Advances in biological production of acetoin: a comprehensive overview.

Acetoin, a high-value-added bio-based platform chemical, is widely used in foods, cosmetics, agriculture, and the chemical industry. It is an important precursor for the synthesis of: 2,3-butanediol, liquid hydrocarbon fuels and heterocyclic compounds. Since the fossil resources are becoming increasingly scarce, biological production of acetoin has received increasing attention as an alternative to chemical synthesis. Although there are excellent reviews on the: application, catabolism and fermentative production of acetoin, little attention has been paid to acetoin production via: electrode-assisted fermentation, whole-cell biocatalysis, and in vitro/cell-free biocatalysis. In this review, acetoin biosynthesis pathways and relevant key enzymes are firstly reviewed. In addition, various strategies for biological acetoin production are summarized including: cell-free biocatalysis, whole-cell biocatalysis, microbial fermentation, and electrode-assisted fermentation. The advantages and disadvantages of the different approaches are discussed and weighed, illustrating the increasing progress toward economical, green and efficient production of acetoin. Additionally, recent advances in acetoin extraction and recovery in downstream processing are also briefly reviewed. Moreover, the current issues and future prospects of diverse strategies for biological acetoin production are discussed, with the hope of realizing the promises of industrial acetoin biomanufacturing in the near future.

Synthesis of Ligustrazine from Acetaldehyde by a Combined Biological-Chemical Approach.

Ligustrazine is an important active alkaloid in medicine and in the food industry. Here, we developed a combined biological-chemical approach to produce ligustrazine from acetaldehyde. First, we constructed a whole-cell biocatalytic system to produce the precursor acetoin from acetaldehyde by overexpressing formolase (FLS). Second, a two-step strategy was developed to enhance protein expression of FLS by codon usage optimization at the first 14 codons and the introduction of an overlapping gene before the start codon. Through expression optimization and directed evolution of FLS, we improved the titer of acetoin about 40 fold when the concentration of acetaldehyde was 1.5 M. Finally, after reaction conditions optimization, the titer of acetoin and ligustrazine reached 222 g L-1 and 94 g L-1, with a 86.5% and 48% conversion rate from acetaldehyde, respectively. The developed one-pot synthesis for acetoin and ligustrazine is expected to be applied to industrial production in the future with the advantages of a green process, high efficiency, and low cost.

Evaluation of the synergistic olfactory effects of diacetyl, acetaldehyde, and acetoin in a yogurt matrix using odor threshold, aroma intensity, and electronic nose analyses.

Despite intensive analyses of yogurt flavor, the synergistic effects of the key aroma compounds on sensory responses and their optimum concentration ranges remain less well-documented. This study investigated the odor thresholds, optimum concentration ranges, and perceptual actions of diacetyl, acetaldehyde, and acetoin in a yogurt matrix. Our results show that the odor thresholds of diacetyl, acetaldehyde, and acetoin in the yogurt matrix were 5.43, 15.4, and 29.0 mg/L, respectively, which were significantly higher than the corresponding values in water. The optimum diacetyl, acetaldehyde, and acetoin concentration ranges were found to be 6.65 to 9.12, 25.9 to 35.5, and 37.3 to 49.9 mg/L, respectively. In Feller's additive model, the addition of each compound led to a significant reduction in their odor threshold in the yogurt matrix, thus demonstrating the synergistic effects of the compounds. In the σ-τ plot, various concentrations of compounds were associated with various degrees of additive behavior with respect to the aroma intensity of the yogurt matrix, thus demonstrating the synergism among these compounds in increasing the overall aroma intensity. The optimal simultaneous concentration ratio of diacetyl:acetaldehyde:acetoin was determined to be 4.00:16.0:32.0 mg/L. The specific synergistic effects were also confirmed by an electronic nose analysis and aroma profile comparison. In summary, these 3 aroma compounds exhibited synergistic effects in a yogurt matrix, thus providing a theoretical basis for the enhancement of flavors in dairy products.

Biological Production of (S)-acetoin: A State-of-the-Art Review.

Acetoin is an important four-carbon compound that has many applications in foods, chemical synthesis, cosmetics, cigarettes, soaps, and detergents. Its stereoisomer (S)-acetoin, a high-value chiral compound, can also be used to synthesize optically active drugs, which could enhance targeting properties and reduce side effects. Recently, considerable progress has been made in the development of biotechnological routes for (S)-acetoin production. In this review, various strategies for biological (S)- acetoin production are summarized, and their constraints and possible solutions are described. Furthermore, future prospects of biological production of (S)-acetoin are discussed.

Acetoin is a precursor to diacetyl in e-cigarette liquids.

