliary activity (AA) family 9 lytic polysaccharide monooxygenase (LPMO) and glycoside hydrolase (GH) family 7 glucanase encoding genes were upregulated when LPH172 was cultivated on cellulosic substrates. Conventional hemicellulose encoding genes (GH10, GH11 and various CEs), as well as AA9 LPMOs, were upregulated when LPH172 was cultivated on xylan. The observed co-expression and co-upregulation of genes encoding AA9 LPMOs, other AA CAZymes, and (hemi)cellulases point to a complex and nuanced degradation strategy.

Conclusions

Our analysis of the genome and transcriptome of T. terrestris LPH172 elucidates the enzyme arsenal that the fungus uses to degrade lignocellulosic substrates. The study provides the basis for future characterization of potential new enzymes for industrial biomass saccharification.

SUBMITTER: Tolgo M

PROVIDER: S-EPMC8176577 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii Furthermore, a facile approach for assessing genomic stability in C. bescii has been established.IMPORTANCECaldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies.

Project description:Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry. Thermobifida fusca was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, T. fusca showed good growth and secreted enzymes when cocultured with Thermomyces lanuginosus. T. lanuginosus grew firstly during the initial stage, whereas T. fusca dominated the system subsequently during cocultivation. The secretome indicated that T. lanuginosus mainly degraded xylan by expressing a GH11 xylanase (g4601.t1, GenBank AAB94633.1; with relative secretion of 4.95 ± 0.65%). T. fusca was induced by xylan mainly to secrete a xylanase from GH11 family (W8GGR4, GenBank AHK22788.1; with relative secretion of 8.71 ± 3.83%) which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations (>0.5%, w/v) of XOS or xylose suppressed the growth of T. fusca; which may be the reason why T. fusca unable to grow alone in corn stalk solid medium. However, T. lanuginosus could utilize the XOS and xylose produced by xylanases secreted by T. fusca. During the synergistic degradation of lignocellulose by T. lanuginosus and T. fusca, xylan was rapidly consumed by T. lanuginosus, the residual cellulose could specifically induced T. fusca to express a GH10 xylanase with a CBM2 domain (Q47KR6, GenBank AAZ56956.1; with relative secretion of 5.03 ± 1.33%) and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, T. lanuginosus increased the secretion of cellulases from T. fusca by 19-25%. The order of T. lanuginosus and T. fusca was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of T. lanuginosus and T. fusca gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation.