Optimal design of a simulated-moving-bed chromatographic process for high-purity separation of acetoin from 2,3-butanediol in a continuous mode.

For the high-purity production of acetoin or 2,3-butanediol (BD) from related fermentation processes, it is essential to accomplish a detailed separation between acetoin and BD in an economical mode. To address this issue, we aimed to develop a highly-efficient simulated-moving-bed (SMB) process for the continuous-mode separation of acetoin from BD with high purity and small loss. As a first step for this task, the adsorption and mass-transfer parameters of acetoin and BD on a proven adsorbent were estimated while assuming that BD isomers (meso-BD and DL-BD) would be identical in adsorption and mass-transfer behaviors. The resultant parameters from such estimation were applied to the optimal design of the acetoin-BD separation SMB. The designed SMB was then experimentally investigated, which revealed that some sign of BD isomerism occurred in the SMB column-profile data and thus had an adverse effect on the SMB separation performance. To resolve this problem, the individual parameters of BD isomers were determined on the basis of the SMB column-profile data and an inverse-method principle. The resulting parameters of BD isomers were used in the re-design of the target SMB, which was then experimentally checked for its separation performance. It was confirmed that such SMB re-designed in consideration of BD isomerism was quite effective in the continuous-mode separation of acetoin from BD with high purity (> 99.2%) and small loss (< 1.52%).

Development and characterization of a field-effect biosensor for the detection of acetoin.

A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.

Acetoin production via unbalanced fermentation in Shewanella oneidensis.

This study describes the realization of an anoxic acetoin production process using the proteobacterium Shewanella oneidensis. Fermentative processes are of high biotechnological relevance since they offer high productivity and a low percentage of substrate consumption for anabolic processes. Nevertheless, the range of compounds that can be produced as sole end product of a fermentative process is limited, since the average oxidation state of substrate and products has to be identical in the absence of an external electron acceptor. This limitation could be overcome by the transfer of the surplus of electrons to a poised electrode surface, which of note is the only known anaerobic electron acceptor that cannot be depleted. In the first genetic engineering step, deletion mutants were developed that are devoid of either one, two, or all three prophages in their genome with the aim to construct a more stable chassis strain for microbe-electrode interaction, due to less prophage induced cell lysis (Gödeke et al., 2011). Current production in a bioelectrochemical system together with the analysis of cells on the anode surface were used as surrogate for the stability assessment. The λ-prophage deletion mutant produced overall 1.34fold more current (6.7 µA cm-2 ) than the wild type and all other constructed strains and showed with 1.1 × 1011 cells the highest cell density on the anode surface (2.3fold more than the wild type). The strain was further modified to contain codon optimized versions of acetolactate synthase and acetolactate decarboxylase derived from Bacillus subtilis. This allowed for the production of a mixture of acetoin and acetate from lactate in an almost 0.4:1 ratio. Further process improvement was reached by the deletion of the acetate kinase and phosphotransacetylase genes ackA/pta. The acetoin yield increased in this mutant from 40 to 86% of the theoretical maximum and acetoin was the only detectable end product. Biotechnol. Bioeng. 2017;114: 1283-1289. © 2017 Wiley Periodicals, Inc.

Synthesis of (3R)-acetoin and 2,3-butanediol isomers by metabolically engineered Lactococcus lactis.

The potential that lies in harnessing the chemical synthesis capabilities inherent in living organisms is immense. Here we demonstrate how the biosynthetic machinery of Lactococcus lactis, can be diverted to make (3R)-acetoin and the derived 2,3-butanediol isomers meso-(2,3)-butanediol (m-BDO) and (2R,3R)-butanediol (R-BDO). Efficient production of (3R)-acetoin was accomplished using a strain where the competing lactate, acetate and ethanol forming pathways had been blocked. By introducing different alcohol dehydrogenases into this strain, either EcBDH from Enterobacter cloacae or SadB from Achromobacter xylosooxidans, it was possible to achieve high-yield production of m-BDO or R-BDO respectively. To achieve biosustainable production of these chemicals from dairy waste, we transformed the above strains with the lactose plasmid pLP712. This enabled efficient production of (3R)-acetoin, m-BDO and R-BDO from processed whey waste, with titers of 27, 51, and 32 g/L respectively. The corresponding yields obtained were 0.42, 0.47 and 0.40 g/g lactose, which is 82%, 89%, and 76% of maximum theoretical yield respectively. These results clearly demonstrate that L. lactis is an excellent choice as a cell factory for transforming lactose containing dairy waste into value added chemicals.

