Effective application of immobilized second generation industrial Saccharomyces cerevisiae strain on consolidated bioprocessing.

Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts.

Whole-Genome Transformation of Yeast Promotes Rare Host Mutations with a Single Causative SNP Enhancing Acetic Acid Tolerance.

Whole-genome (WG) transformation (WGT) with DNA from the same or another species has been used to obtain strains with superior traits. Very few examples have been reported in eukaryotes-most apparently involving integration of large fragments of foreign DNA into the host genome. We show that WGT of a haploid acetic acid-sensitive Saccharomyces cerevisiae strain with DNA from a tolerant strain, but not from nontolerant strains, generated many tolerant transformants, some of which were stable upon subculturing under nonselective conditions. The most tolerant stable transformant contained no foreign DNA but only seven nonsynonymous single nucleotide polymorphisms (SNPs), of which none was present in the donor genome. The SNF4 mutation c.[805G→T], generating Snf4E269*, was the main causative SNP. Allele exchange of SNF4E269* or snf4Δ in industrial strains with unrelated genetic backgrounds enhanced acetic acid tolerance during fermentation under industrially relevant conditions. Our work reveals a surprisingly small number of mutations introduced by WGT, which do not bear any sequence relatedness to the genomic DNA (gDNA) of the donor organism, including the causative mutation. Spontaneous mutagenesis under protection of a transient donor gDNA fragment, maintained as extrachromosomal circular DNA (eccDNA), might provide an explanation. Support for this mechanism was obtained by transformation with genomic DNA of a yeast strain containing NatMX and selection on medium with nourseothricin. Seven transformants were obtained that gradually lost their nourseothricin resistance upon subculturing in nonselective medium. Our work shows that WGT is an efficient strategy for rapidly generating and identifying superior alleles capable of improving selectable traits of interest in industrial yeast strains.

Simultaneous secretion of seven lignocellulolytic enzymes by an industrial second-generation yeast strain enables efficient ethanol production from multiple polymeric substrates.

A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. We have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, ß-glucosidase, cellobiohydrolase I and II, xylanase, ß-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain, reaching 94.5 FPU/g CDW and enabling direct conversion of lignocellulosic substrates into ethanol without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the heterologous enzyme secretion. This strain can therefore serve as a promising industrial platform strain for development of yeast cell factories that can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.

Engineering tolerance to industrially relevant stress factors in yeast cell factories.

The main focus in development of yeast cell factories has generally been on establishing optimal activity of heterologous pathways and further metabolic engineering of the host strain to maximize product yield and titer. Adequate stress tolerance of the host strain has turned out to be another major challenge for obtaining economically viable performance in industrial production. Although general robustness is a universal requirement for industrial microorganisms, production of novel compounds using artificial metabolic pathways presents additional challenges. Many of the bio-based compounds desirable for production by cell factories are highly toxic to the host cells in the titers required for economic viability. Artificial metabolic pathways also turn out to be much more sensitive to stress factors than endogenous pathways, likely because regulation of the latter has been optimized in evolution in myriads of environmental conditions. We discuss different environmental and metabolic stress factors with high relevance for industrial utilization of yeast cell factories and the experimental approaches used to engineer higher stress tolerance. Improving stress tolerance in a predictable manner in yeast cell factories should facilitate their widespread utilization in the bio-based economy and extend the range of products successfully produced in large scale in a sustainable and economically profitable way.