W. H. van Zyl

Deletion of the GRE3 Aldose Reductase Gene and Its Influence on Xylose Metabolism in Recombinant Strains of Saccharomyces cerevisiae Expressing the xylA and XKS1 Genes

ABSTRACT Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified inS. cerevisiae. The GRE3gene, encoding the AR enzyme, was deleted in S.cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI fromT. thermophilus was produced, and endogenous xylulokinase from S.cerevisiae was overproduced in S.cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene fromT. thermophilus on a replicative plasmid generated recombinant xylose utilizing S.cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.

Degradation of Xylan to d-Xylose by Recombinant Saccharomyces cerevisiae Coexpressing the Aspergillus niger β-Xylosidase (xlnD) and the Trichoderma reesei Xylanase II (xyn2) Genes

ABSTRACT The β-xylosidase-encoding xlnD gene ofAspergillus niger 90196 was amplified by the PCR technique from first-strand cDNA synthesized on mRNA isolated from the fungus. The nucleotide sequence of the cDNA fragment was verified to contain a 2,412-bp open reading frame that encodes a 804-amino-acid propeptide. The 778-amino-acid mature protein, with a putative molecular mass of 85.1 kDa, was fused in frame with the Saccharomyces cerevisiae mating factor α1 signal peptide (MFα1s) to ensure correct posttranslational processing in yeast. The fusion protein was designated Xlo2. The recombinant β-xylosidase showed optimum activity at 60°C and pH 3.2 and optimum stability at 50°C. The Ki(app) value ford-xylose and xylobiose for the recombinant β-xylosidase was determined to be 8.33 and 6.41 mM, respectively. TheXLO2 fusion gene and the XYN2 β-xylanase gene from Trichoderma reesei, located on URA3-based multicopy shuttle vectors, were successfully expressed and coexpressed in the yeast Saccharomyces cerevisiae under the control of the alcohol dehydrogenase II gene (ADH2) promoter and terminator. These recombinant S. cerevisiae strains produced 1,577 nkat/ml of β-xylanase activity when expressing only the β-xylanase and 860 nkat/ml when coexpressing the β-xylanase with the β-xylosidase. The maximum β-xylosidase activity was 5.3 nkat/ml when expressed on its own and 3.5 nkat/ml when coexpressed with the β-xylanase. Coproduction of the β-xylanase and β-xylosidase enabled S. cerevisiae to degrade birchwood xylan tod-xylose.

Degradation of aflatoxin B1 by fungal laccase enzymes

The enzymatic degradation of aflatoxin B(1) (AFB(1)) by white rot fungi through laccase production was investigated in different liquid media. A significant (P<0.0001) correlation was observed between laccase activity and AFB(1) degradation exhibited by representatives of Peniophora and Pleurotus ostreatus cultivated in minimal salts (MSM) (r=0.93) and mineral salts - malt extract (MSB-MEB) (r=0.77) liquid media. Peniophora sp. SCC0152 cultured in MSB-MEB liquid medium supplemented with veratryl alcohol and sugarcane bagasse showed high laccase activity (496U/L), as well as 40.45% AFB(1) degradation as monitored using high performance liquid chromatography. P.ostreatus St2-3 cultivated in MSM liquid medium supplemented with veratryl alcohol resulted in laccase activity of 416.39U/L and 35.90% degradation of AFB(1). Aflatoxin B(1) was significantly (P<0.0001) degraded when treated with pure laccase enzyme from Trametes versicolor (1U/ml, 87.34%) and recombinant laccase produced by Aspergillus niger D15-Lcc2#3 (118U/L, 55%). Aflatoxin B(1) degradation by laccase enzyme from T. versicolor and recombinant laccase enzyme produced by A. niger D15-Lcc2#3 coincided with significant (P<0.001) loss of mutagenicity of AFB(1), as evaluated in the Salmonella typhimurium mutagenicity assay. The degradation of AFB(1) by white rot fungi could be an important bio-control measure to reduce the level of this mycotoxin in food commodities.

Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.

Cloning of the Bacillus pumilusβ-xylosidase gene (xynB ) and its expression in Saccharomyces cerevisiae

Abstract A genomic DNA library of the bacterium Bacillus pumilus PLS was constructed and the β-xylosidase gene (xynB) was amplified from a 3-kb genomic DNA fragment with the aid of the polymerase chain reaction technique. The amplified xynB gene was inserted between the yeast alcohol dehydrogenase II gene promoter (ADH2P) and terminator (ADH2T) sequences on a multicopy episomal plasmid (pDLG11). The xynB gene was also fused in-frame to the secretion signal sequence of the yeast mating pheromone α-factor (MFα1S) before insertion between the ADH2P and ADH2T sequences on a similar multicopy episomal plasmid (pDLG12). The resulting construct ADH2P-MFα1S-xynB-ADH2T was designated XLO1. Both plasmids pDLG11 and pDLG12 were introduced into Saccharomyces cerevisiae but only the expression of the XLO1 gene yielded biologically functional β-xylosidase. The total β-xylosidase activity remained cell-associated with a maximum activity of 0.09 nkat/ml obtained when the recombinant S. cerevisiae strain was grown for 143 h in synthetic medium. The temperature and pH optima of the recombinant Xlo1 enzyme were 45–50 °C and pH 6.6 respectively. The enzyme was thermostable at 45 °C; however, at 60 °C most of the Xlo1 was inactive after 5 min.

Xylose isomerase activity influences xylose fermentation with recombinant Saccharomyces cerevisiae strains expressing mutated xylA from Thermus thermophilus.

