Promoters and introns as key drivers for enhanced gene expression in Saccharomyces cerevisiae.

The transcription of genes in the yeast Saccharomyces cerevisiae is governed by multiple layers of regulatory elements and proteins, cooperating to ensure optimum expression of the final protein product based on the cellular requirements. Promoters have always been regarded as the most important determinant of gene transcription, but introns also play a key role in the expression of intron-encoding genes. Some introns can enhance transcription when introduced either promoter-proximal or embedded in the open reading frame of genes. However, the outcome is seldom predictable, with some introns increasing or decreasing transcription depending on the promoter and reporter gene employed. This chapter provides an overview of the general structure and function of promoters and introns and how they may cooperate during transcription to allow intron-mediated enhancement of gene expression. Since S. cerevisiae is a suitable host for recombinant protein production on a commercial level, stronger and more controllable promoters are in high demand. Enhanced gene expression can be achieved via promoter engineering, which may include introns that increase the efficacy of recombinant expression cassettes. Different models for the role of introns in transcription are briefly discussed to show how these intervening sequences can actively interact with the transcription machinery. Furthermore, recent examples of improved protein production via the introduction of promoter-proximal introns are highlighted to showcase the potential value of intron-mediated enhancement of gene expression.

Improvement of cell-tethered cellulase activity in recombinant strains of Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) remains an attractive option for the production of commodity products from pretreated lignocellulose if a process-suitable organism can be engineered. The yeast Saccharomyces cerevisiae requires engineered cellulolytic activity to enable its use in CBP production of second-generation (2G) bioethanol. A promising strategy for heterologous cellulase production in yeast entails displaying enzymes on the cell surface by means of glycosylphosphatidylinositol (GPI) anchors. While strains producing a core set of cell-adhered cellulases that enabled crystalline cellulose hydrolysis have been created, secreted levels of enzyme were insufficient for complete cellulose hydrolysis. In fact, all reported recombinant yeast CBP candidates must overcome the drawback of generally low secretion titers. Rational strain engineering can be applied to enhance the secretion phenotype. This study aimed to improve the amount of cell-adhered cellulase activities of recombinant S. cerevisiae strains expressing a core set of four cellulases, through overexpression of genes that were previously shown to enhance cellulase secretion. Results showed significant increases in cellulolytic activity for all cell-adhered cellulase enzyme types. Cell-adhered cellobiohydrolase activity was improved by up to 101%, ß-glucosidase activity by up to 99%, and endoglucanase activity by up to 231%. Improved hydrolysis of crystalline cellulose of up to 186% and improved ethanol yields from this substrate of 40-50% in different strain backgrounds were also observed. In addition, improvement in resistance to fermentation stressors was noted in some strains. These strains represent a step towards more efficient organisms for use in 2G biofuel production. KEY POINTS: • Cell-surface-adhered cellulase activity was improved in strains engineered for CBP. • Levels of improvement of activity were strain and enzyme dependent. • Crystalline cellulose conversion to ethanol could be improved up to 50%.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains.

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain-heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.

The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains.

The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector-based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae PHAC1 or PKAR2 promoters. The eGFP reporter under control of PKAR2 was identified as the preferred combination due to its superior dynamic range and its greater sensitivity when measuring UPR induction in cellulase producing strains. To our knowledge, we show for the first time that significant UPR activation differences could consistently be observed for different cellulase candidate genes unlike previous RNA-based tests, which were unable to detect these differences. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them favorable for use in CBP yeasts.

Exploring industrial and natural Saccharomyces cerevisiae strains for the bio-based economy from biomass: the case of bioethanol.

Saccharomyces cerevisiae is the preferred microorganism for the production of bioethanol from biomass. Industrial strain development for first-generation ethanol from sugar cane and corn mostly relies on the historical know-how from high gravity beer brewing and alcohol distilleries. However, the recent design of yeast platforms for the production of second-generation biofuels and green chemicals from lignocellulose exposes yeast to different environments and stress challenges. The industrial need for increased productivity, wider substrate range utilization, and the production of novel compounds leads to renewed interest in further extending the use of current industrial strains by exploiting the immense, and still unknown, potential of natural yeast strains. This review describes key metabolic engineering strategies tailored to develop efficient industrial and novel natural yeast strains towards bioethanol production from biomass. Furthermore, it shapes how proof-of-concept studies, often advanced in academic settings on natural yeast, can be upgraded to meet the requirements for industrial applications. Academic and industrial research should continue to cooperate on both improving existing industrial strains and developing novel phenotypes by exploring the vast biodiversity available in nature on the road to establish yeast biorefineries where a range of biomass substrates are converted into valuable compounds.

