Raspberry Ketone Accumulation in and by Expression of Fused Pathway Genes.

Raspberry ketone has generated interest in recent years both as a flavor agent and as a health promoting supplement. Raspberry ketone can be synthesized chemically, but the value of a natural nonsynthetic product is among the most valuable flavor compounds on the market. Coumaroyl-coenzyme A (CoA) is the direct precursor for raspberry ketone but also an essential precursor for flavonoid and lignin biosynthesis in plants and therefore highly regulated.

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.

A Universal Gene Expression System for Novel Yeast Species.

The current progress in sequencing of genomes and characterization of new species provides an increasing list of yeasts that show interesting physiological properties; however, the lack of expression tools for these new hosts is prohibiting their broader use in research or industry. Recently, we developed a universal expression system (SES) functional in broad spectrum of fungal species, which represent a solution for feasible gene expression control and genetic manipulation in these novel hosts. Here, we describe three example approaches for DNA transformation and high-level heterologous gene expression, using the SES system, in three yeast species, where minimal knowledge or prior experience in genetic modifications is available.

Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production.

Background: Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products.

Gene expression engineering in fungi.

Fungi are a highly diverse group of microbial species that possess a plethora of biotechnologically useful metabolic and physiological properties. Important enablers for fungal biology studies and their biotechnological use are well-performing gene expression tools. Different types of gene expression tools exist; however, typically they are at best only functional in one or a few closely related species.

Novel genetic tools that enable highly pure protein production in Trichoderma reesei.

Trichoderma reesei is an established protein production host with high natural capacity to secrete enzymes. The lack of efficient genome engineering approaches and absence of robust constitutive gene expression systems limits exploitation of this organism in some protein production applications. Here we report engineering of T.

Performance of Leuconostoc citreum FDR241 during wheat flour sourdough type I propagation and transcriptional analysis of exopolysaccharides biosynthesis genes.

This study focused on the performance of the dextran producer Leuconostoc citreum as starter culture during 30 days of wheat flour type I sourdough propagation (back-slopping). As confirmed by RAPD-PCR analysis, the strain dominated throughout the propagation procedure, consisting of daily fermentations at 20 °C. The sourdoughs were characterized by consistent lactic acid bacteria cell density and acidification parameters, reaching pH values of 4.

A universal gene expression system for fungi.

Biotechnological production of fuels, chemicals and proteins is dependent on efficient production systems, typically genetically engineered microorganisms. New genome editing methods are making it increasingly easy to introduce new genes and functionalities in a broad range of organisms. However, engineering of all these organisms is hampered by the lack of suitable gene expression tools.

Synthetic Toolkit for Complex Genetic Circuit Engineering in Saccharomyces cerevisiae.

Sustainable production of chemicals, materials, and pharmaceuticals is increasingly performed by genetically engineered cell factories. Engineering of complex metabolic routes or cell behavior control systems requires robust and predictable gene expression tools. In this challenging task, orthogonality is a fundamental prerequisite for such tools.

Production of ethylene glycol or glycolic acid from D-xylose in Saccharomyces cerevisiae.

The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae. The expression of genes encoding D-xylose dehydrogenase (XylB) and D-xylonate dehydratase (XylD) from Caulobacter crescentus and YagE or YjhH aldolase and aldehyde dehydrogenase AldA from Escherichia coli enabled glycolic acid production from D-xylose up to 150 mg/L. In strains expressing only xylB and xylD, 29 mg/L 2-keto-3-deoxyxylonic acid [(S)-4,5-dihydroxy-2-oxopentanoic acid] (2K3DXA) was produced and D-xylonic acid accumulated to ca.

Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae.

This work describes the development and characterization of a modular synthetic expression system that provides a broad range of adjustable and predictable expression levels in S. cerevisiae. The system works as a fixed-gain transcription amplifier, where the input signal is transferred via a synthetic transcription factor (sTF) onto a synthetic promoter, containing a defined core promoter, generating a transcription output signal.

Transcriptome of Saccharomyces cerevisiae during production of D-xylonate.

Background: Production of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process.

Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae.

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S.

Conversion of orange peel to L-galactonic acid in a consolidated process using engineered strains of Aspergillus niger.

Citrus processing waste is a leftover from the citrus processing industry and is available in large amounts. Typically, this waste is dried to produce animal feed, but sometimes it is just dumped. Its main component is the peel, which consists mostly of pectin, with D-galacturonic acid as the main monomer.

The diverse role of Pdr12 in resistance to weak organic acids.

