Draft Genome Sequence of Bacillus sp. strain YC2, an Isolate from the South African Medicinal Plant Pelargonium sidoides.

A rhizosphere-associated Bacillus species was isolated from Pelargonium sidoides DC (Geraniaceae) tubers, whose commercial extracts are used in respiratory tract infection treatment. Genomic data for Bacillus isolates associated with Pelargonium sidoides is lacking. Here, we report the draft genome sequence of Bacillus sp. strain YC2.

Heterologous production of cellulose- and starch-degrading hydrolases to expand Saccharomyces cerevisiae substrate utilization: Lessons learnt.

Selected strains of Saccharomyces cerevisiae are used for commercial bioethanol production from cellulose and starch, but the high cost of exogenous enzymes for substrate hydrolysis remains a challenge. This can be addressed through consolidated bioprocessing (CBP) where S. cerevisiae strains are engineered to express recombinant glycoside hydrolases during fermentation. Looking back at numerous strategies undertaken over the past four decades to improve recombinant protein production in S. cerevisiae, it is evident that various steps in the protein production "pipeline" can be manipulated depending on the protein of interest and its anticipated application. In this review, we briefly introduce some of the strategies and highlight lessons learned with regards to improved transcription, translation, post-translational modification and protein secretion of heterologous hydrolases. We examine how host strain selection and modification, as well as enzyme compatibility, are crucial determinants for overall success. Finally, we discuss how lessons from heterologous hydrolase expression can inform modern synthetic biology and genome editing tools to provide process-ready yeast strains in future. However, it is clear that the successful expression of any particular enzyme is still unpredictable and requires a trial-and-error approach.

Amberlite IRA 900 versus calcium alginate in immobilization of a novel, engineered ß-fructofuranosidase for short-chain fructooligosaccharide synthesis from sucrose.

The immobilization of ß-fructofuranosidase for short-chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered ß-fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food-grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.

The antifungal and Cryptococcus neoformans virulence attenuating activity of Pelargonium sidoides extracts.

ETHNOPHARMACOLOGICAL RELEVANCE: Limitations of clinical antifungal treatments and drug-resistance are drivers of the search for novel antifungal strategies. Extracts prepared from the tubers of the medicinal plant, Pelargonium sidoides, are known for their antiviral and antibacterial activities and are used in ethnomedicine for the treatment of acute respiratory infections. Their impact on fungi has not been well characterised. Here, we provide a first report on the antifungal activity of a P. sidoides aerial tissue extract against Cryptococcus neoformans as well as the effects of both tuber and aerial tissue extracts on selected virulence factors. AIM OF THE STUDY: Novel antimicrobial strategies that target multiple cellular pathways or make use of anti-pathogenic compounds that inhibit virulence factors have been proposed. This work aimed to evaluate P. sidoides plant parts for their anticryptococcal activity and antipathogenic properties on selected virulence factors. MATERIALS AND METHODS: The antifungal activity of crude P. sidoides tuber and aerial tissue extracts (15% m/m ethanol) were compared using a modified colourimetric antifungal susceptibility test. Fungicidal activity of the extracts was confirmed by plate counts. To test yeast resistance to the extracts, it was conditioned by multiple passages in sub-lethal doses followed by antifungal susceptibility testing. Cytotoxicity of the extracts was tested with a blood agar haemolysis assay. Extracts were evaluated for the presence of multiple bioactive compounds by solid-phase fractionation and visualisation by thin-layer chromatography in combination with bioassays. The influence of extracts on the production of the polysaccharide capsule, ergosterol content as well as laccase and urease activities were also evaluated. Cell surface variations after extract exposure were visualised by scanning electron microscopy (SEM). RESULTS: Both tuber and aerial tissue extracts were fungicidal and contained multiple bioactive compounds which constrained the development of antifungal resistance. No haemolytic activity was observed, and the extracts did not appear to target ergosterol biosynthesis. However, the extracts displayed anti-pathogenic potential by significantly inhibiting laccase and urease activity while also significantly reducing capsule size. SEM revealed notable cell surface variations and provided support for the observed reduction in capsule size. CONCLUSIONS: Our results provide support to the exploration of medicinal plants as sources of alternative antifungal therapies and the potential use of multicomponent inhibition and or virulence attenuation for next-generation treatment strategies. Our data also provide relevant information that may support the further use of P. sidoides in traditional medicines as well as in commercialised phytopharmaceuticals.

The heterologous expression potential of an acid-tolerant Talaromyces pinophilus ß-glucosidase in Saccharomyces cerevisiae.

A filamentous fungus displaying high cellulase activity was isolated from a compost heap with triticale (a wheat-rye hybrid) as the main constituent. It was preliminarily identified as a Talaromyces pinophilus species. A 2577 base pair ß-glucosidase gene was cloned from complementary DNA and heterologously expressed in Saccharomyces cerevisiae. The recombinant ß-glucosidase production profile was assessed and compared to that of the Saccharomycopsis fibuligera ß-glucosidase which served as a benchmark. The enzyme was also characterised in terms of pH and temperature tolerance as well as response to inhibitors. Maximal extracellular ß-glucosidase activity of 0.56 nkat/mg total protein was measured using p-nitrophenyl-ß-D-glucopyranoside as substrate. The recombinant protein displayed a pH optimum of 4.0, and good thermostability as 70% of maximal enzyme activity was retained after 1 h at 60 °C. Activity of the recombinant ß-glucosidase was adversely affected by the presence of glucose and ethanol at higher concentrations while xylose had no effect. The expression of the T. pinophilus ß-glucosidase did not reach the same titres as for the benchmark; however, in the context of constructing a yeast strain for bioethanol production in a consolidated bioprocess, the enzyme may still show good potential.

