Starch fermentation by recombinantsaccharomyces cerevisiae strains expressing the ?-amylase and glucoamylase genes fromlipomyces kononenkoae andsaccharomycopsis fibuligera (original) (raw)
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2017
Starch-based bioethanol has emerged as a sustainable and renewable alternative energy source, but exogenous enzymes and heat is required for the conventional liquefaction process. The cost-effective utilisation of raw starch requires consolidated bioprocessing (CBP), which entails starch hydrolysis and glucose fermentation by a single organism. This requires the coexpression of recombinant α-amylases and glucoamylases in a strain of Saccharomyces cerevisiae. The Aureobasidium pullulans ApuA, Aspergillus terreus AteA, Cryptococcus sp. S-2 CryA and Saccharomycopsis fibuligera SfiA α-amylase encoding genes were cloned and expressed in the S. cerevisiae Y294 laboratory strain under the transcriptional control of the enolase 1 promoter [ENO1P] and terminator [ENO1T] sequences. The S. cerevisiae Y294[ApuA] and Y294[AteA] strains were superior to the other strains, producing extracellular α-amylase activities of 2.57 U.ml -1 and 3.20 U.ml -1 , respectively. When co-expressed with the Asper...
Biotechnology and bioengineering, 2015
The development of a yeast strain that converts raw starch to ethanol in one step (called Consolidated Bioprocessing, CBP) could significantly reduce the commercial costs of starch-based bioethanol. An efficient amylolytic Saccharomyces cerevisiae strain suitable for industrial bioethanol production was developed in this study. Codon-optimized variants of the Thermomyces lanuginosus glucoamylase (TLG1) and Saccharomycopsis fibuligera α-amylase (SFA1) genes were δ-integrated into two S. cerevisiae yeast with promising industrial traits, i.e. strains M2n and MEL2. The recombinant M2n[TLG1-SFA1] and MEL2[TLG1-SFA1] yeast displayed high enzyme activities on soluble and raw starch (up to 8118 and 4461 nkat/g dry cell weight, respectively) and produced about 64 g/L ethanol from 200 g/L raw corn starch in a bioreactor, corresponding to 55% of the theoretical maximum ethanol yield (g of ethanol/g of available glucose equivalent). Their starch-to-ethanol conversion efficiencies were even hig...
Raw starch conversion by Saccharomyces cerevisiae expressing Aspergillus tubingensis amylases
Biotechnology for Biofuels, 2013
Background Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol. Results The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l-1 (0.038 and 0.028 g l-1 h-1), respectively, after 10 days. With a substrate load of 200 g l-1 ...
Biotechnology for Biofuels
Background: Consolidated bioprocessing (CBP) combines enzyme production, saccharification and fermentation into a one-step process. This strategy represents a promising alternative for economic ethanol production from starchy biomass with the use of amylolytic industrial yeast strains. Results: Recombinant Saccharomyces cerevisiae Y294 laboratory strains simultaneously expressing an α-amylase and glucoamylase gene were screened to identify the best enzyme combination for raw starch hydrolysis. The codon optimised Talaromyces emersonii glucoamylase encoding gene (temG_Opt) and the native T. emersonii α-amylase encoding gene (temA) were selected for expression in two industrial S. cerevisiae yeast strains, namely Ethanol Red ™ (hereafter referred to as the ER) and M2n. Two δ-integration gene cassettes were constructed to allow for the simultaneous multiple integrations of the temG_Opt and temA genes into the yeasts' genomes. During the fermentation of 200 g l −1 raw corn starch, the amylolytic industrial strains were able to ferment raw corn starch to ethanol in a single step with high ethanol yields. After 192 h at 30 °C, the S. cerevisiae ER T12 and M2n T1 strains (containing integrated temA and temG_Opt gene cassettes) produced 89.35 and 98.13 g l −1 ethanol, respectively, corresponding to estimated carbon conversions of 87 and 94%, respectively. The addition of a commercial granular starch enzyme cocktail in combination with the amylolytic yeast allowed for a 90% reduction in exogenous enzyme dosage, compared to the conventional simultaneous saccharification and fermentation (SSF) control experiment with the parental industrial host strains. Conclusions: A novel amylolytic enzyme combination has been produced by two industrial S. cerevisiae strains. These recombinant strains represent potential drop-in CBP yeast substitutes for the existing conventional and raw starch fermentation processes.
Enzyme and Microbial Technology, 1998
The production of ethanol from starch has been investigated in three genetically modified Saccharomyces cerevisiae strains (YPG/AB, YPG/MM, and YPB-G). Two of the three strains produce the Aspergillus awamori glucoamylase together with either the Bacillus subtilis (YPG/AB) or the mouse (YPG/MM) ␣-amylase as separately secreted polypeptides. YPB-G, on the other hand, secretes a bifunctional fusion protein that contains both the B. subtilis ␣-amylase and the A. awamori glucoamylase activities. Substrate utilization, biomass growth, and ethanol production were all studied in both starch-and glucose-containing media. Much higher growth rates were found when any of the three strains were grown on glucose. YPG/AB showed the most efficient utilization of starch for ethanol production with the lowest levels of reducing sugars accumulating in the medium. The superior performance of YPG/AB as compared to YPB-G was found to correlate with its higher level of ␣-amylase activity. The ethanol production levels of YPG/AB in starch-and glucose-containing media were found to be comparable. YPB-G, which secretes the bifunctional fusion protein, could produce ethanol in media with starch concentrations above 100 g l Ϫ1 while YPG/MM did not produce ethanol from starch because of its negligible secretion of glucoamylase.
2017
Thesis (PhD)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Starchy biomass is an ideal, abundant substrate for bioethanol production. The cost effective conversion of starch requires a fermenting yeast that is able to produce starch hydrolysing enzymes and ferment glucose to ethanol in one step called consolidated bioprocessing (CBP). Despite the advantages, CBP yeasts have not yet been employed for the industrial processing of raw starch during bioethanol production. Molecular biology has enabled the optimised expression of synthetically produced genes in Saccharomyces cerevisiae. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were codon optimised using different strategies and expressed in S. cerevisiae Y294. However, compared to the native coding sequences for the amyA and glaA genes, adapted synonymous codon usage resulted in a decrease in extracellular enzyme activity of 72% (30 nkat.ml-1) and 69% (4 nkat.ml-1), res...
TURKISH JOURNAL OF BIOLOGY, 2016
This study investigates the global transcriptional response of Saccharomyces cerevisiae strain WTPB-G, which was developed by transforming laboratory strain FY23 with the pPB-G plasmid expressing the Bacillus subtilis α-amylase and the Aspergillus awamori glucoamylase as a fusion protein. Genome-wide analysis of transcript levels revealed the cellular mechanisms and the related pathways that were affected by the genetic modification, which conferred the ability of starch utilization to wild-type cells by the introduction of a plasmid-harboring gene encoding the amylolytic activity. Fermentations were carried out in media containing glucose as the only carbon source under aerated and microaerated conditions, and the effect of aeration was also investigated in terms of fermentation properties and transcriptional response. The genome-wide gene expression analysis highlighted that plasmid replication induced cell wall organization and biogenesis, and repressed approximately 20% of the genes involved in ribosome biogenesis and RNA processing. Although oxygen limitation was found to be less effective in the transcriptional changes, a link between oxygen limitation and genes involved in oxidation-reduction, pH reduction, phosphate-containing compounds, and lipid and fatty acid processes was observed.
Proceeding of The International Seminar on Chemistry 2008, 2008
Raw starch digesting amylolytic enzyme is a potential enzyme that can be used in bioethanol production, since it can save energy requirement of starch processing. S. fibuligera R-64 produces two types of amylolytic enzyme, i.e. α-amylase and glucoamylase, which are capable to digest raw starch. This research is aimed to investigate the effect of different carbon sources on amylase production by the yeast S. fibuligera and to investigate the enzyme ability in digesting raw starches. The media that used for enzyme production media were 1% of yeast extract and 1% of raw starch (sago, corn, cassava, and rice). The media was shaked (180 rpm) in room temperature for 72 hours. The crude extract was used for digesting raw starches. The crude extract was added to 5% (w/v) raw starch solution in 25 mM phosphate-citrate buffer pH 5.8. The mixture was then shaked in room temperature for 24 hours. After 24 hours, the sample was taken, and the reducing sugar was determined using colorimetric method using alkaline potassium ferry cyanide reagent. The results showed that sago is the best carbon source, which gave the highest amylolytic activity (305.77units/mg). We also found that sago was the easiest starch digested by the enzyme, followed by rice, corn and cassava.
Frontiers in Microbiology, 2021
Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient ...