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.

Inhibition Control by Continuous Extractive Fermentation Enhances De Novo 2-Phenylethanol Production by Yeast.

Current microbial cell factory methods for producing chemicals from renewable resources primarily rely on (fed-)batch production systems, leading to the accumulation of the desired product. Industrially relevant chemicals like 2-phenylethanol (2PE), a flavor and fragrance compound, can exhibit toxicity at low concentrations, inhibit the host activity, and negatively impact titer, rate, and yield. Batch liquid-liquid (L-L) In Situ Product Removal (ISPR) was employed to mitigate inhibition effects, but was not found sufficient for industrial-scale application.

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production.

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply.

Microbial electrosynthesis from CO reaches productivity of syngas and chain elongation fermentations.

Carbon-based products are essential to society, yet producing them from fossil fuels is unsustainable. Microorganisms have the ability to take up electrons from solid electrodes and convert carbon dioxide (CO) to valuable carbon-based chemicals. However, higher productivities and energy efficiencies are needed to reach a viability that can make the technology transformative.

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.

Draft genome sequence of the Cas9 expressing strain IMX2600, a laboratory and platform strain from the CEN.PK lineage for cell-factory research.

The biobased-economy aims to create a circular biotechnology ecosystem to transition from a fossil fuel-based to a sustainable industry based on biomass. For this, new microbial cell-factories are essential. We present the draft genome of the CEN.

Expanding the genome editing toolbox of Saccharomyces cerevisiae with the endonuclease ErCas12a.

ErCas12a is a class 2 type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S.

Long-read direct RNA sequencing of the mitochondrial transcriptome of Saccharomyces cerevisiae reveals condition-dependent intron abundance.

Mitochondria fulfil many essential roles and have their own genome, which is expressed as polycistronic transcripts that undergo co- or posttranscriptional processing and splicing. Due to the inherent complexity and limited technical accessibility of the mitochondrial transcriptome, fundamental questions regarding mitochondrial gene expression and splicing remain unresolved, even in the model eukaryote Saccharomyces cerevisiae. Long-read sequencing could address these fundamental questions.

CRI-SPA: a high-throughput method for systematic genetic editing of yeast libraries.

Biological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and new research tools allowing simple and rapid mapping of sequence to function are desirable. Here, we describe CRI-SPA, a method allowing the transfer of chromosomal genetic features from a CRI-SPA Donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae.

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts.

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits such as wort α-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S.

Full humanization of the glycolytic pathway in Saccharomyces cerevisiae.

Although transplantation of single genes in yeast plays a key role in elucidating gene functionality in metazoans, technical challenges hamper humanization of full pathways and processes. Empowered by advances in synthetic biology, this study demonstrates the feasibility and implementation of full humanization of glycolysis in yeast. Single gene and full pathway transplantation revealed the remarkable conservation of glycolytic and moonlighting functions and, combined with evolutionary strategies, brought to light context-dependent responses.

Genetic bases for the metabolism of the DMS precursor S-methylmethionine by Saccharomyces cerevisiae.

Dimethyl sulfide (DMS) is a sulfur containing volatile that enhances general fruity aroma and imparts aromatic notes in wine. The most important precursor of DMS is S-methylmethionine (SMM), which is synthesized by grapes and can be metabolized by the yeast S. cerevisiae during wine fermentation.

Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties.

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.

Engineering of molybdenum-cofactor-dependent nitrate assimilation in Yarrowia lipolytica.

Engineering a new metabolic function in a microbial host can be limited by the availability of the relevant cofactor. For instance, in Yarrowia lipolytica, the expression of a functional nitrate reductase is precluded by the absence of molybdenum cofactor (Moco) biosynthesis. In this study, we demonstrated that the Ogataea parapolymorpha Moco biosynthesis pathway combined with the expression of a high affinity molybdate transporter could lead to the synthesis of Moco in Y.

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.

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.

gEL DNA: A Cloning- and Polymerase Chain Reaction-Free Method for CRISPR-Based Multiplexed Genome Editing.

Even for the genetically accessible yeast , the CRISPR-Cas DNA editing technology has strongly accelerated and facilitated strain construction. Several methods have been validated for fast and highly efficient single editing events, and diverse approaches for multiplex genome editing have been described in the literature by means of Cas9 or Cas12a endonucleases and their associated guide RNAs (gRNAs). The gRNAs used to guide the Cas endonuclease to the editing site are typically expressed from plasmids using native Pol II or Pol III RNA polymerases.

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.

A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae.

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S.

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.

Design and Experimental Evaluation of a Minimal, Innocuous Watermarking Strategy to Distinguish Near-Identical DNA and RNA Sequences.

The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast .

Exploring the abundance of oleate hydratases in the genus Rhodococcus-discovery of novel enzymes with complementary substrate scope.

Oleate hydratases (Ohys, EC 4.2.1.