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.

Comparing laboratory and industrial yeast platforms for the direct conversion of cellobiose into ethanol under simulated industrial conditions.

An engineered yeast producing all the cellulases needed for cellulose saccharification could produce ethanol from lignocellulose at a lower cost. This study aimed to express fungal ß-glucosidases in Saccharomyces cerevisiae to convert cellobiose into ethanol. Furthermore, two engineering platforms (laboratory vs industrial strain) have been considered towards the successful deployment of the engineered yeast under simulated industrial conditions. The industrial S. cerevisiae M2n strain was engineered through the δ-integration of the ß-glucosidase Pccbgl1 of Phanerochaete chrysosporium. The most efficient recombinant, M2n[pBKD2-Pccbgl1]-C1, was compared to the laboratory S. cerevisiae Y294[Pccbgl1] strain, expressing Pccbgl1 from episomal plasmids, in terms of cellobiose fermentation in a steam exploded sugarcane bagasse pre-hydrolysate. Saccharomyces cerevisiae Y294[Pccbgl1] was severely hampered by the pre-hydrolysate. The industrial M2n[pBKD2-Pccbgl1]-C1 could tolerate high inhibitors-loading in pre-hydrolysate under aerobic conditions. However, in oxygen limited environment, the engineered industrial strain displayed ethanol yield higher than the laboratory Y294[Pccbgl1] only when supplemented with supernatant containing further recombinant ß-glucosidase. This study showed that the choice of the host strain is crucial to ensure bioethanol production from lignocellulose. A novel cellobiose-to-ethanol route has been developed and the recombinant industrial yeast could be a promising platform towards the future consolidated bioprocessing of lignocellulose into ethanol.

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (ß-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

Engineering of Saccharomyces cerevisiae to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase, xylosidase and a bacterial xylose isomerase.

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.

Constructing recombinant Saccharomyces cerevisiae strains for malic-to-fumaric acid conversion.

Saccharomyces cerevisiae with its robustness and good acid tolerance, is an attractive candidate for use in various industries, including waste-based biorefineries where a high-value organic acid is produced, such as fumaric acid could be beneficial. However, this yeast is not a natural producer of dicarboxylic acids, and genetic engineering of S. cerevisiae strains is required to achieve this outcome. Disruption of the natural FUM1 gene and the recombinant expression of fumarase and malate transporter genes improved the malic acid-to-fumaric acid conversion by engineered S. cerevisiae strains. The efficacy of the strains was significantly influenced by the source of the fumarase gene (yeast versus bacterial), the presence of the XYNSEC signal secretion signal and the available oxygen in synthetic media cultivations. The ΔFUM1Ckr_fum + mae1 and ΔFUM1(ss)Ckr_fum + mae1 strains converted extracellular malic acid into 0.98 and 1.11 g/L fumaric acid under aerobic conditions.

Valorization of apple and grape wastes with malic acid-degrading yeasts.

It is estimated that more than 20% of processed apples and grapes are discarded as waste, which is dominated by pomace rich in malic acid that could be converted to high-value organic acids or other chemicals. A total of 98 yeast strains isolated from apple, grape, and plum wastes were evaluated for their ability to degrade malic acid relative to known yeast strains. Most (94%) of the new isolates degraded malic acid efficiently (> 50%) in the presence and absence of exogenous glucose, whereas only 14% of the known strains could do so, thus confirming the value of exploring (and exploiting) natural biodiversity. The best candidates were evaluated in synthetic media for their ability to convert malic acid to other valuable products under aerobic and oxygen-limited conditions, with two strains that produced ethanol and acetic acid as potential biorefinery products during aerobic cultivations and oxygen-limited fermentations on sterilized apple and grape pomace. Noteworthy was the identification of a Saccharomyces cerevisiae strain that is more efficient in degrading malic acid than other members of the species. This natural strain could be of value in the wine-making industry that often requires pH corrections due to excess malic acid.