Resistance to weak organic acids is important relative to both weak organic acid preservatives and the development of inhibitor tolerant yeast as industrial production organisms. The ABC transporter Pdr12 is important for resistance to sorbic and propionic acid, but its role in tolerance to other weak organic acids with industrial relevance is not well established. In this study, yeast strains with altered expression of PDR12 and/or CMK1, a protein kinase associated with post-transcriptional negative regulation of Pdr12, were exposed to seven weak organic acids: acetic, formic, glycolic, lactic, propionic, sorbic and levulinic acid.

Noninvasive high-throughput single-cell analysis of the intracellular pH of Saccharomyces cerevisiae by ratiometric flow cytometry.

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology.

A novel L-xylulose reductase essential for L-arabinose catabolism in Trichoderma reesei.

L-Xylulose reductases belong to the superfamily of short chain dehydrogenases and reductases (SDRs) and catalyze the NAD(P)H-dependent reduction of L-xylulose to xylitol in L-arabinose and glucuronic acid catabolism. Here we report the identification of a novel L-xylulose reductase LXR3 in the fungus Trichoderma reesei by a bioinformatic approach in combination with a functional analysis. LXR3, a 31 kDa protein, catalyzes the reduction of L-xylulose to xylitol via NADPH and is also able to convert D-xylulose, D-ribulose, L-sorbose, and D-fructose to their corresponding polyols.

Engineering filamentous fungi for conversion of D-galacturonic acid to L-galactonic acid.

D-Galacturonic acid, the main monomer of pectin, is an attractive substrate for bioconversions, since pectin-rich biomass is abundantly available and pectin is easily hydrolyzed. l-Galactonic acid is an intermediate in the eukaryotic pathway for d-galacturonic acid catabolism, but extracellular accumulation of l-galactonic acid has not been reported. By deleting the gene encoding l-galactonic acid dehydratase (lgd1 or gaaB) in two filamentous fungi, strains were obtained that converted d-galacturonic acid to l-galactonic acid.

L-xylo-3-hexulose reductase is the missing link in the oxidoreductive pathway for D-galactose catabolism in filamentous fungi.

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol.

Sorbitol dehydrogenase of Aspergillus niger, SdhA, is part of the oxido-reductive D-galactose pathway and essential for D-sorbitol catabolism.

In filamentous fungi D-galactose can be catabolised through the oxido-reductive and/or the Leloir pathway. In the oxido-reductive pathway D-galactose is converted to d-fructose in a series of steps where the last step is the oxidation of d-sorbitol by an NAD-dependent dehydrogenase. We identified a sorbitol dehydrogenase gene, sdhA (JGI53356), in Aspergillus niger encoding a medium chain dehydrogenase which is involved in D-galactose and D-sorbitol catabolism.

Identification of the galactitol dehydrogenase, LadB, that is part of the oxido-reductive D-galactose catabolic pathway in Aspergillus niger.

For the catabolism of D-galactose three different metabolic pathways have been described in filamentous fungi. Apart from the Leloir pathway and the oxidative pathway, there is an alternative oxido-reductive pathway. This oxido-reductive pathway has similarities to the metabolic pathway of L-arabinose, and in Trichoderma reesei (Hypocrea jecorina) and Aspergillus nidulans the same enzyme is employed for the oxidation of L-arabitol and galactitol.

Bioconversion of D-galacturonate to keto-deoxy-L-galactonate (3-deoxy-L-threo-hex-2-ulosonate) using filamentous fungi.

Background: The D-galacturonic acid derived from plant pectin can be converted into a variety of other chemicals which have potential use as chelators, clarifiers, preservatives and plastic precursors. Among these is the deoxy-keto acid derived from L-galactonic acid, keto-deoxy-L-galactonic acid or 3-deoxy-L-threo-hex-2-ulosonic acid. The keto-deoxy sugars have been found to be useful precursors for producing further derivatives.

The 'true' L-xylulose reductase of filamentous fungi identified in Aspergillus niger.

L-Xylulose reductase is part of the eukaryotic pathway for l-arabinose catabolism. A previously identified L-xylulose reductase in Hypocrea jecorina turned out to be not the 'true' one since it was not upregulated during growth on L-arabinose and the deletion strain showed no reduced L-xylulose reductase activity but instead lost the D-mannitol dehydrogenase activity. In this communication we identified the 'TRUE'L-xylulose reductase in Aspergillus niger.

Identification of an L-arabinose reductase gene in Aspergillus niger and its role in L-arabinose catabolism.

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source.

Metabolic engineering of fungal strains for conversion of D-galacturonate to meso-galactarate.

D-galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of D-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical.