Identification and characterisation of moderately thermostable diisobutyl phthalate degrading esterase from a Great Artesian Basin Bacillus velezensis NP05.

Phthalate esters are known to be endocrine disrupting chemicals and are documented to pollute environments. Enzymatic degradation of PAEs is a potential bioremedial strategy to manage contamination. Thermostable bioremedial enzymes have advantages in enzyme manufacturing and storage. In this study, we identified, overexpressed, and characterised a moderately thermostable para-nitrobenzyl esterase from whole genome sequencing of a Bacillus velezensis NP05 from the Great Artesian Basin, capable of sequential 2-step hydrolysis of diisobutyl phthalate. The pnbA enzyme has a molecular weight of 55.14 kDa and pI of 5.31. It preferentially degrades para-nitrophenyl butanoate and has an optimal pH of 7-8. The pnbA esterase has an optimal temperature of 55 °C with a half-life of 4 h. Using HPLC we found that pnbA (0.122 U) can hydrolyse 0.83 mM of DIBP within 25 min. Lastly, pnbA is potentially a more economically viable candidate for enzymatic bioremediation of diisobutyl phthalate as a free enzyme.

Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the ß-1,3-glucan paramylon under varying light conditions.

Euglena gracilis Z and a "sugar loving" variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a ß-1,3-glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose-yeast extract medium supporting high-level paramylon production. Both strains produced the highest paramylon yields (7.4-8 g · L-1 , respectively) while grown in the dark, but the maximum yield was achieved faster by E. gracilis var. saccharophila (48 h vs. 72 h). The glucose-to-paramylon yield coefficient Ypar/glu  = 0.46 ± 0.03 in the E. gracilis var. saccharophila cultivation, obtained in this study, is the highest reported to date. Proteomic analysis of the metabolic pathways provided molecular clues for the strain behavior observed during cultivation. For example, overexpression of enzymes in the gluconeogenesis/glycolysis pathways including fructokinase-1 and chloroplastic fructose-1,6-bisphosphatase (FBP) may have contributed to the faster rate of paramylon accumulation in E. gracilis var. saccharophila. Differentially expressed proteins in the early steps of chloroplastogenesis pathway including plastid uroporphyrinogen decarboxylases, photoreceptors, and a highly abundant (68-fold increase) plastid transketolase may have provided the E. gracilis var. saccharophila strain an advantage in paramylon production during diurnal cultivations. In conclusion, the variant strain E. gracilis var. saccharophila seems to be well suited for producing large amounts of paramylon. This work has also resulted in the identification of molecular targets for future improvement of paramylon production in E. gracilis, including the FBP and phosophofructokinase 1, the latter being a key regulator of glycolysis.

Expression of the mammalian peptide hormone obestatin in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is an expression host widely exploited for the production of recombinant proteins. However, its capacity for expressing small peptides (<20 kDa) has remained largely uncharted to date. In this work, we have produced the hormone peptide obestatin fused to the hydrophobin I tag (Obe-HFBI), using the T. reesei cellobiohydrolase I core (CBHI) or xylanase 2 (XYN2) pro-region as a carrier and the cbh1 promoter for gene expression, in high protein-low protease producing mutant strains T. reesei Rut-C30 and HEPI. The yield of obestatin was improved from about 300 ng/ml to up to 5.5 µg/ml through adaptive laboratory evolution and modifications to the cultivation strategy, which included adjustments of the type and ratio of carbon and nitrogen sources used in the medium. The successful expression of Obe-HFBI demonstrated the potential of T. reesei as an expression host for small peptides and further enhancement of the recombinant yield through modification of culture conditions.

Ultrastructural features of the early secretory pathway in Trichoderma reesei.

We have systematically analysed the ultrastructure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase-overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24 h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24 h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFP fusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

Methods for isolation and cultivation of filamentous fungi.

Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology.

A gene encoding a new cold-active lipase from an Antarctic isolate of Penicillium expansum.

Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.

Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30.

Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein-secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHIΔ4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields.

Proteome mapping of the Trichoderma reesei 20S proteasome.

The 26S proteasome, a multicatalytic protease comprising the catalytic 20S core particle and the 19S regulatory particle has a crucial role in cellular protein quality control. We have used a chromatography-based approach to purify and map the protein content of the 20S core particle from the industrially-exploited filamentous fungus Trichoderma reesei. There are no previous reports on the isolation or proteomic mapping of the proteasome from any filamentous fungus. From the reference map, 13 of the 14 20S proteasome subunits and many related proteins that co-purified with the 20S proteasome have been identified. These include 78 kDa glucose-regulated protein (BIP) and several chaperones including heat shock proteins involved in the unfolded protein response (UPR). Some proteasome interacting proteins (PIPs) were also identified on the proteome map and included 14-3-3-like protein, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, actin, translation elongation factor, enolase, ATPase in the ER (CDC48), and eukaryotic initiation factor. We present here a master map for the 20S catalytic core to pave the way for future differential display studies addressing intracellular degradation of endogenous and foreign proteins in filamentous fungi.