Mating of natural Saccharomyces cerevisiae strains for improved glucose fermentation and lignocellulosic inhibitor tolerance.

Natural Saccharomyces cerevisiae isolates from vineyards in the Western Cape, South Africa were evaluated for ethanol production in industrial conditions associated with the production of second-generation biofuels. The strains displayed high phenotypic diversity including the ability to grow at 45 °C and in the presence of 20% (v/v) ethanol, strain YI13. Strains HR4 and YI30 were inhibitor-tolerant under aerobic and oxygen-limited conditions, respectively. Spore-to-spore hybridization generated progeny that displayed heterosis, including increased ethanol productivity and improved growth in the presence of a synthetic inhibitor cocktail. Hybrid strains HR4/YI30#6 and V3/YI30#6 were able to grow at a high salt concentration (2 mol/L NaCl) with V3/YI30#6 also able to grow at a high temperature (45 °C). Strains HR4/YI30#1 and #3 were inhibitor-tolerant, with strain HR4/YI30#3 having similar productivity (0.36 ± 0.0036 g/L per h) as the superior parental strain, YI30 (0.35 ± 0.0058 g/L per h). This study indicates that natural S. cerevisiae strains display phenotypic variation and heterosis can be achieved through spore-to-spore hybridization. Several of the phenotypes (temperature-, osmo-, and inhibitor tolerance) displayed by both the natural strains and the generated progeny were at the maximum conditions reported for S. cerevisiae strains.

Production of bioethanol from multiple waste streams of rice milling.

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20%w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20%w/v), mainly composed by lignocellulose, was pre-treated at 55°C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic® CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20%w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production.

Special issue from the 20th International Symposium on Alcohol Fuels (ISAF 2013): alcohol fuels enabling sustainable future development.

This Editorial introduces a special issue from the 20th International Symposium on Alcohol Fuels (ISAF 2013) on alcohol fuels enabling sustainable future development.

A global conversation about energy from biomass: the continental conventions of the global sustainable bioenergy project.

The global sustainable bioenergy (GSB) project was formed in 2009 with the goal of providing guidance with respect to the feasibility and desirability of sustainable, bioenergy-intensive futures. Stage 1 of this project held conventions with a largely common format on each of the world's continents, was completed in 2010, and is described in this paper. Attended by over 400 persons, the five continental conventions featured presentations, breakout sessions, and drafting of resolutions that were unanimously passed by attendees. The resolutions highlight the potential of bioenergy to make a large energy supply contribution while honouring other priorities, acknowledge the breadth and complexity of bioenergy applications as well as the need to take a systemic approach, and attest to substantial intra- and inter-continental diversity with respect to needs, opportunities, constraints and current practice relevant to bioenergy. The following interim recommendations based on stage 1 GSB activities are offered: - Realize that it may be more productive, and also more correct, to view the seemingly divergent assessments of bioenergy as answers to two different questions rather than the same question. Viewed in this light, there is considerably more scope for reconciliation than might first be apparent, and it is possible to be informed rather than paralysed by divergent assessments.- Develop established and advanced bioenergy technologies such that each contributes to the other's success. That is, support and deploy in the near-term meritorious, established technologies in ways that enhance rather than impede deployment of advanced technologies, and support and deploy advanced technologies in ways that expand rather than contract opportunities for early adopters and investors.- Be clear in formulating policies what mix of objectives are being targeted, measure the results of these policies against these objectives and beware of unintended consequences.- Undertake further exploration of land efficiency levers and visions for multiply-beneficial bioenergy deployment. This should be unconstrained by current practices, since we cannot hope to achieve a sustainable and a secure future by continuing the practices that have led to the unsustainable and insecure present. It should also be approached from a global perspective, based on the best science available, and consider the diverse realities, constraints, needs and opportunities extant in different regions of the world.The future trajectory of the GSB project is also briefly considered.