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.

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.

Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems.

Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts.

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, ß-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. Higher secretion titers were achieved by cellulolytic strains with the YI13 genetic background and cellulolytic transformants produced up to 1.34 fold higher glucose concentrations (g/L) than a control composed of equal amounts of each enzyme type. The transformant co-producing BGLI and EGII in a secreted ratio of 1:15 (cellulase activity unit per gram dry cell weight) converted 56.5% of the cellulose present in corn cob to glucose in hydrolysis experiments and yielded 4.05 g/L ethanol in fermentations. We demonstrate that the choice of optimal genetic background and cellulase activity secretion ratio can improve cellulosic ethanol production by consolidated bioprocessing yeast strains.