Specific growth rates and growth stoichiometries of Saccharomycotina yeasts on ethanol as sole carbon and energy substrate.

Emerging low-emission production technologies make ethanol an interesting substrate for yeast biotechnology, but information on growth rates and biomass yields of yeasts on ethanol is scarce. Strains of 52 Saccharomycotina yeasts were screened for growth on ethanol. The 21 fastest strains, among which representatives of the Phaffomycetales order were overrepresented, showed specific growth rates in ethanol-grown shake-flask cultures between 0.

Quantitative physiology and biomass composition of Cyberlindnera jadinii in ethanol-grown cultures.

Background: Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilizing and energy-dense. The yeast C.

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth.

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S.

Optimizing the balance between heterologous acetate- and CO2-reduction pathways in anaerobic cultures of Saccharomyces cerevisiae strains engineered for low-glycerol production.

In anaerobic Saccharomyces cerevisiae cultures, NADH (reduced form of nicotinamide adenine dinucleotide)-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as 'redox-sink' and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions.

Quantification and mitigation of byproduct formation by low-glycerol-producing Saccharomyces cerevisiae strains containing Calvin-cycle enzymes.

Background: Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures.

An engineered non-oxidative glycolytic bypass based on Calvin-cycle enzymes enables anaerobic co-fermentation of glucose and sorbitol by Saccharomyces cerevisiae.

Background: Saccharomyces cerevisiae is intensively used for industrial ethanol production. Its native fermentation pathway enables a maximum product yield of 2 mol of ethanol per mole of glucose. Based on conservation laws, supply of additional electrons could support even higher ethanol yields.

Identification of fungal dihydrouracil-oxidase genes by expression in Saccharomyces cerevisiae.

Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete Alternaria alternata and the basidiomycete Schizophyllum commune, only supported growth of an S. cerevisiae ura1Δ mutant when synthetic media were supplemented with dihydrouracil.

Respiratory reoxidation of NADH is a key contributor to high oxygen requirements of oxygen-limited cultures of Ogataea parapolymorpha.

While thermotolerance is an attractive trait for yeasts used in industrial ethanol production, oxygen requirements of known thermotolerant species are incompatible with process requirements. Analysis of oxygen-sufficient and oxygen-limited chemostat cultures of the facultatively fermentative, thermotolerant species Ogataea parapolymorpha showed its minimum oxygen requirements to be an order of magnitude larger than those reported for the thermotolerant yeast Kluyveromyces marxianus. High oxygen requirements of O.

Pathway engineering strategies for improved product yield in yeast-based industrial ethanol production.

Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast . This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism.

Class-II dihydroorotate dehydrogenases from three phylogenetically distant fungi support anaerobic pyrimidine biosynthesis.

Background: In most fungi, quinone-dependent Class-II dihydroorotate dehydrogenases (DHODs) are essential for pyrimidine biosynthesis. Coupling of these Class-II DHODHs to mitochondrial respiration makes their in vivo activity dependent on oxygen availability. Saccharomyces cerevisiae and closely related yeast species harbor a cytosolic Class-I DHOD (Ura1) that uses fumarate as electron acceptor and thereby enables anaerobic pyrimidine synthesis.

Elimination of aromatic fusel alcohols as by-products of strains engineered for phenylpropanoid production by 2-oxo-acid decarboxylase replacement.

Engineered strains of the yeast are intensively studied as production platforms for aromatic compounds such as hydroxycinnamic acids, stilbenoids and flavonoids. Heterologous pathways for production of these compounds use l-phenylalanine and/or l-tyrosine, generated by the yeast shikimate pathway, as aromatic precursors. The Ehrlich pathway converts these precursors to aromatic fusel alcohols and acids, which are undesirable by-products of yeast strains engineered for production of high-value aromatic compounds.

A squalene-hopene cyclase in represents a eukaryotic adaptation to sterol-limited anaerobic environments.

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate.

Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth.

Current large-scale, anaerobic industrial processes for ethanol production from renewable carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer significant economic benefits. However, in contrast to S.

Identification of Oxygen-Independent Pathways for Pyridine Nucleotide and Coenzyme A Synthesis in Anaerobic Fungi by Expression of Candidate Genes in Yeast.

Neocallimastigomycetes are unique examples of strictly anaerobic eukaryotes. This study investigates how these anaerobic fungi bypass reactions involved in synthesis of pyridine nucleotide cofactors and coenzyme A that, in canonical fungal pathways, require molecular oxygen. Analysis of Neocallimastigomycetes proteomes identified a candidate l-aspartate-decarboxylase (AdcA) and l-aspartate oxidase (NadB) and quinolinate synthase (NadA), constituting putative oxygen-independent bypasses for coenzyme A synthesis and pyridine nucleotide cofactor synthesis.

Critical parameters and procedures for anaerobic cultivation of yeasts in bioreactors and anaerobic chambers.

All known facultatively fermentative yeasts require molecular oxygen for growth. Only in a small number of yeast species, these requirements can be circumvented by supplementation of known anaerobic growth factors such as nicotinate, sterols and unsaturated fatty acids. Biosynthetic oxygen requirements of yeasts are typically small and, unless extensive precautions are taken to minimize inadvertent entry of trace amounts of oxygen, easily go unnoticed in small-scale laboratory cultivation systems.

Engineering oxygen-independent biotin biosynthesis in Saccharomyces cerevisiae.

An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae.

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae.

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S.

Uncoupling growth and succinic acid production in an industrial Saccharomyces cerevisiae strain.

This study explores the relation between biomass-specific succinic acid (SA) production rate and specific growth rate of an engineered industrial strain of Saccharomyces cerevisiae, with the aim to investigate the extent to which growth and product formation can be uncoupled. Ammonium-limited aerobic chemostat and retentostat cultures were grown at different specific growth rates under industrially relevant conditions, that is, at a culture pH of 3 and with sparging of a 1:1 CO -air mixture. Biomass-specific SA production rates decreased asymptotically with decreasing growth rate.

Improving Industrially Relevant Phenotypic Traits by Engineering Chromosome Copy Number in .

The lager-brewing yeast is a hybrid between and with an exceptional degree of aneuploidy. While chromosome copy number variation (CCNV) is present in many industrial strains and has been linked to various industrially-relevant traits, its impact on the brewing performance of remains elusive. Here we attempt to delete single copies of chromosomes which are relevant for the production of off-flavor compound diacetyl by centromere silencing.

Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae.

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene.

Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of .

The thermotolerant yeast (formerly ) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate.

Adaptive Laboratory Evolution and Reverse Engineering of Single-Vitamin Prototrophies in Saccharomyces cerevisiae.

Quantitative physiological studies on commonly use synthetic media (SM) that contain a set of water-soluble growth factors that, based on their roles in human nutrition, are referred to as B vitamins. Previous work demonstrated that in CEN.PK113-7D, requirements for biotin were eliminated by laboratory evolution.

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae.

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms.