Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56 g/L ethanol from 20% w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Medium optimization for enhanced production of recombinant lignin peroxidase in Pichia pastoris.

OBJECTIVES: Different cultivation conditions and parameters were evaluated to improve the production and secretion of a recombinant Phanerochaete chrysosporium lipH8 gene in Komagataella phaffii (Pichia pastoris). RESULTS: The recombinant lipH8 gene with its native secretion signal was successfully cloned and expressed in Komagataella phaffii (Pichia pastoris) under the control of the alcohol oxidase 1 promoter (PAOX1). The results revealed that co-feeding with sorbitol and methanol increased rLiP secretion by 5.9-fold compared to the control conditions. The addition of 1 mM FeSO4 increased LiP activity a further 6.0-fold during the induction phase. Moreover, the combination of several optimal conditions and parameters yielded an extracellular rLiP activity of 20.05 U l-1, which is more than ten-fold higher relative to standard growth conditions (BMM10 medium, pH 6 and 30 °C). CONCLUSION: Extracellular activity of a recombinant LiP expressed in P. pastoris increased more than ten-fold when co-feeding sorbitol and methanol as carbon sources, together with urea as nitrogen source, FeSO4 supplementation, lower pH and lower cultivation temperature.

Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance.

The fermentation of spent sulphite liquor (SSL) from the pulping of hardwoods is limited by the combination of xylose, the primary fermentable sugar and high concentrations of microbial inhibitors that decrease the yeast fermentation ability. The inhibitor resistance phenotypes of xylose-capable Saccharomyces cerevisiae strains were therefore enhanced by combining rational engineering for multi-inhibitor tolerance, with adaptation in concentrated hardwood SSL as selective pressure. The adapted strains were assessed in fermentations with 60-80% v/v concentrated SSL under industrially relevant fermentation conditions. During adaptation, strains produced ethanol concentrations between 11.0 and 15.4 g/L in the range of that reported in literature. The adapted TFA40 and TP50 strains displayed enhanced inhibitor resistance phenotypes and were able to ferment xylose-rich SSL at pH below 5, exhibiting improved ethanol yields relative to the reference strain. Using yeast extract and peptone as nitrogen source in concentrated SSL fermentations further improved ethanol yields. However, strains exhibited a trade-off between resistance and ethanol productivity, indicating a carbon/energy cost for the expression of this inhibitor tolerance phenotype. KEY POINTS : • Achieved fermentation of xylose-rich hardwood spent sulphite liquor at pH below 5.0 • Adaptation of xylose-capable S. cerevisiae in concentrated spent sulphite liquor • Adapted strains exhibited enhanced inhibitor resistance phenotypes.

Improved raw starch amylase production by Saccharomyces cerevisiae using codon optimisation strategies.

Amylases are used in a variety of industries that have a specific need for alternative enzymes capable of hydrolysing raw starch. Five α-amylase and five glucoamylase-encoding genes were expressed in the Saccharomyces cerevisiae Y294 laboratory strain to select for recombinant strains that best hydrolysed raw corn starch. Gene variants of four amylases were designed using codon optimisation and different secretion signals. The significant difference in activity levels among the gene variants confirms that codon optimisation of fungal genes for expression in S. cerevisiae does not guarantee improved recombinant protein production. The codon-optimised glucoamylase variant from Talaromyces emersonii (temG_Opt) yielded 3.3-fold higher extracellular activity relative to the native temG, whereas the codon-optimised T. emersonii α-amylase (temA_Opt) yielded 1.6-fold more extracellular activity than the native temA. The effect of four terminator sequences was also investigated using temG and temG_Opt as reporter genes, with the ALY2T terminator resulting in a 14% increase in glucoamylase activity relative to the gene cassettes containing the ENO1T terminator. This is the first report of engineered S. cerevisiae strains to express T. emersonii amylase variants, and these enzymes may have potential applications in the industrial conversion of raw starch under fermentation conditions.

Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha.