Integrating biocompatible chemistry and manipulating cofactor partitioning in metabolically engineered Lactococcus lactis for fermentative production of (3S)-acetoin.

Biocompatible chemistry (BC), that is, non-enzymatic chemical reactions compatible with living organisms, is increasingly used in conjunction with metabolically engineered microorganisms for producing compounds that do not usually occur naturally. Here we report production of one such compound, (3S)-acetoin, a valuable precursor for chiral synthesis, using a metabolically engineered Lactococcus lactis strain growing under respiratory conditions with ferric iron serving as a BC component. The strain used has all competing product pathways inactivated, and an appropriate cofactor balance is achieved by fine-tuning the respiratory capacity indirectly via the hemin concentration. We achieve high-level (3S)-acetoin production with a final titer of 66 mM (5.8 g/L) and a high yield (71% of the theoretical maximum). To the best of our knowledge, this is the first report describing production of (3S)-acetoin from sugar by microbial fermentation, and the results obtained confirm the potential that lies with BC for producing useful chemicals. Biotechnol. Bioeng. 2016;113: 2744-2748. © 2016 Wiley Periodicals, Inc.

Avian influenza infection alters fecal odor in mallards.

Changes in body odor are known to be a consequence of many diseases. Much of the published work on disease-related and body odor changes has involved parasites and certain cancers. Much less studied have been viral diseases, possibly due to an absence of good animal model systems. Here we studied possible alteration of fecal odors in animals infected with avian influenza viruses (AIV). In a behavioral study, inbred C57BL/6 mice were trained in a standard Y-maze to discriminate odors emanating from feces collected from mallard ducks (Anas platyrhynchos) infected with low-pathogenic avian influenza virus compared to fecal odors from non-infected controls. Mice could discriminate odors from non-infected compared to infected individual ducks on the basis of fecal odors when feces from post-infection periods were paired with feces from pre-infection periods. Prompted by this indication of odor change, fecal samples were subjected to dynamic headspace and solvent extraction analyses employing gas chromatography/mass spectrometry to identify chemical markers indicative of AIV infection. Chemical analyses indicated that AIV infection was associated with a marked increase of acetoin (3-hydroxy-2-butanone) in feces. These experiments demonstrate that information regarding viral infection exists via volatile metabolites present in feces. Further, they suggest that odor changes following virus infection could play a role in regulating behavior of conspecifics exposed to infected individuals.

Biodegradation-inspired bioproduction of methylacetoin and 2-methyl-2,3-butanediol.

Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l(-1) for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.

Selective production of 2,3-butanediol and acetoin by a newly isolated bacterium Klebsiella oxytoca M1.

A newly isolated bacterium, designated as Klebsiella oxytoca M1, produced 2,3-butanediol (2,3-BDO) or acetoin selectively as a major product depending on temperature in a defined medium. K. oxytoca M1 produced 2,3-BDO mainly (0.32~0.34 g/g glucose) at 30 °C while acetoin was a major product (0.32~0.38 g/g glucose) at 37 °C. To investigate factors affecting product profiles according to temperature, the expression level of acetoin reductase (AR) that catalyzes the conversion of acetoin to 2,3-BDO was analyzed using crude protein extracted from K. oxytoca M1 grown at 30 and 37 °C. The AR expression at 37 °C was 12.8-fold lower than that at 30 °C at the stationary phase and reverse transcription PCR (RT-PCR) analysis of the budC (encoding AR) was also in agreement with the AR expression results. When AR was overexpressed using K. oxytoca M1 harboring pUC18CM-budC, 2,3-BDO became a major product at 37 °C, indicating that the AR expression level was a key factor determining the major product of K. oxytoca M1 at 37 °C. The results in this study demonstrate the feasibility of using K. oxytoca M1 for the production of not only 2,3-BDO but also acetoin as a major product.

Isolation of acetoin-producing Bacillus strains from Japanese traditional food-natto.

In this study, Bacillus strains with an ability to produce acetoin were isolated from a Japanese traditional food, natto, on the basis of the Voges-Proskauer (VP) reaction, and strain SF4-3 was shown to be a predominant strain in acetoin production. Based on a variety of morphological, physiological, and biochemical characteristics as well as the nucleotide sequence analysis of 16S rDNA, the strain SF4-3 was identified as Bacillus subtilis. When it was incubated at 37°C with a speed of 180 rpm for 96 hr in the flasks, the maximum acetoin concentration was up to 33.90 g/L. The fermentation broths were determined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses; the results showed that the major metabolite was acetoin, and the purity could reach more than 95% without butanedione and 2,3-butanediol, which were usually produced together with acetoin in other strains. A novel aqueous two-phase system (ATPS) composed of hydrophilic solvents and inorganic salts was developed for the extraction of acetoin from fermentation broths. The ethanol and dipotassium hydrogen phosphate system could be used to extract acetoin from fermentation broths. The influences of phase composition on partition of acetoin were investigated. The maximum partition coefficient (9.68) and recovery (94.6%) of acetoin were obtained, when 25% (w/w) dipotassium hydrogen phosphate and 24% (w/w) ethanol were used.

