6 research outputs found
Enhancing xylose utilisation during fermentation by engineering recombinant Saccharomyces cerevisiae strains
Dissertation (DPhil)--University of Stellenbosch, 2007.ENGLISH ABSTRACT: Xylose is the second most abundant sugar present in plant biomass. Plant biomass is
the only potential renewable and sustainable source of energy available to mankind at
present, especially in the production of transportation fuels. Transportation fuels such as
gasoline can be blended with or completely replaced by ethanol produced exclusively
from plant biomass, known as bio-ethanol. Bio-ethanol has the potential to reduce
carbon emissions and also the dependence on foreign oil (mostly from the Middle East
and Africa) for many countries.
Bio-ethanol can be produced from both starch and cellulose present in plants,
even though cellulosic ethanol has been suggested to be the more feasible option.
Lignocellulose can be broken down to cellulose and hemicellulose by the hydrolytic
action of acids or enzymes, which can, in turn, be broken down to monosaccharides
such as hexoses and pentoses. These simple sugars can then be fermented to ethanol
by microorganisms. Among the innumerable microorganisms present in nature, the
yeast Saccharomyces cerevisiae is the most efficient ethanol producer on an industrial
scale. Its unique ability to efficiently synthesise and tolerate alcohol has made it the
‘workhorse’ of the alcohol industry.
Although S. cerevisiae has arguably a relatively wide substrate utilisation range,
it cannot assimilate pentose sugars such as xylose and arabinose. Since xylose
constitutes at least one-third of the sugars present in lignocellulose, the ethanol yield
from fermentation using S. cerevisiae would be inefficient due to the non-utilisation of
this sugar. Thus, several attempts towards xylose fermentation by S. cerevisiae have
been made. Through molecular cloning methods, xylose pathway genes from the
natural xylose-utilising yeast Pichia stipitis and an anaerobic fungus, Piromyces, have
been cloned and expressed separately in various S. cerevisiae strains. However,
recombinant S. cerevisiae strains expressing P. stipitis genes encoding xylose
reductase (XYL1) and xylitol dehydrogenase (XYL2) had poor growth on xylose and
fermented this pentose sugar to xylitol.
The main focus of this study was to improve xylose utilisation by a recombinant
S. cerevisiae expressing the P. stipitis XYL1 and XYL2 genes under anaerobic
fermentation conditions. This has been approached at three different levels: (i) by
creating constitutive carbon catabolite repression mutants in the recombinant
S. cerevisiae background so that a glucose-like environment is mimicked for the yeast
cells during xylose fermentation; (ii) by isolating and cloning a novel xylose reductase
gene from the natural xylose-degrading fungus Neurospora crassa through functional
complementation in S. cerevisiae; and (iii) by random mutagenesis of a recombinant
XYL1 and XYL2 expressing S. cerevisiae strain to create haploid xylose-fermenting
mutant that showed an altered product profile after anaerobic xylose fermentation. From
the data obtained, it has been shown that it is possible to improve the anaerobic xylose utilisation of recombinant S. cerevisiae to varying degrees using the strategies followed,
although ethanol formation appears to be a highly regulated process in the cell.
In summary, this work exposits three different methods of improving xylose
utilisation under anaerobic conditions through manipulations at the molecular level and
metabolic level. The novel S. cerevisiae strains developed and described in this study
show improved xylose utilisation. These strains, in turn, could be developed further to
encompass other polysaccharide degradation properties to be used in the so-called
consolidated bioprocess.AFRIKAANSE OPSOMMING: Xilose is die tweede volopste suiker wat in plantbiomassa teenwoordig is.
Plantbiomassa is die enigste potensiële hernubare en volhoubare bron van energie wat
tans vir die mensdom beskikbaar is, veral vir die produksie van vervoerbrandstowwe.
