Inducible Synthetic Growth Regulation Using the ClpXP Proteasome Enhances cis,cis-Muconic Acid and Glycolic Acid Yields in .

Engineered microbial cells can produce sustainable chemistry, but the production competes for resources with growth. Inducible synthetic control over the resource use would enable fast accumulation of sufficient biomass and then divert the resources to production. We developed inducible synthetic resource-use control over by expressing a bacterial ClpXP proteasome from an inducible promoter.

Use of ambr250 to assess mucic acid production in fed-batch cultures of a marine Trichoderma sp. D-221704.

Mucic acid, a diacid with potential use in the food, cosmetic, chemical and pharmaceutical industries, can be produced by microbial conversion of D-galacturonic acid, which is abundant in pectin. Using the ambr250 bioreactor system, we found that a recently generated transformant (D-221704, formerly referred to as T2) of a marine Trichoderma species produced up to 53 g L mucic acid in glucose-limited fed-batch culture with D-galacturonic acid in the feed at pH 4, with a yield of 0.99 g mucic acid per g D-galacturonic acid consumed.

Lager Yeast Design Through Meiotic Segregation of a × Hybrid.

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial × tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed.

Engineering marine fungi for conversion of D-galacturonic acid to mucic acid.

Background: Two marine fungi, a Trichoderma sp. and a Coniochaeta sp., which can grow on D-galacturonic acid and pectin, were selected as hosts to engineer for mucic acid production, assessing the suitability of marine fungi for production of platform chemicals.

Diacetyl control during brewery fermentation via adaptive laboratory engineering of the lager yeast Saccharomyces pastorianus.

Diacetyl contributes to the flavor profile of many fermented products. Its typical buttery flavor is considered as an off flavor in lager-style beers, and its removal has a major impact on time and energy expenditure in breweries. Here, we investigated the possibility of lowering beer diacetyl levels through evolutionary engineering of lager yeast for altered synthesis of α-acetolactate, the precursor of diacetyl.

Trans-regulation and localization of orthologous maltose transporters in the interspecies lager yeast hybrid.

In the interspecies lager yeast hybrid there are MAL loci involved in maltose and maltotriose utilization derived from each parent (Saccharomyces cerevisiae and Saccharomyces eubayanus). We show that trans-regulation across hybrid subgenomes occurs for MAL genes. However, gene expression is less efficient with non-native activators (trans-activation) compared to native activators (cis-activation).

Overexpressed maltose transporters in laboratory and lager yeasts: Localization and competition with endogenous transporters.

Plain and fluorescently tagged versions of Agt1, Mtt1 and Malx1 maltose transporters were overexpressed in two laboratory yeasts and one lager yeast. The plain and tagged versions of each transporter supported similar transport activities, indicating that they are similarly trafficked and have similar catalytic activities. When they were expressed under the control of the strong constitutive PGK1 promoter only minor proportions of the fluorescent transporters were associated with the plasma membrane, the rest being found in intracellular structures.

Improved cider fermentation performance and quality with newly generated Saccharomyces cerevisiae × Saccharomyces eubayanus hybrids.

Yeast cryotolerance may be advantageous for cider making, where low temperatures are usually employed. Here, we crossed the cryotolerant S. eubayanus with a S.

Novel brewing yeast hybrids: creation and application.

The natural interspecies Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid yeast is responsible for global lager beer production and is one of the most important industrial microorganisms. Its success in the lager brewing environment is due to a combination of traits not commonly found in pure yeast species, principally low-temperature tolerance, and maltotriose utilization. Parental transgression is typical of hybrid organisms and has been exploited previously for, e.

Maltose and maltotriose utilisation by group I strains of the hybrid lager yeast Saccharomyces pastorianus.

Brewer's wort is a challenging environment for yeast as it contains predominantly α-glucoside sugars. There exist two subgroups of the lager yeast Saccharomyces pastorianus which differ in sugar utilisation. We performed wort fermentations and compared representative strains from both groups with respect to their ability to transport and ferment maltose and maltotriose.

Ploidy influences the functional attributes of de novo lager yeast hybrids.

The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification.

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose.

Genes encoding L-arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on L-arabinose was dependent on a functioning L-arabinose transporter, or by screening a differential display library, respectively. These transporters also transport D-xylose and were designated KmAXT1 (arabinose-xylose transporter) and PgAXT1, respectively. Transport assays using L-arabinose showed that KmAxt1p has K(m) 263 mM and V(max) 57 nM/mg/min, and PgAxt1p has K(m) 0.

