Nokuthula Peace Mchunu

Draft genome sequence of the rubber tree Hevea brasiliensis

BackgroundHevea brasiliensis, a member of the Euphorbiaceae family, is the major commercial source of natural rubber (NR). NR is a latex polymer with high elasticity, flexibility, and resilience that has played a critical role in the world economy since 1876.ResultsHere, we report the draft genome sequence of H. brasiliensis. The assembly spans ~1.1 Gb of the estimated 2.15 Gb haploid genome. Overall, ~78% of the genome was identified as repetitive DNA. Gene prediction shows 68,955 gene models, of which 12.7% are unique to Hevea. Most of the key genes associated with rubber biosynthesis, rubberwood formation, disease resistance, and allergenicity have been identified.ConclusionsThe knowledge gained from this genome sequence will aid in the future development of high-yielding clones to keep up with the ever increasing need for natural rubber.

Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli

The alkaline stability of the xylanase from Thermomyces lanuginosus was further improved by directed evolution using error-prone PCR mutagenesis. Positive clones were selected by their ability to produce zones of clearing on pH 9 and 12 xylan agar plates. Variant NC38 was able to withstand harsh alkaline conditions retaining 84% activity after exposure at pH 10 for 90 min at 60 degrees C, while the parent enzyme had 22% activity after 60 min. The alkaline stable variant NC38 was cloned into pBGP1 under the control GAP promoter and pET22b(+) for expression in Pichia pastoris and Escherichia coli BL21, respectively. Best extracellular expression of the recombinant xylanase was observed in P. pastoris (261.7+/-0.61 U ml(-1)) whereas intracellular activity was observed in E. coli (47.9+/-0.28 U ml(-1)) was low. Total activity obtained in P. pastoris was 545-fold higher than E. coli. The mutated alkaline stable xylanase from P. pastoris was secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.

Xylanase Superproducer: Genome Sequence of a Compost-Loving Thermophilic Fungus, Thermomyces lanuginosus Strain SSBP

ABSTRACT We report here the draft genome sequence of Thermomyces lanuginosus strain SSBP, which was isolated from soil in South Africa. This fungus produces the largest amount of xylanase ever reported in the literature.

Improved electroporation-mediated non-integrative transformation of Thermomyces lanuginosus.

The development of an efficient fungal expression system for recombinant proteins requires an improved transformation system for the host organism. We report a facile, efficient and highly reproducible electroporation-mediated transformation system for Thermomyces lanuginosus with a transformation efficiency of 1.27 x 10(3) transformants/microg DNA. Conidia of T. lanuginosus were stably transformed to hygromycin B resistance using the pBC-hygro plasmid construct. Optimal electroporation conditions for maximum transformation of 10(8) conidia ml(-1) in 1.2 M sorbitol buffer (15 mM DTT, 5% DMSO) were a field strength of 5.5 kV/cm for 10 ms and a DNA concentration of 0.5 microg microl(-1). Transformants were recovered in prewarmed potato dextrose broth supplemented with 1.2 M sorbitol for 1-2 h at 50 degrees C. The presence of the hygromycin B phosphotransferase (hph) gene and non-integrative transformation was confirmed by PCR, Southern hybridization analysis and plasmid recovery. Transformants exhibited altered phenotype with reduced pigmentation and transformants were found to be mitotically stable after 15 sequential transfers on nonselective media without selective pressure.

Tandem mass tag-based quantitative proteomics analyses reveal the response of Bacillus licheniformis to high growth temperatures

As the optimal growth temperature of Bacillus licheniformis is relatively higher than many other industrial bacteria, its use for industrial production can reduce contamination and minimize cooling and product recovery costs during fermentation processes. However, little is known about the thermotolerance of this important bacterial species. To investigate the underlying mechanism, strains B. licheniformis ATCC 14580 and B186 were cultivated at their own optimal growth temperature (42 °C and 50 °C) and higher temperature (60 °C), respectively, and tandem mass tags (TMT)-based quantitative proteome analysis and bioinformatics tools were employed to identify differentially expressed proteins. A total of 21 differential proteins were identified and shown to participate in a wide range of biological processes, including protein refolding, amino acid and fatty acid metabolism, etc. Hence, the ability of B. licheniformis to exhibit optimal growth at high temperatures may depend on invoking its intrinsic “heat-against” proteomic mechanism for long-term viability. Our results may assist the genetic improvement of industrial strains of this important Bacillus specie.

Secretory expression of recombinant small laccase from Streptomyces coelicolor A3(2) in Pichia pastoris

This work reports for the first time the secretory expression of the small laccase (SLAC) from Streptomyces coelicolor A3(2) in Pichia pastoris. Using an AOX1 promoter and α factor as a secretion signal, the recombinant P. pastoris harbouring the laccase gene (rSLAC) produced high titres of extracellular laccase (500 ± 10 U/l), which were further increased seven fold by pre-incubation at 80 °C for 30 min. The enzyme (∼38 kDa) had an optimum activity at 80 °C, but optimum pH varied with substrate used. Km values for ABTS, SGZ and 2,6-DMP were 142.85 μM, 10 μM and 54.55 μM and the corresponding kcat values were 60.6 s-1, 25.36 s-1 and 27.84 s-1, respectively. The t1/2 values of the rSLAC at 60 °C, 70 °C, 80 °C were 60 h, 32 h and 10 h, respectively. The enzyme deactivation energy (Ed) was 117.275 kJ/mol while ΔG, ΔH and ΔS for thermal inactivation of the rSLAC were all positive. The rSLAC decolourised more than 90% of Brilliant Blue G and Trypan Blue dye in 6 h without the addition of a mediator. High titres of SLAC expressed in P. pastoris enhance its potential for various industrial applications.