Vervoerbrandstowwe soos petrol kan vermeng word met etanol wat uitsluitlik van
plantbiomassa vervaardig is, bekend as bio-etanol, of heeltemal daardeur vervang
word. Bio-etanol het die potensiaal om koolstofuitlatings te verminder en vir baie lande
ook afhanklikheid op buitelandse olie (hoofsaaklik afkomstig van die Midde-Ooste en
Afrika) te verminder.
Bio-etanol kan vanaf beide die stysel en sellulose in plante vervaardig word,
maar sellulosiese etanol word as die meer praktiese opsie beskou. Lignosellulose kan
deur die hidrolitiese aksie van sure of ensieme in sellulose en hemisellulose afgebreek
word en dit kan op hulle beurt weer in monosakkariede soos heksoses en pentoses
afgebreek word. Hierdie eenvoudige suikers kan dan deur mikro-organismes tot etanol
gegis word. Onder die tallose mikro-organismes wat in die natuur teenwoordig is, is die
gis Saccharomyces cerevisiae die doeltreffendste etanolprodusent in die bedryf. Sy
unieke vermoë om alkohol te vervaardig en te weerstaan het dit die werksperd van die
alkoholbedryf gemaak.
Hoewel S. cerevisiae ‘n taamlike breë spektrum van substrate kan benut, kan dit
nie pentosesuikers soos xilose en arabinose assimileer nie. Aangesien xilose ten
minste ‘n derde van die suikers wat in lignosellulose teenwoordig is, uitmaak, sou die
etanolopbrengs uit gisting met S. cerevisiae onvoldoende wees omdat hierdie suiker nie
benut word nie. Verskeie pogings is dus aangewend om xilosegisting deur S. cerevisiae
te bewerkstellig. Deur middel van molekulêre kloneringsmetodes is gene van die xiloseweg
uit ‘n gis wat xilose natuurlik benut, Pichia stipitis, en ‘n anaërobiese swam,
Piromyces, afsonderlik in S. cerevisiae-rasse gekloneer en uitgedruk. ‘n Rekombinante
ras wat P. stipitis- se XYL1-xilosereduktase- en XYL2-xilitoldehidrogenase gene uitdruk,
het egter swak groei op xilose getoon en het dié pentosesuiker tot xilitol gegis.
Die hooffokus van hierdie ondersoek was om die benutting van xilose deur ‘n
rekombinante S. cerevisiae-ras wat P. stipitis se XYL1 en XYL2-gene uitdruk onder
anaërobiese gistingstoestande te verbeter. Dit is op drie verskillende vlakke benader:
(i) deur konstitutiewe koolstofkataboliet-onderdrukkende mutante in die rekombinante
S. cerevisiae-agtergrond te skep sodat ‘n glukose-agtige omgewing tydens xilosegisting
vir die gisselle nageboots word; (ii) deur ‘n nuwe xilose-reduktasegeen uit die natuurlike
xilose-afbrekende swam Neurospora crassa te isoleer en deur funksionele
komplementasie in S. cerevisiae te kloneer; en (iii) deur willekeurige mutagenese van
die rekombinante S. cerevisiae-ras ‘n haploïede xilose-gistende mutant te skep wat ‘n
gewysigde produkprofiel ná anaërobiese xilosegisting vertoon. Deur hierdie drieledige
benadering te volg, is dit bewys dat dit moontlik is om die anaërobiese xilosebenutting
van rekombinante S. cerevisiae-rasse in wisselende mate deur die aangepaste metodes te verbeter, hoewel etanolvorming ‘n hoogs gereguleerde proses in die sel blyk
te wees.