New lager yeast strains generated by interspecific hybridization.

The interspecific hybrid Saccharomyces pastorianus is the most commonly used yeast in brewery fermentations worldwide. Here, we generated de novo lager yeast hybrids by mating a domesticated and strongly flocculent Saccharomyces cerevisiae ale strain with the Saccharomyces eubayanus type strain. The hybrids were characterized with respect to the parent strains in a wort fermentation performed at temperatures typical for lager brewing (12 °C).

Three Agt1 transporters from brewer's yeasts exhibit different temperature dependencies for maltose transport over the range of brewery temperatures (0–20 °C).

Zero-trans rates of maltose transport by brewer's yeasts exert strong control over fermentation rates and are strongly temperature-dependent over the temperature range (20–0 °C) of brewery fermentations. Three α-glucoside transporters, ScAgt1(A60) (a Saccharomyces cerevisiae version of Agt1 from an ale strain), ScAgt1-A548V (a variant of ScAgt1(A60) with a single amino acid change in a transmembrane domain), and SbAgt1 (a Saccharomyces (eu)bayanus version from a lager strain), were compared. When expressed in the same laboratory yeast, grown at 24 °C and assayed at 0, 10, and 20 °C, SbAgt1 had the lowest absolute maltose uptake activity at 20 °C but smallest temperature dependence, ScAgt1-A548V had the highest activity but greatest temperature dependence, and ScAgt1(A60) had intermediate properties.

Variation in α-acetolactate production within the hybrid lager yeast group Saccharomyces pastorianus and affirmation of the central role of the ILV6 gene.

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation.

Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus.

Two distinct genetic groups (Saaz and Frohberg) exist within the hybrid Saccharomyces pastorianus (S. cerevisiae × S. eubayanus) taxon.

Adaptive evolution of the lager brewing yeast Saccharomyces pastorianus for improved growth under hyperosmotic conditions and its influence on fermentation performance.

An adaptive evolution method to obtain stable Saccharomyces pastorianus brewing yeast variants with improved fermentation capacity is described. The procedure involved selection for rapid growth resumption at high osmotic strength. It was applied to a lager strain and to a previously isolated ethanol-tolerant strain.

Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast.

Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods.

The temperature dependence of maltose transport in ale and lager strains of brewer's yeast.

Lager beers are traditionally made at lower temperatures (6-14 degrees C) than ales (15-25 degrees C). At low temperatures, lager strains (Saccharomyces pastorianus) ferment faster than ale strains (Saccharomyces cerevisiae). Two lager and two ale strains had similar maltose transport activities at 20 degrees C, but at 0 degrees C the lager strains had fivefold greater activity.

Selection from industrial lager yeast strains of variants with improved fermentation performance in very-high-gravity worts.

There are economic and other advantages if the fermentable sugar concentration in industrial brewery fermentations can be increased from that of currently used high-gravity (ca. 14 to 17 degrees P [degrees Plato]) worts into the very-high-gravity (VHG; 18 to 25 degrees P) range. Many industrial strains of brewer's yeast perform poorly in VHG worts, exhibiting decreased growth, slow and incomplete fermentations, and low viability of the yeast cropped for recycling into subsequent fermentations.

Improved fermentation performance of a lager yeast after repair of its AGT1 maltose and maltotriose transporter genes.

The use of more concentrated, so-called high-gravity and very-high-gravity (VHG) brewer's worts for the manufacture of beer has economic and environmental advantages. However, many current strains of brewer's yeasts ferment VHG worts slowly and incompletely, leaving undesirably large amounts of maltose and especially maltotriose in the final beers. alpha-Glucosides are transported into Saccharomyces yeasts by several transporters, including Agt1, which is a good carrier of both maltose and maltotriose.

Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression.

Brewer's yeast experiences constantly changing environmental conditions during wort fermentation. Cells can rapidly adapt to changing surroundings by transcriptional regulation. Changes in genomic expression can indicate the physiological condition of yeast in the brewing process.

Characterization and functional analysis of the MAL and MPH Loci for maltose utilization in some ale and lager yeast strains.

Maltose and maltotriose are the major sugars in brewer's wort. Brewer's yeasts contain multiple genes for maltose transporters. It is not known which of these express functional transporters.