Cervical cancer is ranked the fourth most common cancer in women worldwide. Despite two prophylactic vaccines being commercially available, they are unaffordable for most women in developing countries. We compared the optimized expression of monomers of the unique HPV type 16 L1-L2 chimeric protein (SAF) in two yeast strains of Pichia pastoris, KM71 (Muts ) and GS115 (Mut+ ), with Hansenula polymorpha NCYC 495 to determine the preferred host in bioreactors. SAF was uniquely created by replacing the h4 helix of the HPV-16 capsid L1 protein with an L2 peptide. Two different feeding strategies in fed-batch cultures of P. pastoris Muts were evaluated: a predetermined feed rate vs. feeding based on the oxygen consumption by maintaining constant dissolved oxygen levels (DO stat). All cultures showed a significant increase in biomass when methanol was fed using the DO stat method. In P. pastoris the SAF concentrations were higher in the Muts strains than in the Mut+ strains. However, H. polymorpha produced the highest level of SAF at 132.10 mg L-1 culture while P. pastoris Muts only produced 23.61 mg L-1 . H. polymorpha showed greater potential for the expression of HPV-16 L1/L2 chimeric proteins despite the track record of P. pastoris as a high-level producer of heterologous proteins.

Expression and comparison of codon optimised Aspergillus tubingensis amylase variants in Saccharomyces cerevisiae.

The expression of codon optimised genes is a popular genetic engineering approach for the production of industrially relevant proteins. This study investigates and compares the expression of codon optimised and codon adapted amylase variants. The Aspergillus tubingensis raw starch hydrolysing α-amylase (amyA) and glucoamylase (glaA) encoding genes were redesigned using synonymous codons and expressed in Saccharomyces cerevisiae Y294. Codon optimisation to favour S. cerevisiae codon bias resulted in a decrease in extracellular enzyme activity of 72% (30.28 nkat ml-1) and 68% (4.08 nkat ml-1) compared to the expression of the native amyA and glaA genes, respectively, after 96 h of growth. However, a lower cultivation temperature and co-expression with the PDI1 gene increased extracellular activity levels of the codon optimised α-amylase and glucoamylase, respectively. Despite the identical amino acid sequence of GlaA, GlaA_Opt and GlaA_CBI, differential scanning fluorimetry revealed changes in the glucoamylase proteins' melting temperatures (>3°C). Shifts in the fluorescence curves suggest changes in glucoamylase tertiary structure. Results indicate that synonymous codon changes resulting from codon optimisation of amyA and glaA genes does not guarantee increased recombinant protein production and that there is crucial translational information present within the coding sequence that controls protein folding and secretion.

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae.

Crystalline cellulose is one of the major contributors to the recalcitrance of lignocellulose to degradation, necessitating high dosages of cellulase to digest, thereby impeding the economic feasibility of cellulosic biofuels. Several recombinant cellulolytic yeast strains have been developed to reduce the cost of enzyme addition, but few of these strains are able to efficiently degrade crystalline cellulose due to their low cellulolytic activities. Here, by combining the cellulase ratio optimization with a novel screening strategy, we successfully improved the cellulolytic activity of a Saccharomyces cerevisiae strain displaying four different synergistic cellulases on the cell surface. The optimized strain exhibited an ethanol yield from Avicel of 57% of the theoretical maximum, and a 60% increase of ethanol titer from rice straw. To our knowledge, this work is the first optimization of the degradation of crystalline cellulose by tuning the cellulase ratio in a cellulase cell-surface display system. This work provides key insights in engineering the cellulase cocktail in a consolidated bioprocessing yeast strain. Biotechnol. Bioeng. 2017;114: 1201-1207. © 2017 Wiley Periodicals, Inc.

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase.

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, Km  = 0.43 mM, Kcat = 0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50 °C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86 mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.74 U/ml/h) in fed-batch than batch fermentation.

Expression of rotavirus VP6 protein: a comparison amongst Escherichia coli, Pichia pastoris and Hansenula polymorpha.

During this study. we successfully expressed a codon-optimized gene for rotavirus VP6 protein intracellularly in two methylotrophic yeasts, Pichia pastoris and Hansenula polymorpha, during methanol induction. Expressions were performed in shake flasks and subsequently scaled-up to 1.3 L bioreactors. The yields obtained in the yeasts were compared with that observed in Escherichia coli. Despite producing the lowest biomass levels of all the expression systems in shake flasks, the highest VP6 concentration was obtained with E. coli. In shake flasks, P. pastoris yielded higher volumetric levels of VP6 than H. polymorpha, but specific production of VP6 was approximately similar in both yeasts. In the controlled environment of bioreactors, yeast strains attained typical high cell densities, but also increased VP6 production compared to all shake flask cultures. Unlike in shake flask expressions, H. polymorpha outperformed both P. pastoris as well as E. coli during bioreactor cultivation. VP6 production was in all three expression systems growth-associated. In contrast to yeast expressions, bacterial expressed VP6 protein was found to be insoluble upon analysis. This is the first report of VP6 expressed in methylotrophic yeast and holds the promise for the inexpensive production of VP6 as a possible vaccine candidate or drug delivery mechanism.