Enhanced acetoin production by Serratia marcescens H32 with expression of a water-forming NADH oxidase.

Cofactor engineering was employed to enhance production of acetoin by Serratia marcescens H32. 2,3-Butanediol was a major byproduct of acetoin fermentation by S. marcescens H32. In order to decrease 2,3-butanediol formation and achieve a high efficiency of acetoin production, nox gene encoding a water-forming NADH oxidase from Lactobacillus brevis was expressed. Batch fermentations suggested the expression of the NADH oxidase could increase the intracellular NAD(+) concentration (1.5-fold) and NAD(+)/NADH ratio (2.9-fold). Meanwhile, 2,3-butanediol was significantly decreased (52%), and the accumulation of acetoin was enhanced (33%) accordingly. By fed-batch culture of the engineered strain, the final acetoin titer up to 75.2g/l with the productivity of 1.88 g/(lh) was obtained. To the best of our knowledge, these results were new records on acetoin fermentation ever reported.

(R)-acetoin-female sex pheromone of the summer chafer Amphimallon solstitiale (L.).

Extracts of Amphimallon solstitiale (L.), a well known, widely distributed and rather common European scarab beetle, were analyzed by GC-MS and GC-EAD. Acetoin - (R):(S) > 9:1 - as well as 2,3-butanediol - (2R,3R): (2S,3S):meso = 1:1:9 - were present in extracts of both males and females. Although (2S,3S)-butanediol did not show any EAD activity, the other compounds elicited strong responses exclusively with male antennae. In contrast, several EAD active green leaf volatiles were detected equally well by male and female antennae. During preliminary field bioassays, (R)-acetoin was highly attractive to swarming males, whereas neither rac-acetoin nor the 2,3-butanediols showed activity. Therefore, (R)-acetoin is the female sex pheromone of A. solstitiale.

Evaluation of solid-phase microextraction for the isotopic analysis of volatile compounds produced during fermentation by lactic acid bacteria.

The use of solid-phase microextraction (SPME) coupled with isotope ratio mass spectrometry (IRMS) for the analysis of flavor compounds produced by lactic acid bacteria has been evaluated using both liquid and headspace sampling modes. Initially, it was necessary to optimize the conditions for the SPME extraction of flavors-diacetyl and acetoin-in standard aqueous solutions. The effects of salt, headspace versus liquid sampling, and coating phase were tested. Second, the suitability of the coupling of SPME and gas chromatography-combustion interface-IRMS (GC-C-IRMS) for the determination of delta(13)C values was assessed. It is shown that neither the analyte concentration nor the period of fiber exposure has an effect on the delta(13)C values. Finally, having verified that there are no matrix effects from the fermentation medium, it is reported for the first time that flavor compounds can be extracted directly from culture supernatant by SPME and their delta(13)C values can be obtained by GC-C-IRMS.

Brewers' yeast pyruvate decarboxylase produces acetoin from acetaldehyde: a novel tool to study the mechanism of steps subsequent to carbon dioxide loss.

A gas-liquid chromatographic technique was developed for the determination of both acetaldehyde and the 3-4% acetoin side product that results from the brewers' yeast pyruvate decarboxylase (EC 4.1.1.1) catalyzed reaction of pyruvic acid. Employing this method enabled the demonstration of the catalysis of acetaldehyde condensation to acetoin by the enzyme. It was found that the acetoin produced enzymatically from pyruvic acid or from acetaldehyde was optically active, thus providing stereochemical information about the reaction. Deuterium kinetic isotope effects (employing CH3CHO and CH3CDO) were determined on the steady-state kinetic parameters to be 4.5 (Vmax) and 3.2 (Vmax/Kappm), respectively. This enabled, for the first time, the estimation of relative kinetic barriers for steps past decarboxylation. It could be concluded that (a) C-H bond scission was part of rate limitation in the enzyme-catalyzed condensation of acetaldehyde to acetoin and that (b) among the steps leading to the release of acetaldehyde, protonation of the key enamine intermediate was part of rate limitation. This latter finding is also directly applicable to the mechanism of pyruvate decarboxylation.