Opsommend kan gesê word dat hierdie werk drie verskillende metodes uiteensit om
xilosebenutting onder anaërobiese toestande te verbeter deur manipulasies op die
molekulêre en metaboliese vlak. Die nuwe S. cerevisiae-rasse wat in hierdie studie
ontwikkel en beskryf word, toon verbeterde xilosebenutting. Hierdie rasse kan op hulle
beurt verder ontwikkel word om ander polisakkariedafbrekende eienskappe in te sluit
wat in die sogenaamde gekonsolideerde bioproses gebruik kan word
Fungal endophytes from two orchid species pointer towards organ specificity
Fungal endophytes may influence plant communities by altering the host's fitness either positively or negatively. Little is known, however, about their host/organ specificity, life style and role in plantfungus symbiosis under varying environmental conditions. We compared the leaf and root endophyte assemblages of two orchids (Bulbophyllum neilgherrense and Pholidota pallida) from natural forests and greenhouse conditions. Xylariaceae species were consistently associated with leaf and root tissues, while Guignardia and Pestalotiopsis were found predominantly in the leaf tissues of both orchids. Correspondence analysis of the endophyte assemblages showed that the endophytes exhibited distinct organ but little host specificity. More endophytes were shared by the two different orchids growing in the same location when compared to endophyte assemblages of a single orchid from different locations. Considering the influence of endophytes in shaping the host's community, diverse habitats must be screened vigorously to address questions regarding the role of endophytes in hostendophyte interactions. Rostlinná společenstva mohou být ovlivněna působením houbových endofytů, které se odráží ve změnách fitness jejich hostitelů, ať už pozitivních či negativních. Avšak málo je známo o hostitelské či orgánové specificitě endofytů, jejich životním stylu a roli v symbiotickém vztahu s rostlinou při růz-ných podmínkách prostředí. Autoři srovnávali společenstva listových a kořenových endofytů dvou orchidejí (Bulbophyllum neilgherrense a Pholidota pallida), rostoucích v přirozených lesích a sklení-kových podmínkách. Druhy z čeledi Xylariaceae byly pevně spjaty s listovými i kořenovými pletivy, zatímco druhy rodů Guignardia a Pestalotiopsis jsou nacházeny převážně v listových pletivech obou orchidejí. Korespondenční analýza společenstev endofytů ukázala, že tyto houby vykazují zřetelnou orgánovou specificitu, ale nízkou specificitu hostitelskou. Vice společných endofytů měly dvě různé orchideje rostoucí na společné lokalitě ve srovnání se společenstvy endofytů jednotlivých rostlin z různých lokalit. Vzhledem k vlivu endofytů na utváření společenstev svých hostitelů bude ještě potřebný důsledný průzkum různých substrátů pro ozřejmení role endofytů v interakcích s jejich hostiteli
Molecular cloning and functional expression of a novel Neurospora crassa xylose reductase in Saccharomyces cerevisiae in the development of a xylose fermenting strain
The development of a xylose-fermenting Saccharomyces cerevisiae yeast would be of great benefit to the bioethanol industry. The conversion of xylose to ethanol involves a cascade of enzymatic reactions and processes. Xylose (aldose) reductases catalyse the conversion of xylose to xylitol. The aim of this study was to clone, characterise and express a cDNA copy of a novel aldose reductase {NCAR-X) from the filamentous fungus Neurospora crassa in S. cerevisiae. NCAR-X harbours an open reading frame (ORF) of 900 nucleotides. This ORF encodes a protein (NCAR-X, assigned NCBI protein accession ID: XP_956921) consisting of 300 amino acids, with a predicted molecular weight of 34 kDa. The NVAR-X-encoded aldose reductase showed significant homology to the xylose reductases of Candida tenuis and Pichia stipitis. When NCAR-X was expressed under the control of phosphoglycerate kinase I gene (PGK1) regulatory sequences in S. cerevisiae, its expression resulted In the production of biologically active xylose reductase. Small-scale oxygen-limited xylose fermentation with the NCAR-X containing S. cerevisiae strains resulted In the production of less xylitol and at least 15% more ethanol than the strains transformed with the P. stipitis xylose reductase gene (PsXYL1). The NCAR-X-encoded enzyme produced by S. cerevisiae was NADPH-dependent and no activity was observed in the presence of NADH. The co-expression of the NCAR-X and PsXYL1 gene constructs in S. cerevisiae constituted an important part of an extensive research program aimed at the development of xylolytic yeast strains capable of producing ethanol from plant biomass.9 page(s