Three xylose isomerase enzymes (XI) [Eur. J. Biochem. 269 (2002) 157], encoded by mutated xylA genes from Thermus thermophilus, were produced at two different levels in Saccharomyces cerevisiae; xylA genes were chromosomally integrated and expressed from multicopy plasmids, respectively. An extra copy of the endogenous xylulokinase gene (XKS1) was chromosomally integrated and the aldose reductase (AR) GRE3 gene was deleted. Ethanol was formed from xylose only when xylA was expressed from multicopy plasmids and when the specific XI activity was higher than 30 mU/mg protein. Deletion of the GRE3 gene was crucial for ethanol formation, possibly because reduced xylitol formation caused less inhibition of XI.

Cloning and expression of the α–L-arabinofuranosidase gene (ABF2) of Aspergillus niger in Saccharomyces cerevisiae

Abstract First-strand cDNA was prepared from mRNA of Aspergillus niger MRC11624 induced on oat spelts xylan. Using the cDNA as a template, the α-L-arabinofuranosidase gene (abf B) was amplified with the polymerase chain reaction technique. The abf B DNA fragment was inserted between the yeast phosphoglycerate kinase I gene promoter (PGK1P) and terminator (PGK1T) sequences on a multicopy episomal plasmid. The resulting construct PGK1P-abf B-PGK1T was designated ABF2. The ABF2 gene was expressed successfully in Saccharomyces cerevisiae and functional α-L-arabinofuranosidase was secreted from the yeast cells. The ABF2 nucleotide sequence was determined and verified to encode a 449-amino-acid protein (Abf 2) that is 94% identical to the α-L-arabinofuranosidase B of A. niger N400. Maximum α-L-arabinofuranosidase activities of 0.020 U/ml and 1.40 U/ml were obtained with autoselective recombinant S. cerevisiae strains when grown for 48 h in synthetic and complex medium respectively.

Enhanced xylan degradation and utilisation by Pichia stipitis overproducing fungal xylanolytic enzymes

Abstract β-Xylanase encoding genes of Trichoderma reesei ( xyn2 ) and Aspergillus kawachii ( xynC ) were cloned as cDNA copies under transcriptional control of the inducible Pichia stipitis xylose reductase gene ( XYL1 ) promoter on episomal plasmids pRDH12 and pRDH16, respectively. A cDNA copy of the β-xylosidase encoding gene of Aspergillus niger ( xlnD ) was cloned as an in-reading-frame fusion with the Saccharomyces cerevisiae MFα1 secretion signal under transcriptional control of the constitutive P. stipitis transketolase ( TKL ) gene promoter on an episomal plasmid (pRDH21). Combinations of the individual β-xylanase encoding genes and β-xylosidase expression cassette were also cloned onto episomal plasmids (pRDH22 and pRDH26). All of the plasmids were subsequently transformed to P. stipitis TJ26 and the β-xylanase activity, β-xylosidase activity and growth of the recombinant strains on xylan as sole carbon source were monitored. The strains expressing the A. kawachii xynC gene reached the highest maximum levels of β-xylanase activity and the activity was sustained for a longer period than the T. reesei xyn2 expressing strains where the levels of activity declined rapidly after reaching a maximum. All recombinant strains as well as the control strain were shown to produce extracellular protease activity. All strains expressing the A. niger xlnD gene reached similar levels of β-xylosidase activity, markedly higher than the control strains. The recombinant xylanolytic enzymes, whether produced alone or simultaneously, lead to an increase in biomass production of the recombinant strains when grown on medium containing xylan as sole carbon source. Simultaneous expression of the A. kawachii xynC gene and the A. niger xlnD gene gave the highest level of biomass production of any of the recombinant strains.

Cloning of two β-xylanase-encoding genes from Aspergillus niger and their expression in Saccharomyces cerevisiae

Two endo-β-1,4-xylanase-encoding genes were amplified from Aspergillus niger ATCC 90196 mRNA, inserted between the yeast ADH2 promoter and terminator sequences (genes designated XYN4 and XYN5) and expressed in Saccharomyces cerevisiae. The nucleotide sequences of the XYN4 and XYN5 genes revealed that both genes encode 211-amino acid proteins that are 92% identical to each other. Both the Xyn4 and Xyn5 enzymes have pH and temperature optima of pH 4 and 60°C, respectively. Autoselective S. cerevisiae strains were developed that allowed β-xylanase production and secretion in complex medium.

Next-generation cellulosic ethanol technologies and their contribution to a sustainable Africa

The world is currently heavily dependent on oil, especially in the transport sector. However, rising oil prices, concern about environmental impact and supply instability are among the factors that have led to greater interest in renewable fuel and green chemistry alternatives. Lignocellulose is the only foreseeable renewable feedstock for sustainable production of transport fuels. The main technological impediment to more widespread utilization of lignocellulose for production of fuels and chemicals in the past has been the lack of low-cost technologies to overcome the recalcitrance of its structure. Both biological and thermochemical second-generation conversion technologies are currently coming online for the commercial production of cellulosic ethanol concomitantly with heat and electricity production. The latest advances in biological conversion of lignocellulosics to ethanol with a focus on consolidated bioprocessing are highlighted. Furthermore, integration of cellulosic ethanol production into existing bio-based industries also using thermochemical processes to optimize energy balances is discussed. Biofuels have played a pivotal yet suboptimal role in supplementing Africas energy requirements in the past. Capitalizing on sub-Saharan Africas total biomass potential and using second-generation technologies merit a fresh look at the potential role of bioethanol production towards developing a sustainable Africa while addressing food security, human needs and local wealth creation.