Recombinant Arginine Deiminase from Levilactobacillus brevis Inhibits the Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway.

The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.

Construction and Evaluation of Peptide-Linked Lactobacillus brevis ß-Galactosidase Heterodimers.

BACKGROUND: ß-galactosidases are enzymes that are utilized to hydrolyze lactose into galactose and glucose, and are is widely used in the food industry. OBJECTIVE: We describe the recombinant expression of an unstudied, heterodimeric ß-galactosidase originating from Lactobacillus brevis ATCC 367 in Escherichia coli. Furthermore, six different constructs, in which the two protein subunits were fused with different peptide linkers, were also investigated. METHODS: The heterodimeric subunits of the ß-galactosidase were cloned in expressed in various expression constructs, by using either two vectors for the independent expression of each subunit, or using a single Duet vector for the co-expression of the two subunits. RESULTS: The co-expression in two independent expression vectors only resulted in low ß-galactosidase activities, whereas the co-expression in a single Duet vector of the independent and fused subunits increased the ß-galactosidase activity significantly. The recombinant ß-galactosidase showed comparable hydrolyzing properties towards lactose, N-acetyllactosamine, and pNP-ß-D-galactoside. CONCLUSION: The usability of the recombinant L. brevis ß-galactosidase was further demonstrated by the hydrolysis of human, bovine, and goat milk samples. The herein presented fused ß-galactosidase constructs may be of interest for analytical research as well as in food- and biotechnological applications.

Mitochondrial ubiquinol oxidation is necessary for tumour growth.

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1-6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7-9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP-that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)-an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.

Improving the Thermostability of Glutamate Decarboxylase from Lactobacillus brevis by Consensus Mutagenesis.

γ-Aminobutyrate (GABA) is an important bioactive compound synthesized through decarboxylation of L-glutamate by the glutamate decarboxylase (GAD). In this study, stabilized variants of the GAD from Lactobacillus brevis were constructed by consensus mutagenesis. Using Consensus Finder ( http://cbs-kazlab.oit.umn.edu/ ), eight positions with the most prevalent amino acid (over 60% threshold) among the homologous family members were identified. Subsequently, these eight residues were individually mutated to match the consensus sequence using site-directed mutagenesis. Compared to the wild-type, T383K variant displayed the largest shift in thermostability among the single variants, with a 3.0 °C increase in semi-inactivation temperature (T5015), a 1.7-fold improvement of half-life (t1/2) at 55 °C, and a 1.2-fold improvement of t1/2 at 37 °C, respectively, while its catalytic efficiency (kcat/Km) was reduced. To obtain the mutant with improvement in both thermostability and catalytic activity, we performed a site-saturation mutation at T383. Notably, mutants T383V and T383G exhibited an increasement in thermostability and kcat/Km than that of wild-type. This study not only emphasizes the value of consensus mutagenesis for improving the thermostability of GAD but also sheds a powerful guidance to study the thermal stability of other enzymes.

Expression of two glutamate decarboxylase genes in Lactobacillus brevis during gamma-aminobutyric acid production with date residue extract.

Gamma-aminobutyric acid (GABA) is produced by Lactobacillus brevis using date residue fermentation. In this study, the GABA production method was improved, for which L. brevis strain JCM 1059T was the most efficient among the four L. brevis strains examined. This was presumably due to a difference in the expression level of the gene encoding glutamate decarboxylase that catalyzes GABA synthesis.Abbreviation: GABA: gamma-aminobutyric acid.

A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis.

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

Bioconversion of Gamma-Aminobutyric Acid from Monosodium Glutamate by Lactobacillus brevis Bmb5.

