Browsing by Author "Van Rooyen, Ronel, 1976-"

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    Cloning of a novel Bacillus pumilus cellobiose-utilising system : functional expression in Escherichia coli
    (Stellenbosch : Stellenbosch University, 2002-12) Van Rooyen, Ronel, 1976-; Van Zyl, Willem Heber; La Grange, D. C.; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source on earth. It is well established that efficient degradation of cellulose requires the synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases (CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that display broad substrate specificity with respect to hydrolysis of cellobiose and different aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate- dependent phosphotransferase systems (PTS) have been reported in various bacteria, including: Bacillus species. In this study, we have used a cellobiose chromophore analog, p-nitrophenyl- ~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose utilization genes that are functionally expressed in Escherichia coli. Cloning and sequencing of the most active clone with subsequent sequence analysis allowed the identification of four adjacent open reading frames. An operon of four genes (celBACH), encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence data. The amino acid sequence of the celH gene displayed good homology with ~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene product belongs to family 1 of the glycosyl hydrolases, which employ a retaining mechanism of enzymatic bond hydrolysis. In vivo PTS activity assays concluded that the optimal temperature and pH at which the recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively. Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a significant problem. Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was only achievable in the presence ofO.01% glucose. This phenomenon could be explained by the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated transport. Glucose supplementation induced the glycolytic pathway and subsequently the availability of PEP. Furthermore, it could be concluded that the general PTS components . (enzyme I and HPr) of E. coli must have complemented the CelBACH system from B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host.
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    Genetic engineering of the yeast Saccharomyces cerevisiae to ferment cellobiose
    (Stellenbosch : Stellenbosch University, 2007-03) Van Rooyen, Ronel, 1976-; Van Zyl, Willem Heber; Stellenbosch University. Faculty of Science. Dept. of Microbiology.
    ENGLISH ABSTRACT: The conversion of cellulosic biomass into fuels and chemicals has the potential to positively impact the South African economy, but is reliant on the development of low-cost conversion technology. Perhaps the most important progress to be made is the development of “consolidated bioprocessing” (CBP). CBP refers to the conversion of pretreated biomass into desired product(s) in a single process step with either a single organism or consortium of organisms and without the addition of cellulase enzymes. Among the microbial hosts considered for CBP development, Saccharomyces cerevisiae has received significant interest from the biotechnology community as the yeast preferred for ethanol production. The major advantages of S. cerevisiae include high ethanol productivity and tolerance, as well as a well-developed gene expression system. Since S. cerevisiae is non-cellulolytic, the functional expression of at least three groups of enzymes, namely endoglucanases (EC 3.2.1.4); exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) is a prerequisite for cellulose conversion via CBP. The endo- and exoglucanases act synergistically to efficiently degrade cellulose to soluble cellodextrins and cellobiose, whereas the β-glucosidases catalyze the conversion of the soluble cellulose hydrolysis products to glucose. This study focuses on the efficient utilization of cellobiose by recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Since it is generally accepted that S. cerevisiae do not produce a dedicated cellobiose permease/transporter, the obvious strategy was to produce a secretable β-glucosidase that will catalyze the hydrolysis of cellobiose to glucose extracellularly. β-Glucosidase genes of various fungal origins were isolated and heterologously expressed in S. cerevisiae. The mature peptide sequence of the respective β-glucosidases were fused to the secretion signal of the Trichoderma reesei xyn2 gene and expressed constitutively from a multi-copy yeast expression vector under transcriptional control of the S. cerevisiae PGK1 promoter and terminator. The resulting recombinant enzymes were characterized with respect to pH and temperature optimum, as well as kinetic properties. The maximum specific growth rates (μmax) of the recombinant strains were compared during batch cultivation in high-performance bioreactors. S. cerevisiae secreting the recombinant Saccharomycopsis fibuligera BGL1 enzyme was identified as the best strain and grew at 0.23 h-1 on cellobiose (compared to 0.29 h-1 on glucose). More significantly, was the ability of this strain to anaerobically ferment cellobiose at 0.18 h-1 (compared to 0.25 h-1 on glucose). However, extracellular cellobiose hydrolysis has two major disadvantages, namely glucose’s inhibitory effect on the activity of cellulase enzymes as well as the increased risk of contamination associated with external glucose release. In an alternative approach, the secretion signal from the S. fibuligera β-glucosidase (BGL1) was removed and expressed constitutively from the above-mentioned multi-copy yeast expression vector. Consequently, the BGL1 enzyme was functionally produced within the intracellular space of the recombinant S. cerevisiae strain. A strategy employing continuous selection pressure was used to adapt the native S. cerevisiae disaccharide transport system(s) for cellobiose uptake and subsequent intracellular utilization. RNA Bio-Dot results revealed the induction of the native α-glucoside (AGT1) and maltose (MAL) transporters in the adapted strain, capable of transporting and utilizing cellobiose intracellularly. Aerobic batch cultivation of the strain resulted in a μmax of 0.17 h-1 and 0.30 h-1 when grown in cellobiose- and cellobiose/maltose-medium, respectively. The addition of maltose significantly improved the uptake of cellobiose, suggesting that cellobiose transport (via the combined action of the maltose permease and α-glucosidase transporter) is the rate-limiting step when the adapted strain is grown on cellobiose as sole carbon source. In agreement with the increased μmax value, the substrate consumption rate also improved significantly from 0.25 g.g DW-1.h-1 when grown on cellobiose to 0.37 g.g DW-1.h-1 upon addition of maltose to the medium. The adapted strain also displayed several interesting phenotypical characteristics, for example, flocculation, pseudohyphal growth and biofilm-formation. These features resemble some of the properties associated with the highly efficient cellulase enzyme systems of cellulosome-producing anaerobes. Recombinant S. cerevisiae strains that can either hydrolyse cellobiose extracellularly or transport and utilize cellobiose intracellularly. Both recombinant strains are of particular interest when the final goal of industrial-scale ethanol production from cellulosic waste is considered. However, the latter strain’s ability to efficiently remove cellobiose from the extracellular space together with its flocculating, pseudohyphae- and biofilm-forming properties can be an additional advantage when the recombinant S. cerevisiae strain is considered as a potential host for future CBP technology.