Michael Rey

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

BackgroundBacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.ResultsWe determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.ConclusionsDespite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase

Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The enzyme was stable at 60°C for 30 min. The Km and kcat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a Km of 0.33±0.07 mM and a kcat of (5.5±0.5)×103 min−1 for maltotriose. The temperature dependence of kcat/Km indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.

Complete Genome Sequence for the Fusarium Head Blight Antagonist Bacillus amyloliquefaciens Strain TrigoCor 1448

ABSTRACT We present the complete genome sequence for Bacillus amyloliquefaciens TrigoCor 1448 (ATCC 202152), a bacterial biological control agent for Fusarium head blight in wheat. We compare it to its closest relative, B. amyloliquefaciens strain AS43.3.

12 - Combination of Suppression Subtractive Hybridization and Microarray Technologies to Enumerate Biomass-Induced Genes in the Cellulolytic Fungus Trichoderma reesei

The concerted action of many enzymes is required for the hydrolysis of cellulosic biomass. However, only a few have been identified and characterized in detail. Mixtures of isolated cellulase components have been shown to be less efficient than crude culture filtrates for the conversion of biomass to fermentable sugars. This may suggest that additional components are required for complete saccharification of biomass. Trichoderma reesei is the best-studied cellulolytic fungus. To identify new T. reesei genes involved in biomass conversion, we have employed the high throughput analysis of expression of subtractive cDNA libraries by DNA microarray technology. The cDNA libraries have been generated by suppression subtractive hybridization (SSH), which allowed not only the selection of differentially expressed mRNAs, but also the enrichment for rare mRNAs and equalization of cDNA in a pool. Messenger RNA pools from T. reesei cells grown on glucose, cellulose, or acid-pretreated corn stover (PCS) was isolated and used for construction of the SSH cDNA libraries. Three SSH libraries representing cellulose-induced, PCS-induced and PCS minus cellulose-induced transcripts were constructed. Approximately 3600 cDNA clones from three SSH libraries were amplified by high throughput rolling circle amplification (RCA) to produce DNA for microarray printing. Microarray hybridization with tester and driver probes revealed728. DNA sequence analysis and bioinformatics were used to assemble these clones into approximately 90 previously unrecognized genes/proteins. Among them we have identified a number of novel enzymes/proteins with potential direct benefit for improving biomass degradation. Thus, the combination of SSH and cDNA microarray technologies has proven to be a powerful tool for discovery of new differentially expressed genes involved in biomass utilization.