Gamma-aminobutyric acid (GABA) plays important roles in host physiology. However, the effects of GABA are greatly restricted due to its low bioavailability in the human body. Here, a high acid-tolerance GABA-producing strain, Lactobacillus brevis Bmb5, was isolated from kimchi. Bmb5 converted glutamate to GABA (7.23 ± 0.68 µg/µl) at a rate of 72.3%. The expression of gadB gene, encoding the enzyme involved in the decarboxylation of glutamate to GABA, was decreased upon incubation. Our findings indicate GABA production in Bmb5 is not directly correlated with gadB gene expression, providing new insight into the mechanisms underlying GABA production in Lactobacillus.

Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, Lactobacillus brevis KB290.

We demonstrate here that exopolysaccharide (EPS) production, cell aggregation, and bile resistance in Lactobacillus brevis KB290 are conferred by three eps genes (gtf27, gtf28, and orf29) located on the 42.4-kb plasmid pKB290-1. The predicted products of gtf27 and gtf28 belong to the membrane-bound glycosyltransferase family whereas the orf29 gene product showed homology with the ABC transporter. On in silico analysis, these genes were found to be widely distributed among lactobacilli from publicly available genomes and metagenomes, and their function is not yet elucidated. RT-PCR analysis showed that the eps genes were organised in an operon and their expression was markedly lower in arabinose- and xylose-containing media than in a glucose-containing medium. The three eps genes were cloned and expressed in homologous and heterologous strains. Considerably less EPS was produced by the plasmid-cured KB1802 strain than by the parental KB290 strain, whereas a similar amount was produced by the KB1802 strain expressing the three eps genes. The KB1802 strain expressing gtf27 and gtf28 but not orf29 did not produce EPS. Cell aggregation and bile resistance were also decreased in KB1802 strains but were complemented by eps genes. Moreover, the three eps genes conferred these phenotypes to a Lactobacillus plantarum strain. In conclusion, the three eps genes in pKB290-1 were sufficient for EPS biosynthesis with glucose and N-acetylglucosamine, and were responsible for cell aggregation and bile resistance. We consider these phenotypes to be at least partly responsible for KB290-specific properties.

Improving Soluble Expression of Tyrosine Decarboxylase from Lactobacillus brevis for Tyramine Synthesis with High Total Turnover Number.

The soluble expression of tyrosine decarboxylase (TDC) in heterologous host is often challenging. Here, acidic condition was found to be favorable for improving the soluble expression of TDC from Lactobacillus brevis in Escherichia coli, while addition of carbohydrates (such as glucose, arabinose, and fructose) was vital for decreasing the insoluble fraction. By simple pH control and addition of glucose, the specific activity of TDC in crude extract was enhanced to 46.3 U mg-1, 3.67-fold of that produced from LB medium. Optimization of the reaction conditions revealed that Tween-80 was effective in improving the tyramine production catalyzed by TDC, especially at high tyrosine loadings. As much as 400 mM tyrosine could be completely converted into tyramine with a substrate to catalyst ratio of 29.0 g g-1 and total turnover number of 23,300. This study provides efficient strategies for the highly soluble expression of TDC and biocatalytic production of tyramine.

Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase-NADPH Oxidase Fusions for Continuous Oxidations.

To expand the arsenal of industrially applicable oxidative enzymes, fusions of alcohol dehydrogenases with an NADPH-oxidase were designed. Three different alcohol dehydrogenases (LbADH, TbADH, ADHA) were expressed with a thermostable NADPH-oxidase fusion partner (PAMO C65D) and purified. The resulting bifunctional biocatalysts retained the catalytic properties of the individual enzymes, and acted essentially like alcohol oxidases: transforming alcohols to ketones by using dioxygen as mild oxidant, while merely requiring a catalytic amount of NADP+ . In small-scale reactions, the purified fusion enzymes show good performances, with 69-99 % conversion, 99 % ee with a racemic substrate, and high cofactor and enzyme total turnover numbers. As the fusion enzymes essentially act as oxidases, we found that commonly used high-throughput oxidase-activity screening methods can be used. Therefore, if needed, the fusion enzymes could be easily engineered to tune their properties.

Neutron and X-ray crystal structures of Lactobacillus brevis alcohol dehydrogenase reveal new insights into hydrogen-bonding pathways.