Sequence and structure-based prediction of fructosyltransferase activity for functional subclassification of fungal GH32 enzymes.

Sucrolytic enzymes catalyse sucrose hydrolysis or the synthesis of fructooligosaccharides (FOSs), a prebiotic in human and animal nutrition. FOS synthesis capacity differs between sucrolytic enzymes. Amino-acid-sequence-based classification of FOS synthesizing enzymes would greatly facilitate the in silico identification of novel catalysts, as large amounts of sequence data lie untapped. The development of a bioinformatics tool to rapidly distinguish between high-level FOSs synthesizing predominantly sucrose hydrolysing enzymes from fungal genomic data is presented. Sequence comparison of functionally characterized enzymes displaying low- and high-level FOS synthesis revealed conserved motifs unique to each group. New light is shed on the sequence context of active site residues in three previously identified conserved motifs. We characterized two enzymes predicted to possess low- and high-level FOS synthesis activities based on their conserved motif sequences. FOS data for the enzymes confirmed our successful prediction of their FOS synthesis capacity. Structural comparison of enzymes displaying low- and high-level FOS synthesis identified steric hindrance between nystose and a long loop region present only in low-level FOS synthesizers. This loop is proposed to limit the synthesis of FOS species with higher degrees of polymerization, a phenomenon observed among enzymes displaying low-level FOS synthesis. Conserved sequence motifs surrounding catalytic residues and a distant structural determinant were identifiers of FOS synthesis capacity and allow for functional annotation of sucrolytic enzymes directly from amino acid sequence. The tool presented may also be useful to study the structure-function relationships of ß-fructofuranosidases by identifying mutations present in a group of closely related enzymes displaying similar function.

Direct, simultaneous quantification of fructooligosaccharides by FT-MIR ATR spectroscopy and chemometrics for rapid identification of superior, engineered ß-fructofuranosidases.

Fructooligosaccharides (FOS) are popular components of functional foods produced by the enzymatic transfer of fructose units to sucrose. Improving ß-fructofuranosidase traits by protein engineering is restricted by the absence of a rapid, direct screening method for the fructooligosaccharide products produced by enzyme variants. The use of standard high-performance liquid chromatography (HPLC) methods involves time-consuming sample preparation and chromatographic and data analysis steps. To overcome these limitations, this work presents a rapid method for screening ß-fructofuranosidase variant libraries using Fourier transform mid-infrared attenuated total reflectance (FT-MIR ATR) spectroscopy and calibration using partial least squares (PLS) regression. The method offers notable improvements in terms of sample analysis times and cost, with the added benefit of the absence of toxic eluents. Wavenumber interval selection methods were tested to develop optimised PLS regression models that were successfully applied to quantify of glucose, fructose, sucrose, 1-kestose and nystose, the substrates and products of ß-fructofuranosidase activity. To the best of our knowledge, this is the first report on the use of infrared spectroscopy and PLS calibration for the quantification of 1-kestose and nystose. Independent test set-validated results indicated that optimal wavenumber selection by interval PLS (iPLS) served to provide the best models for all sugars, bar glucose. Application of this screening method will facilitate the engineering of ß-fructofuranosidases and other glycosyltransferase enzymes by random mutagenesis strategies, as it provides, for the first time, a rapid, direct assay for transferase products that may be adapted to a high-throughput set-up.

Identification of the gene for ß-fructofuranosidase from Ceratocystis moniliformis CMW 10134 and characterization of the enzyme expressed in Saccharomyces cerevisiae.

BACKGROUND: ß-Fructofuranosidases (or invertases) catalyse the commercially-important biotransformation of sucrose into short-chain fructooligosaccharides with wide-scale application as a prebiotic in the functional foods and pharmaceutical industries. RESULTS: We identified a ß-fructofuranosidase gene (CmINV) from a Ceratocystis moniliformis genome sequence using protein homology and phylogenetic analysis. The predicted 615 amino acid protein, CmINV, grouped with an existing clade within the glycoside hydrolase (GH) family 32 and showed typical conserved motifs of this enzyme family. Heterologous expression of the CmINV gene in Saccharomyces cerevisiae BY4742∆suc2 provided further evidence that CmINV indeed functions as a ß-fructofuranosidase. Firstly, expression of the CmINV gene complemented the inability of the ∆suc2 deletion mutant strain of S. cerevisiae to grow on sucrose as sole carbohydrate source. Secondly, the recombinant protein was capable of producing short-chain fructooligosaccharides (scFOS) when incubated in the presence of 10% sucrose. Purified deglycosylated CmINV protein showed a molecular weight of ca. 66 kDa and a Km and Vmax on sucrose of 7.50 mM and 986 µmol/min/mg protein, respectively. Its optimal pH and temperature conditions were determined to be 6.0 and 62.5°C, respectively. The addition of 50 mM LiCl led to a 186% increase in CmINV activity. Another striking feature was the relatively high volumetric production of this protein in S. cerevisiae as one mL of supernatant was calculated to contain 197 ± 6 International Units of enzyme. CONCLUSION: The properties of the CmINV enzyme make it an attractive alternative to other invertases being used in industry.