Overexpression of native Saccharomyces cerevisiae ER-to-Golgi SNARE genes increased heterologous cellulase secretion.

Soluble N-ethylmaleimide-sensitive factor attachment receptor proteins (SNAREs) are essential components of the yeast protein-trafficking machinery and are required at the majority of membrane fusion events in the cell, where they facilitate SNARE-mediated fusion between the protein transport vesicles, the various membrane-enclosed organelles and, ultimately, the plasma membrane. We have demonstrated an increase in secretory titers for the Talaromyces emersonii Cel7A (Te-Cel7A, a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (Sf-Cel3A, a ß-glucosidase) expressed in Saccharomyces cerevisiae through single and co-overexpression of some of the endoplasmic reticulum (ER)-to-Golgi SNAREs (BOS1, BET1, SEC22 and SED5). Overexpression of SED5 yielded the biggest improvements for both of the cellulolytic reporter proteins tested, with maximum increases in extracellular enzyme activity of 22 % for the Sf-Cel3A and 68 % for the Te-Cel7A. Co-overexpression of the ER-to-Golgi SNAREs yielded proportionately smaller increases for the Te-Cel7A (46 %), with the Sf-Cel3A yielding no improvement. Co-overexpression of the most promising exocytic SNARE components identified in literature for secretory enhancement of the cellulolytic proteins tested (SSO1 for Sf-Cel3A and SNC1 for Te-Cel7A) with the most effective ER-to-Golgi SNARE components identified in this study (SED5 for both Sf-Cel3A and Te-Cel7A) yielded variable results, with Sf-Cel3A improved by 131 % and Te-Cel7A yielding no improvement. Improvements were largely independent of gene dosage as all strains only integrated single additional SNARE gene copies, with episomal variance between the most improved strains shown to be insignificant. This study has added further credence to the notion that SNARE proteins fulfil an essential role within a larger cascade of secretory machinery components that could contribute significantly to future improvements to S. cerevisiae as protein production host.

Consolidated bioprocessing of starchy substrates into ethanol by industrial Saccharomyces cerevisiae strains secreting fungal amylases.

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 higher on natural sorghum and triticale substrates (62 and 73% of the theoretical yield, respectively). This is the first report of direct ethanol production from natural starchy substrates (without any pre-treatment or commercial enzyme addition) using industrial yeast strains co-secreting both a glucoamylase and α-amylase.

Over-expression of native Saccharomyces cerevisiae exocytic SNARE genes increased heterologous cellulase secretion.

SNAREs (soluble NSF [N-ethylmaleimide-sensitive factor] attachment receptor proteins) are required at the majority of fusion events during intracellular membrane transport and play crucial roles in facilitating protein trafficking between the various membrane-enclosed organelles and the plasma membrane. We demonstrate increases in the secretion of the Talaromyces emersonii Cel7A (a cellobiohydrolase) and the Saccharomycopsis fibuligera Cel3A (a ß-glucosidase), through the separate and simultaneous over-expression of different components of the exocytic SNARE complex in Saccharomyces cerevisiae. Over-expression of SNC1 yielded the biggest improvement in Te-Cel7A secretion (71 %), whilst SSO1 over-expression lead to the highest increases in Sf-Cel3A secretion (43.8 %). Simultaneous over-expression of differential combinations of these SNARE components yielded maximal increases of ~52 % and ~49 % for the secretion of Te-Cel7A and Sf-Cel3A, respectively. These increases generally did not cause deleterious growth effects, whilst differential improvement patterns were observed for the two reporter proteins (Sf-Cel3A and Te-Cel7A). Simultaneous over-expression of up to three of these components, in strains secreting the more efficiently expressed Sf-Cel3A, illustrated a slight decrease in osmotic tolerance at elevated NaCl concentrations, as well as a detectable decrease in ethanol tolerance at increased concentrations. This work illustrates the potential of engineering components of the anterograde secretory pathway, particularly its SNARE components, for the improvement of heterologous cellulase secretion.

Production of cellulosic ethanol and enzyme from waste fiber sludge using SSF, recycling of hydrolytic enzymes and yeast, and recombinant cellulase-producing Aspergillus niger.

Bioethanol and enzymes were produced from fiber sludges through sequential microbial cultivations. After a first simultaneous saccharification and fermentation (SSF) with yeast, the bioethanol concentrations of sulfate and sulfite fiber sludges were 45.6 and 64.7 g/L, respectively. The second SSF, which included fresh fiber sludges and recycled yeast and enzymes from the first SSF, resulted in ethanol concentrations of 38.3 g/L for sulfate fiber sludge and 24.4 g/L for sulfite fiber sludge. Aspergillus niger carrying the endoglucanase-encoding Cel7B gene of Trichoderma reesei was grown in the spent fiber sludge hydrolysates. The cellulase activities obtained with spent hydrolysates of sulfate and sulfite fiber sludges were 2,700 and 2,900 nkat/mL, respectively. The high cellulase activities produced by using stillage and the significant ethanol concentrations produced in the second SSF suggest that onsite enzyme production and recycling of enzyme are realistic concepts that warrant further attention.

Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

Engineered yeast for the efficient hydrolysis of polylactic acid.

Polylactic acid (PLA) is a major contributor to the global bioplastic production capacity. However, post-consumer PLA waste is not fully degraded during non-optimal traditional organic waste treatment processes and can persist in nature for many years. Efficient enzymatic hydrolysis of PLA would contribute to cleaner, more energy-efficient, environmentally friendly waste management processes. However, high costs and a lack of effective enzyme producers curtail the large-scale application of such enzymatic systems. This study reports the recombinant expression of a fungal cutinase-like enzyme (CLE1) in the yeast Saccharomyces cerevisiae, which produced a crude supernatant that efficiently hydrolyses different types of PLA materials. The codon-optimised Y294[CLEns] strain delivered the best enzyme production and hydrolysis capabilities, releasing up to 9.44 g/L lactic acid from 10 g/L PLA films with more than 40% loss in film weight. This work highlights the potential of fungal hosts producing PLA hydrolases for future commercial applications in PLA recycling.

Enzymatic hydrolysis of single-use bioplastic items by improved recombinant yeast strains.

Single-use bioplastic items pose new challenges for a circular plastics economy as they require different processing than petroleum-based plastics items. Microbial and enzymatic recycling approaches could address some of the pitfalls created by the influx of bioplastic waste. In this study, the recombinant expression of a cutinase-like-enzyme (CLE1) was improved in the yeast Saccharomyces cerevisiae to efficiently hydrolyse several commercial single-use bioplastic items constituting blends of poly(lactic acid), poly(1,4-butylene adipate-co-terephthalate), poly(butylene succinate) and mineral fillers. The hydrolysis process was optimised in controlled bioreactor configurations to deliver substantial monomer concentrations and, ultimately, 29 to 78% weight loss. Product inhibition studies and molecular docking provided insights into potential bottlenecks of the enzymatic hydrolysis process, while FT-IR analysis showed the preferential breakdown of specific polymers in blended commercial bioplastic items. This work constitutes a step towards implementing enzymatic hydrolysis as a circular economy approach for the valorisation of end-of-life single-use bioplastic items.

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 ß-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.

Heterologous Expression of Plantaricin 423 and Mundticin ST4SA in Saccharomyces cerevisiae.

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals.

Additional glucoamylase genes increase ethanol productivity on rice and potato waste streams by a recombinant amylolytic yeast.

The implementation of consolidated bioprocessing for converting starch to ethanol relies on a robust yeast that produces enough amylases for rapid starch hydrolysis. Furthermore, using low-cost substrates will assist with competitive ethanol prices and support a bioeconomy, especially in developing countries. This paper addresses both challenges with the expression of additional glucoamylase gene copies in an efficient amylolytic strain (Saccharomyces cerevisiae ER T12) derived from the industrial yeast, Ethanol Red™. Recombinant ER T12 was used as a host to increase ethanol productivity during raw starch fermentation; the ER T12.7 variant, selected from various transformants, displayed enhanced raw starch conversion and a 36% higher ethanol concentration than the parental strain after 120 h. Unripe rice, rice bran, potato waste and potato peels were evaluated as alternative starchy substrates to test ER T12.7's fermenting ability. ER T12.7 produced high ethanol yields at significantly improved ethanol productivity, key criteria for its industrial application.

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture.

Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and ß-glucosidase activities of 0.038 and 0.30 U mg dry cell weight(-1), respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.