Lactobacillus brevis alcohol dehydrogenase (LbADH) is a well studied homotetrameric enzyme which catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. LbADH is stable and enzymatically active at elevated temperatures and accepts a broad range of substrates, making it a valuable tool in industrial biocatalysis. Here, the expression, purification and crystallization of LbADH to generate large, single crystals with a volume of up to 1 mm3 suitable for neutron diffraction studies are described. Neutron diffraction data were collected from an H/D-exchanged LbADH crystal using the BIODIFF instrument at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany to a resolution dmin of 2.15 Šin 16 days. This allowed the first neutron crystal structure of LbADH to be determined. The neutron structure revealed new details of the hydrogen-bonding network originating from the ion-binding site of LbADH and provided new insights into the reasons why divalent magnesium (Mg2+) or manganese (Mn2+) ions are necessary for its activity. X-ray diffraction data were obtained from the same crystal at the European Synchrotron Radiation Facility (ESRF), Grenoble, France to a resolution dmin of 1.48 Å. The high-resolution X-ray structure suggested partial occupancy of Mn2+ and Mg2+ at the ion-binding site. This is supported by the different binding affinity of Mn2+ and Mg2+ to the tetrameric structure calculated via free-energy molecular-dynamics simulations.

Engineering a Coenzyme A Detour To Expand the Product Scope and Enhance the Selectivity of the Ehrlich Pathway.

The Ehrlich pathway is a major route for the renewable production of higher alcohols. However, the product scope of the Ehrlich pathway is restricted, and the product selectivity is suboptimal. Here, we demonstrate that a Coenzyme A (CoA) detour, which involves conversion of the 2-keto acids into acyl-CoAs, expands the biological toolkit of reaction chemistries available in the Ehrlich pathway to include the gamut of CoA-dependent enzymes. As a proof-of-concept, we demonstrated the first biosynthesis of a tertiary branched-alcohol, pivalcohol, at a level of ∼10 mg/L from glucose in Escherichia coli, using a pivalyl-CoA mutase from Xanthobacter autotrophicus. Furthermore, engineering an enzyme in the CoA detour, the Lactobacillus brevis CoA-acylating aldehyde dehydrogenase, allowed stringent product selectivity. Targeted production of 3-methyl-1-butanol (3-MB) in E. coli mediated by the CoA detour showed a 3-MB:side-product (isobutanol) ratio of >20, an increase over the ratios previously achieved using the conventional Ehrlich pathway.

Exploring the contributions of two glutamate decarboxylase isozymes in Lactobacillus brevis to acid resistance and γ-aminobutyric acid production.

BACKGROUND: The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. However, direct experimental evidence for detailed contributions of gad genes to acid tolerance and GABA production is lacking. RESULTS: Molecular analysis revealed that gadB is cotranscribed in tandem with upstream gadC, and that expression of gadCB is greatly upregulated in response to low ambient pH when cells enter the late exponential growth phase. In contrast, gadA is located away from the other gad genes, and its expression was consistently lower and not induced by mild acid treatment. Analysis of deletion mutations in the gad genes of L. brevis demonstrated a decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild-type> ΔgadA> ΔgadB> ΔgadC> ΔgadAB, indicating that the GAD activity mainly endowed by GadB rather than GadA is an indispensable step in the GadCB mediated acid resistance of this organism. Moreover, engineered strains with higher GAD activities were constructed by overexpressing key GAD system genes. With the proposed two-stage pH and temperature control fed-batch fermentation strategy, GABA production by the engineered strain L. brevis 9530: pNZ8148-gadBC continuously increased reaching a high level of 104.38 ± 3.47 g/L at 72 h. CONCLUSIONS: This is the first report of the detailed contribution of gad genes to acid tolerance and GABA production in L. brevis. Enhanced production of GABA by engineered L. brevis was achieved, and the resulting GABA level was one of the highest among lactic acid bacterial species grown in batch or fed-batch culture.

