29 research outputs found
Adapting the yeast consolidated bioprocessing paradigm for biorefineries
Despite decades long development, no natural or engineered organism has been isolated that can produce commodity products at the rates and yields required by industry via direct microbial conversion. However, new genomic editing tools and systems level knowledge of metabolism
provides opportunities to develop yeast strains for second-generation biorefineries.IS
Improved cellulase expression in diploid yeast strains enhanced consolidated bioprocessing of pretreated corn residues
In an effort to find a suitable genetic background for efficient cellulolytic secretion, genetically diverse strains were transformed to produce core fungal cellulases namely, β-glucosidase (BGLI), endoglucanase (EGII) and cellobiohydrolase (CBHI) in various combinations and expression configurations. The secreted enzyme activity levels, gene copy number, substrate specificities, as well as hydrolysis and fermentation yields of the transformants were analysed. The effectiveness of the partially cellulolytic yeast transformants to convert two different pre-treated corn residues, namely corn cob and corn husk was then explored
Supplementation of recombinant cellulases with LPMOs and CDHs improves consolidated bioprocessing of cellulose
The increased demand for energy has sparked a global search for renewable energy sources that could partly replace fossil fuel resources and help mitigate climate change. Cellulosic biomass is an ideal feedstock for renewable bioethanol production, but the process is not currently economically feasible due to the high cost of pretreatment and enzyme cocktails to release fermentable sugars. Lytic polysaccharide monooxygenases (LPMOs) and cellobiose dehydrogenases (CDHs) are auxiliary enzymes that can enhance cellulose hydrolysis. In this study, four LPMO and two CDH genes were subcloned and expressed in the Saccharomyces cerevisiae Y294 laboratory strain. SDS-PAGE analysis confirmed the extracellular production of the LPMOs and CDHs in the laboratory S. cerevisiae Y294 strain. A rudimentary cellulase cocktail (cellobiohydrolase 1 and 2, endoglucanase and β-glucosidase) was expressed in the commercial CelluX™ 4 strain and extracellular production of the individual cellulases was confirmed by SDS-PAGE analysis. In vitro cooperation of the CDHs and LPMOs with the rudimentary cellulases produced by strain CelluX™ 4[F4–1] was demonstrated on Whatman filter paper. The significant levels of soluble sugars released from this crystalline cellulose substrate indicated that these auxiliary enzymes could be important components of the CBP yeast cellulolytic system
Developing Organisms for Consolidated Bioprocessing of Biomass to Ethanol
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Crispr-based multi-gene integration strategies to create saccharomyces cerevisiae strains for consolidated bioprocessing
Significant engineering of Saccharomyces cerevisiae is required to enable consolidated bioprocessing (CBP) of lignocellulose to ethanol. Genome modification in S. cerevisiae has been successful partly due to its efficient homology-directed DNA repair machinery, and CRISPR technology has made multi-gene editing even more accessible. Here, we tested the integration of cellulase encoding genes to various sites on the yeast genome to inform the best strategy for creating cellulolytic strains for CBP. We targeted endoglucanase (EG) or cellobiohydrolase (CBH) encoding genes to discreet chromosomal sites for single-copy integration or to the repeated delta sites for multi-copy integration. CBH1 activity was significantly higher when the gene was targeted to the delta sequences compared to single gene integration loci. EG production was comparable, though lower when the gene was targeted to a chromosome 10 site. We subsequently used the information to construct a strain containing three cellulase encoding genes. While individual cellulase activities could be assayed and cellulose conversion demonstrated, it was shown that targeting specific genes to specific loci had dramatic effects on strain efficiency. Since marker-containing plasmids could be cured from these strains, additional genetic changes can subsequently be made to optimize strains for CBP conversion of lignocellulose
Identification of superior cellulase secretion phenotypes in haploids derived from natural Saccharomyces cerevisiae isolates
The yeast Saccharomyces cerevisiae is considered an important host for consolidated bioprocessing and the production of
high titres of recombinant cellulases is required for efficient hydrolysis of lignocellulosic substrates to fermentable sugars.
Since recombinant protein secretion profiles vary highly among different strain backgrounds, careful selection of robust
strains with optimal secretion profiles is of crucial importance. Here, we construct and screen sets of haploid derivatives,
derived from natural strain isolates YI13, FINI and YI59, for improved general cellulase secretion. This report details a novel
approach that combines secretion profiles of strains and phenotypic responses to stresses known to influence the secretion
pathway for the development of a phenotypic screen to isolate strains with improved secretory capacities. A clear
distinction was observed between the YI13 haploid derivatives and industrial and laboratory counterparts, Ethanol Red and
S288c, respectively. By using sub-lethal concentrations of the secretion stressor tunicamycin and cell wall stressor Congo
Red, YI13 haploid derivative strains demonstrated tolerance profiles related to their heterologous secretion profiles. Our
results demonstrated that a new screening technique combined with a targeted mating approach could produce a pool of
novel strains capable of high cellulase secretion
High level secretion of cellobiohydrolases by Saccharomyces cerevisiae
<p>Abstract</p> <p>Background</p> <p>The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of <it>Saccharomyces cerevisiae </it>for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.</p> <p>Results</p> <p>We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by <it>S. cerevisiae</it>. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of <it>cbh1 </it>and <it>cbh2 </it>genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing <it>S. cerevisiae </it>strains with the addition of beta-glucosidase.</p> <p>Conclusions</p> <p>Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.</p
Engineering of Pichia stipitis for enhanced xylan utilization
Thesis (PhD)--Stellenbosch University, 2003.ENGLISH ABSTRACT: Plant biomass, the most abundant renewable resource in nature, consists of matrices of
mainly lignin, cellulose, hemicellulose as well as inorganic components. Xylan, the
major hemicellulose component in plant cell walls, is the most abundant polysaccharide
after cellulose. This makes the main constituent sugar of xylan, D-xylose, the second
most abundant renewable monosaccharide in nature. Very few hemicelluloses are either
homopolymeric or entirely linear. Therefore, the variety of enzymes involved in their
hydrolysis is more complex than the enzyme group responsible for the hydrolysis of
cellulose. Although the ability to degrade xylan is common among bacteria and
filamentous fungi, this trait is relatively rare among yeasts. However, some strains of the
yeast Pichia stipitis are, amongst others, able to degrade xylan. As P. stipitis is also one
of the best D-xylose fermenting yeasts thus far described, this yeast has the potential of
fermenting polymeric xylan directly to ethanol. However, it was shown that the natural
xylanolytic ability of this yeast is very weak.
In this study, xylanolytic genes were expressed in P. stipitis to test the ability of the yeast
to produce heterologous proteins, and to determine the enhancement of xylan utilisation
by the recombinant strain. The native xylose reductase gene (XYLl) and transketolase
gene (TKL) and the heterologous Saccharomyces cerevisiae phosphoglycerate kinase
(PGKl) gene promoter were cloned into P. stipitis transformation vectors and used to
express the Trichoderma reesei ~-xylanase encoding gene (xyn2) as reporter gene. It was
shown that the XYLl promoter was induced in the presence of D-xylose and that the TKL
promoter was constitutively expressed. The PGKl promoter of S. cerevisiae did not
function in P. stipitis .
When the T reesei xyn2 gene and the Aspergillus kawachii ~-xylanase encoding gene
(xynC) were expressed under control of the XYLl promoter, extracellular ~-xylanase
activity of up to 136 nkat/ml and 171 nkatlml was observed, respectively. This activity
declined over time due to the presence of extracellular proteases, secreted by P. stipitis.
Growing the cultures in a fermentor and controlling the pH level to pH 6 did not alleviate the reduction of heterologous l3-xylanase activity. When the Aspergillus niger
l3-xylosidase encoding gene (xlnD) was expressed as a fusion gene (designated XL02)
with the S. cerevisiae mating factor secretion signal (MFal) under control of the
P. stipitis TKL promoter, extracellular l3-xylosidase activity of 0.132 nkatlml was
observed. Co-expression of the xyn2 and XL02 genes led to B-xylanase and l3-xylosidase
activities of 128 nkatlml and 0.113 nkat/ml, respectively. Co-expression of the xynC and
XL02 genes led to l3-xylanase and l3-xylosidase activities of 165 nkat/ml and 0.124
nkatlml, respectively.
The expression of the fungal xylanolytic genes in P. stipitis also led to an increased
biomass yield when the recombinant strains were cultured on birchwood xylan as sole
carbon source. The strain co-expressing the A. kawachii l3-xylanase and A. niger
l3-xylosidase encoding genes was the most successful, yielding a 3.2-fold higher biomass
level than the control strain. Biomass levels of the recombinant strains were further
improved on average by 85% by growing them in a fermentor under conditions of high
oxygenation. The strains were also tested for direct conversion of xylan to ethanol and
the strain co-expressing the A. kawachii l3-xylanase and A. niger l3-xylosidase encoding
genes produced 1.35 giL ethanol, which represents a 3.6-fold increase in ethanol yield
over the reference strain. These strains represent a step towards the efficient degradation
and utilisation of hemicellulosic materials by ethanol-producing yeasts.AFRIKAANSE OPSOMMING: Plant biomassa, die volopste hernubare koolstotbron in die natuur, bestaan uit matrikse
van lignien, sellulose en hemisellulose. Xilaan, die hoof hemisellulose komponent in
plantselwande, is na sellulose die volopste polisakkaried. Gevolglik is die hoof
suikerkomponent van xilaan, naamlik D-xilose, die tweede volopste hernubare
monosakkaried in die natuur. Baie min hemisellulose molekules is homopolimere of
heeltemal linieĂŞr. Daarom is die ensieme betrokke by die atbraak van hemiselluloses
meer kompleks as die ensieme betrokke by die atbraak van sellulose. Bakterieë en
filamentagtige fungi wat oor die vermoë om xilaan af te breek beskik, kom wydversprei
voor maar relatief min giste kan xilaan benut. Sommige rasse van die gisspesie Pichia
stipitis het egter beperkte vermoë om xilaan af te breek. P. stipitis is ook een van die
beste D-xilose fermenterende giste wat tot dusver beskryf is en het dus die potensiaalom
etanol vanafpolimeriese xilaan te produseer.
In hierdie studie is gene wat kodeer vir xilaanatbrekende ensieme in P. stipitis uitgedruk
om die vermoë van die gis as heteroloë uitdrukking sisteem te evalueer. Verder is die
effek van die heteroloë xilaanatbrekende ensieme tydens groei op xilaan as enigste
koolstotbron getoets. Die promoters van die xilosereduktasegeen (XYLl), die
transketolasegeen (TKL) van P. stipitis en die fosfogliseraatkinasegeen (PGKl) van
Saccharomyces cerevisiae is in P. stipitis transformasie vektore gekloneer en gebruik om
die Trichoderma reesei ~-xilanasegeen (xyn2) as verklikkergeen uit te druk. Dit het
bewys dat die XYLI promotor induseerbaar is in die teenwoordigheid van D-xilose terwyl
die TKL geen konstant uitgedruk was. Die PGKI promotor van S. cerevisiae was nie
funksioneel in P. stipitis nie.
EkstrasellulĂŞre ~-xilanase aktiwiteit van onderskeidelik 136 nkatlml en 171 nkatlml kon
waargeneem word wanneer die T reesei xyn2 geen of die Aspergillus kawachii
~-xilanasegeen (xynC) onder beheer van die XYLI promotor uitgedruk is. Hierdie
aktiwiteit het afgeneem na gelang van tyd a.g.v. die teenwoordigheid van ekstrasellulĂŞre
proteases wat deur P. stipitis uitgeskei word. Die afname van ekstrasellulĂŞre ~-xilanase aktiwiteit kon nie voorkom word deur die kulture in 'n fermentor te groei en die pH vlak
tot pH 6 te beheer nie. Tydens uitdrukking van die Aspergillus niger ~-xilosidase geen
(xlnD) as 'n fusiegeen (genoem XL02) met die paringsfaktor sekresiesein (MFal) van
S. cerevisiae onder transkripsionele beheer van die P. stipitis TKL promotor, kon
ekstrasellulĂŞre ~-xilosidase aktiwiteit van 0.132 nkatlml waargeneem word.
Gesamentlike uitdrukking van die xyn2 en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 128 nkatlml and 0.113 nkat/ml, onderskeidelik.
Gesamentlike uitdrukking van die xynC en XL02 gene het gelei tot ~-xilanase en
~-xilosidase aktiwiteite van 165 nkatlml and 0.124 nkatlml, onderskeidelik.
Die uitdrukking van xilaanatbrekende ensieme III P. stipitis het verhoogbe
biomassaproduksie teweeg gebring wanneer die rekombinante gisrasse op birchwood
xilaan as enigste koolstotbron gegroei het. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitdruk, was die mees
suksesvolle ras en het 3.2-voudig hoër biomassa as die kontrole ras opgelewer. Die
biomassa van die rekombinante rasse tydens groei op xilaan as enigste koolstotbron kon
gemiddeld met 85% verhoog word deur die giste onder hoë suurstotkonsentrase in 'n
fermentor te kweek. Die rekombinante rasse is verder ook getoets vir hul vermoë om
xilaan direk tot etanol om te skakel. Die rekombinante ras wat die A. kawachii
~-xilanasegeen en die A. niger ~-xilosidase geen gesamentlik uitgedruk het, het 'n 3.6-
voudige verhoging in etanolproduksie getoon en 1.35 gIL ethanol gelewer. Hierdie
rekombinante gisrasse verteenwoordig 'n stap nader aan die doeltreffende atbraak en
benutting van hemisellulose deur etanolproduserende giste
Engineering cellulolytic ability into bioprocessing organisms
The original publication is available at www.springerlink.com.Includes bibliography.Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates
Overexpression of native PSE1 and SOD1 in Saccharomyces cerevisiae improved heterologous cellulase secretion
Engineering cellulolytic ability into the yeast Saccharomyces cerevisiae to create an organism for consolidated bioprocessing (CBP) will require the simultaneous production and secretion of a number of heterologous cellulases. In addition, the generally low secretion titers achieved by this yeast will have to be overcome. To this end two native S. cerevisiae genes, PSE1 and SOD1, were individually overexpressed by placing each gene under the transcriptional control of the constitutive PGK1 promoter. The effect of these genes on heterologous protein secretion of three cellulases – an exoglucanase encoded by cel6A of Neocallimastix patriciarum, a β-glucosidase encoded by cel3A of Saccharomycopsis fibuligera and an endoglucanase encoded by cel7B of Trichoderma reesei was investigated by integrating the PGK1P/T–PSE1 and PGK1P/T–SOD1 cassettes into S. cerevisiae strains producing the relevant cellulases. Transformants were obtained that showed significantly higher secreted protein yield, with a resulting heterologous protein activity that ranged between 10% and 373% higher compared to the parental strains when grown in complex media. When both PSE1 and SOD1 were overexpressed in the yeast that produced Cel3A, a dramatic 447% increase in β-glucosidase activity was observed. This study shows that cellulase secretion in S. cerevisiae could be greatly improved with strain engineering. However, it also demonstrated that such strain engineering may have very enzyme specific effects as the induction of Cel3A secretion was far greater than that of the other cellulases investigated. Identifying cellulases amenable to expression in S. cerevisiae and engineering strains to maximize heterologous protein secretion may be imperative to creating optimal strains for CBP and may have wider implications for heterologous protein secretion in S. cerevisiae in general.7 page(s