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'...

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. However, this option is disallowed by the...

From Visionary Tale to Application The Bright and Dark Side of Photo-Biocatalysis

Redox biocatalysis is a promising approach to carry out redox reactions in industry. The chemical nature of redox reactions requires a stoichiometric supply of redox equivalents. The outstanding class of enzymes “Oxidoreductases” require specific redox equivalents for their function. We envision more environmentally friendly sources for these...

An NADH preferring acetoacetyl-CoA reductase is engaged in poly-3-hydroxybutyrate accumulation in Escherichia coli

Oxygen supply implies higher production cost and reduction of maximum theoretical yields. Thus, generation of fermentation products is more cost-effective. Aiming to find a key piece for the production of (poly)-3-hydroxybutyrate (PHB) as a fermentation product, here we characterize an acetoacetyl-CoA reductase, isolated from a Candidatus...

Engineering Saccharomyces cerevisiae for Succinic Acid Production From Glycerol and Carbon Dioxide

Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol’s higher reducing power (compared to sugars) for the production of the platform chemical succinic acid ...

Light-Harvesting Dye-Alginate Hydrogel for Solar-Driven, Sustainable Biocatalysis of Asymmetric Hydrogenation

We report visible light-driven, asymmetric hydrogenation of C=C bonds using an ene-reductase from Thermus scotoductus SA-01 (TsOYE) and a light-harvesting dye (rose bengal, RB) co-immobilized in an alginate hydrogel. Highly efficient encapsulation of RB in alginate hydrogel was achieved using the intrinsic affinity between TsOYE and RB,...

Straightforward Regeneration of Reduced Flavin Adenine Dinucleotide Required for Enzymatic Tryptophan Halogenation

Flavin-dependent halogenases are known to regioselectively introduce halide substituents into aromatic moieties, for example, the indole ring of tryptophan. The process requires halide salts and oxygen instead of molecular halogen in the chemical halogenation. However, the reduced cofactor flavin adenine dinucleotide (FADH₂) has to...

Optimizing anaerobic growth rate and fermentation kinetics in Saccharomyces cerevisiae strains expressing Calvin-cycle enzymes for improved ethanol yield

Background: Reduction or elimination of by-product formation is of immediate economic relevance in fermentation processes for industrial bioethanol production with the yeast Saccharomyces cerevisiae. Anaerobic cultures of wild-type S. cerevisiae require formation of glycerol to maintain the intracellular NADH/NAD⁺ balance....

Biocatalytic C=C Bond Reduction through Carbon Nanodot-Sensitized Regeneration of NADH Analogues

Light-driven activation of redox enzymes is an emerging route for sustainable chemical synthesis. Among redox enzymes, the family of Old Yellow Enzyme (OYE) dependent on the nicotinamide adenine dinucleotide cofactor (NADH) catalyzes the stereoselective reduction of α,β-unsaturated hydrocarbons. Here, we report OYE-catalyzed asymmetric...

Engineering NADH/NAD⁺ ratio in Halomonas bluephagenesis for enhanced production of polyhydroxyalkanoates (PHA)

Halomonas bluephagenesis has been developed as a platform strain for the next generation industrial biotechnology (NGIB) with advantages of resistances to microbial contamination and high cell density growth (HCD), especially for production of polyhydroxyalkanoates (PHA) including poly(3-hydroxybutyrate) (PHB), poly(3-hydroxybutyrate-co-4...

Metabolic engineering strategies for optimizing acetate reduction, ethanol yield and osmotolerance in Saccharomyces cerevisiae

Background: Glycerol, whose formation contributes to cellular redox balancing and osmoregulation in Saccharomyces cerevisiae, is an important by-product of yeast-based bioethanol production. Replacing the glycerol pathway by an engineered pathway for NAD⁺-dependent acetate reduction has been shown to improve ethanol yields and...

Improving ethanol yield in acetate-reducing Saccharomyces cerevisiae by cofactor engineering of 6-phosphogluconate dehydrogenase and deletion of ALD6

Background: Acetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation...

Carbon dioxide fixation by Calvin-Cycle enzymes improves ethanol yield in yeast

Background Redox-cofactor balancing constrains product yields in anaerobic fermentation processes. This challenge is exemplified by the formation of glycerol as major by-product in yeast-based bioethanol production, which is a direct consequence of the need to reoxidize excess NADH and causes a loss of conversion efficiency. Enabling the use of...

Immobilized redox mediators for electrochemical NAD(P)+ regeneration

The applicability of dissolved redox mediators for NAD(P)+ regeneration has been demonstrated several times. Nevertheless, the use of mediators in solutions for sensor applications is not a very convenient strategy since the analysis is not reagentless and long stabilization times occur. The most important drawbacks of dissolved mediators in...