A gut microbial factor modulates locomotor behaviour in Drosophila.

While research into the biology of animal behaviour has primarily focused on the central nervous system, cues from peripheral tissues and the environment have been implicated in brain development and function1. There is emerging evidence that bidirectional communication between the gut and the brain affects behaviours including anxiety, cognition, nociception and social interaction1-9. Coordinated locomotor behaviour is critical for the survival and propagation of animals, and is regulated by internal and external sensory inputs10,11. However, little is known about how the gut microbiome influences host locomotion, or the molecular and cellular mechanisms involved. Here we report that germ-free status or antibiotic treatment results in hyperactive locomotor behaviour in the fruit fly Drosophila melanogaster. Increased walking speed and daily activity in the absence of a gut microbiome are rescued by mono-colonization with specific bacteria, including the fly commensal Lactobacillus brevis. The bacterial enzyme xylose isomerase from L. brevis recapitulates the locomotor effects of microbial colonization by modulating sugar metabolism in flies. Notably, thermogenetic activation of octopaminergic neurons or exogenous administration of octopamine, the invertebrate counterpart of noradrenaline, abrogates the effects of xylose isomerase on Drosophila locomotion. These findings reveal a previously unappreciated role for the gut microbiome in modulating locomotion, and identify octopaminergic neurons as mediators of peripheral microbial cues that regulate motor behaviour in animals.

Enhanced d-tagatose production by spore surface-displayed l-arabinose isomerase from isolated Lactobacillus brevis PC16 and biotransformation.

In the present study, a new strain of Lactobacillus brevis producing d-tagatose was isolated and identified. Then, the l-arabinose isomerase (L-AI) of this strain was displayed on the spore surface of Bacillus subtilis DB403 by using an anchoring protein CotG and a peptide linker (Gly-Gly-Gly-Gly-Ser). This displayed L-AI with high specific activity and stability was used as a novel immobilized biocatalyst for producing d-tagatose through batch and semi-continuous biotransformation. The conversion rate of d-tagatose from 125 g/L d-galactose was achieved 79.7% at 28 h, and the volumetric productivity reached 4.3 g/L/h at 20 h. Furthermore, the displayed L-AI showed a good performance on the reusability and remained 87% of the specific activity and 40.7% of the conversion rate after five recycles. A high efficient immobilized method for producing food-grade d-tagatose was established using spore surface-displayed L-AI.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Šresolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.

Expression and characterization of glutamate decarboxylase from Lactobacillus brevis HYE1 isolated from kimchi.

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni-NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.

Different strategies for multi-enzyme cascade reaction for chiral vic-1,2-diol production.

The stereoselective three-enzyme cascade for the one-pot synthesis of (1S,2S)-1-phenylpropane-1,2-diol ((1S,2S)-1-PPD) from inexpensive starting substrates, benzaldehyde and acetaldehyde, was explored. By coupling stereoselective carboligation catalyzed by benzoylformate decarboxylase (BFD), L-selective reduction of a carbonyl group with alcohol dehydrogenase from Lactobacillus brevis (ADHLb) as well as the coenzyme regeneration by formate dehydrogenase (FDH), enantiomerically pure diastereoselective 1,2-diol was produced. Two different multi-enzyme system approaches were applied: the sequential two-step one-pot and the simultaneous one-pot cascade. All enzymes were kinetically characterized. The impact of acetaldehyde on the BFD and ADHLb stability was investigated. To overcome the kinetic limitation of acetaldehyde in the carboligation reaction and to reduce its influence on the enzyme stability, experiments were performed in two different excesses of acetaldehyde (100 and 300%). Due to the ADHLb deactivation by acetaldehyde, the simultaneous one-pot cascade proved not to be the first choice for the investigated three-enzyme system. In the sequential cascade with 300% acetaldehyde excess a 100% yield of vic 1,2-diol was reached.