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, β-glucosidase, cellobiohydrolase I and II, xylanase, β-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.status: publishe

Polygenic Analysis in Absence of Major Effector ATF1 Unveils Novel Components in Yeast Flavor Ester Biosynthesis

Flavor production in yeast fermentation is of paramount importance for industrial production of alcoholic beverages. Although major enzymes of flavor compound biosynthesis have been identified, few specific mutations responsible for strain diversity in flavor production are known. The ATF1-encoded alcohol acetyl coenzyme A (acetyl-CoA) transferase (AATase) is responsible for the majority of acetate ester biosynthesis, but other components affecting strain diversity remain unknown. We have performed parallel polygenic analysis of low production of ethyl acetate, a compound with an undesirable solvent-like off-flavor, in strains with and without deletion of ATF1 We identified two unique causative mutations, eat1K179fs and snf8E148*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence of ATF1EAT1 encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that of EAT1K179fs , and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced when eat1K179fs was present together with ATF1 in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction of snf8E148* lowered ethyl acetate levels also in the presence of ATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering of snf8E148* in three industrial yeast strains (for production of wine, saké, and ale beer) and fermentation in an application-relevant medium showed a high but strain-dependent potential for flavor enhancement. Our work has identified EAT1 and SNF8 as new genetic elements determining ethyl acetate production diversity in yeast strains.IMPORTANCE Basic research with laboratory strains of the yeast Saccharomyces cerevisiae has identified the structural genes of most metabolic enzymes, as well as genes encoding major regulators of metabolism. On the other hand, more recent work on polygenic analysis of yeast biodiversity in natural and industrial yeast strains is revealing novel components of yeast metabolism. A major example is the metabolism of flavor compounds, a particularly important property of industrial yeast strains used for the production of alcoholic beverages. In this work, we have performed polygenic analysis of production of ethyl acetate, an important off-flavor compound in beer and other alcoholic beverages. To increase the chances of identifying novel components, we have used in parallel a wild-type strain and a strain with a deletion of ATF1 encoding the main enzyme of acetate ester biosynthesis. This revealed a new structural gene, EAT1, encoding a putative mitochondrial enzyme, which was recently identified as an ethanol acetyl-CoA transferase in another yeast species. We also identified a novel regulatory gene, SNF8, which has not previously been linked to flavor production. Our results show that polygenic analysis of metabolic traits in the absence of major effector genes can reveal novel structural and regulatory genes. The mutant alleles identified can be used to affect the flavor profile in industrial yeast strains for production of alcoholic beverages in more subtle ways than by deletion or overexpression of the already known major effector genes and without significantly altering other industrially important traits. The effect of the novel variants was dependent on the genetic background, with a highly desirable outcome in the flavor profile of an ale brewing yeast.status: publishe

Looking beyond Saccharomyces: the potential of non-conventional yeast species for desirable traits in bioethanol fermentation

Saccharomyces cerevisiae has been used for millennia in the production of food and beverages and is by far the most studied yeast species. Currently, it is also the most used microorganism in the production of first-generation bioethanol from sugar or starch crops. Second-generation bioethanol, on the other hand, is produced from lignocellulosic feedstocks that are pretreated and hydrolyzed to obtain monomeric sugars, mainly D-glucose, D-xylose and L-arabinose. Recently, S. cerevisiae recombinant strains capable of fermenting pentose sugars have been generated. However, the pretreatment of the biomass results in hydrolysates with high osmolarity and high concentrations of inhibitors. These compounds negatively influence the fermentation process. Therefore, robust strains with high stress tolerance are required. Up to now, more than 2000 yeast species have been described and some of these could provide a solution to these limitations because of their high tolerance to the most predominant stress conditions present in a second-generation bioethanol reactor. In this review, we will summarize what is known about the non-conventional yeast species showing unusual tolerance to these stresses, namely Zygosaccharomyces rouxii (osmotolerance), Kluyveromyces marxianus and Ogataea (Hansenula) polymorpha (thermotolerance), Dekkera bruxellensis (ethanol tolerance), Pichia kudriavzevii (furan derivatives tolerance) and Z. bailii (acetic acid tolerance).status: publishe

The molecular biology of fruity and floral aromas in beer and other alcoholic beverages

Aroma compounds provide attractiveness and variety to alcoholic beverages. We discuss the molecular biology of a major subset of beer aroma volatiles, fruity and floral compounds, originating from raw materials (malt and hops), or formed by yeast during fermentation. We introduce aroma perception, describe the most aroma-active, fruity and floral compounds in fruits and their presence and origin in beer. They are classified into categories based on their functional groups and biosynthesis pathways: (1) higher alcohols and esters, (2) polyfunctional thiols, (3) lactones and furanones, and (4) terpenoids. Yeast and hops are the main sources of fruity and flowery aroma compounds in beer. For yeast, the focus is on higher alcohols and esters, and particularly the complex regulation of the alcohol acetyl transferase ATF1 gene. We discuss the release of polyfunctional thiols and monoterpenoids from cysteine- and glutathione-S-conjugated compounds and glucosides, respectively, the primary biological functions of the yeast enzymes involved, their mode of action and mechanisms of regulation that control aroma compound production. Furthermore, we discuss biochemistry and genetics of terpenoid production and formation of non-volatile precursors in Humulus lupulus (hops). Insight in these pathways provides a toolbox for creating innovative products with a diversity of pleasant aromas.status: publishe

Repeated batches as a strategy for high 2G ethanol production from undetoxified hemicellulose hydrolysate using immobilized cells of recombinant Saccharomyces cerevisiae in a fixed-bed reactor

Background: The search for sustainable energy sources has become a worldwide issue, making the development of efficient biofuel production processes a priority. Immobilization of second-generation (2G) xylose-fermenting Saccharomyces cerevisiae strains is a promising approach to achieve economic viability of 2G bioethanol production from undetoxified hydrolysates through operation at high cell load and mitigation of inhibitor toxicity. In addition, the use of a fixed-bed reactor can contribute to establish an efficient process because of its distinct advantages, such as high conversion rate per weight of biocatalyst and reuse of biocatalyst. Results: This work assessed the influence of alginate entrapment on the tolerance of recombinant S. cerevisiae to acetic acid. Encapsulated GSE16-T18SI.1 (T18) yeast showed an outstanding performance in repeated batch fermentations with cell recycling in YPX medium supplemented with 8 g/L acetic acid (pH 5.2), achieving 10 cycles without significant loss of productivity. In the fixed-bed bioreactor, a high xylose fermentation rate with ethanol yield and productivity values of 0.38 gethanol/gsugars and 5.7 g/L/h, respectively were achieved in fermentations using undetoxified sugarcane bagasse hemicellulose hydrolysate, with and without medium recirculation. Conclusions: The performance of recombinant strains developed for 2G ethanol production can be boosted strongly by cell immobilization in alginate gels. Yeast encapsulation allows conducting fermentations in repeated batch mode in fixed-bed bioreactors with high xylose assimilation rate and high ethanol productivity using undetoxified hemicellulose hydrolysate.status: publishe