Development and characterisation of a recombinant Saccharomyces cerevisiae mutant strain with enhanced xylose fermentation properties
The purpose of this study was to help lay the foundation for further development of xylose-fermenting Saccharomyces cerevisiae yeast strains through an approach that combined metabolic engineering and random mutagenesis in a recombinant hap-loid strain that overexpressed only two genes of the xylose pathway. Previously, S. cerevisiae strains, overexpressing heterologous genes encoding xylose reductase, xylitol dehydrogenase and the endogenous XKS1 xylulokinase gene, were randomly mutagenised to develop improved xylose-fermenting strains. In this study, two gene cassettes (ADHI p-PsXYL1-ADH1T- and PGKlP-PsXYL2-PGK1T) containing the xylose reductase (PsXYL1) and xylitol dehydrogenase (PsXYL2) genes from the xylose-fermenting yeast, Pichia stipitis, were integrated into the genome of a haploid S. cerevisiae strain (CEN.PK 2-1D). The resulting recombinant strain (YUSM 1001) over-expressing the P. stipitis XYL1 and XYL2 genes (but not the endogenous XKS1 gene) was subjected to ethyl methane sulfonate (EMS) mutagenesis. The resulting mutants were screened for faster growth rates on an agar medium containing xylose as the sole carbon source. A mutant strain (designated Y-X) that showed 20-fold faster growth in xylose medium in shake-flask cultures was isolated and characterised. In anaerobic batch fermentation, the Y-X mutant strain consumed 2.5-times more xylose than the YUSM 1001 parental strain and also produced more ethanol and glycerol. The xylitol yield from the mutant strain was lower than that from the parental strain, which did not produce glycerol and ethanol from xylose. The mutant also showed a 50% reduction in glucose consumption rate. Transcript levels of XYL1, XYL2 and XKS1 and the GPD2 glycerol 3-phosphate dehydrogenase gene from the two strains were compared with real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The mutant showed 10-40 times higher relative expression of these four genes, which corresponded with either the higher activities of their encoded enzymes or by-product formation during fermentation. Furthermore, no mutations were observed in the mutant's promoter sequences or the open reading frames of some of its key genes involved in carbon catabolite repression, glycerol production and redox balancing. The data suggest that the enhancement of the xylose fermentation properties of the Y-X mutant was made possible by increased expression of the xylose pathway genes, especially the XKS1 xylulokinase gene.9 page(s
A constitutive catabolite repression mutant of a recombinant Saccharomyces cerevisiae strain improves xylose consumption during fermentation
Efficient xylose utilisation by microorganisms is of importance to the lignocellulose fermentation industry. The aim of this work was to develop constitutive catabolite repression mutants in a xylose-utilising recombinant Saccharomyces cerevisiae strain and evaluate the differences in xylose consumption under fermentation conditions. S. cerevisiae YUSM was constitutively catabolite repressed through specific disruptions within theMIG1 gene. The strains were grown aerobically in synthetic complete medium with xylose as the sole carbon source. Constitutive catabolite repressed strain YCR17 grew four-fold better on xylose in aerobic conditions than the control strain YUSM. Anaerobic batch fermentation in minimal medium with glucose-xylose mixtures and N-limited chemostats with varying sugar concentrations were performed. Sugar utilisation and metabolite production during fermentation were monitored. YCR17 exhibited a faster xylose consumption rate than YUSM under high glucose conditions in nitrogen-limited chemostat cultivations. This study shows that a constitutive catabolite repressed mutant could be used to enhance the xylose consumption rate even in the presence of high glucose in the fermentation medium. This could help in reducing fermentation time and cost in mixed sugar fermentation.Vasudevan Thanvanthri Gururajan, Marie-F. Gorwa-Grauslund, Bärbel Hahn-Hägerdal, Isak S. Pretorius and Ricardo R. Cordero Oter