Use of the e-liquid flavourings diacetyl and acetyl propionyl has raised concerns that they might cause respiratory diseases amongst vapers. Product surveys show that these compounds, plus a less toxic alternative, acetoin, are widely used in e-liquids. We have investigated the chemistry of acetoin, acetyl propionyl and diacetyl in e-liquids. They are reactive, with concentrations falling substantially over time. Acetyl propionyl is the most reactive, diacetyl less so, and acetoin significantly more stable. Their reactivity is pH-enhanced when nicotine is present in the e-liquid. Of major concern, we found that acetoin generates diacetyl in e-liquids. We found diacetyl formation in all acetoin-containing e-liquids, but it is not an acetoin-contaminant. Diacetyl concentrations were proportional to acetoin content, grew over time, and formation was accelerated by nicotine. E-liquids stored for up to 18 months contained significant diacetyl, and reduced acetoin levels, showing that acetoin is a long-term diacetyl source. Other reaction pathways operate, and we advance mechanisms to explain this area of e-liquid chemistry. Acetoin use in e-liquids is an inevitable source of diacetyl exposure for e-cigarette users. Acetoin, acetyl propionyl and diacetyl are avoidable hazards for vapers, and we recommend e-liquid manufacturers move away from their use in e-liquid formulations.

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.

Systematic Characterization of the Metabolism of Acetoin and Its Derivative Ligustrazine in Bacillus subtilis under Micro-Oxygen Conditions.

Bacillus subtilis is an important microorganism for brewing of Chinese Baijiu, which contributes to the formation of flavor chemicals including acetoin and its derivative ligustrazine. The first stage of Baijiu brewing process is under micro-oxygen conditions; however, there are few studies about B. subtilis metabolism under these conditions. Effects of various factors on acetoin and ligustrazine metabolism were investigated under these conditions, including key genes and fermentation conditions. Mutation of bdhA (encoding acetoin reductase) or overexpression of glcU (encoding glucose uptake protein) increased acetoin concentration. Addition of Vigna angularis powder to the culture medium also promoted acetoin production. Optimal culture conditions for ligustrazine synthesis were pH 6.0 and 42 °C. Ammonium phosphate was shown to promote ligustrazine synthesis in situ. This is the first report of acetoin and ligustrazine metabolism in B. subtilis under micro-oxygen conditions, which will ultimately promote the application of B. subtilis for maintaining Baijiu quality.

Rational design of Meso-2,3-butanediol dehydrogenase by molecular dynamics simulation and experimental evaluations.

Meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) catalyzes NAD+ -dependent conversion of meso-2,3-butanediol to acetoin, a crucial external energy storage molecule in fermentive bacteria. In this study, the active tunnel of meso-2,3-BDH was identified. The two short α helixes positioned away from the α4-helix possibly expose the hydrophobic ligand-binding cavity, gating the exit of product and cofactor from the activity pocket. Further MM/GBSA-binding free energy analysis shows that Phe212 and Asn146 function as the key product-release sites. Site-directed mutagenesis experiments targeted to the sites show that the kcat of Phe212Tyr is enhanced up to (4-8)-fold. The original activity of Asn146Gln is retained, but the activity of Asn146Ala mutation is lost. These results could provide helpful guidance on rational design of short-chain dehydrogenases/reductases.

Functional genomics provides insights into the role of Propionibacterium freudenreichii ssp. shermanii JS in cheese ripening.

Propionibacterium freudenreichii is a commercially important bacterium that is essential for the development of the characteristic eyes and flavor of Swiss-type cheeses. These bacteria grow actively and produce large quantities of flavor compounds during cheese ripening at warm temperatures but also appear to contribute to the aroma development during the subsequent cold storage of cheese. Here, we advance our understanding of the role of P. freudenreichii in cheese ripening by presenting the 2.68-Mbp annotated genome sequence of P. freudenreichii ssp. shermanii JS and determining its global transcriptional profiles during industrial cheese-making using transcriptome sequencing. The annotation of the genome identified a total of 2377 protein-coding genes and revealed the presence of enzymes and pathways for formation of several flavor compounds. Based on transcriptome profiling, the expression of 348 protein-coding genes was altered between the warm and cold room ripening of cheese. Several propionate, acetate, and diacetyl/acetoin production related genes had higher expression levels in the warm room, whereas a general slowing down of the metabolism and an activation of mobile genetic elements was seen in the cold room. A few ripening-related and amino acid catabolism involved genes were induced or remained active in cold room, indicating that strain JS contributes to the aroma development also during cold room ripening. In addition, we performed a comparative genomic analysis of strain JS and 29 other Propionibacterium strains of 10 different species, including an isolate of both P. freudenreichii subspecies freudenreichii and shermanii. Ortholog grouping of the predicted protein sequences revealed that close to 86% of the ortholog groups of strain JS, including a variety of ripening-related ortholog groups, were conserved across the P. freudenreichii isolates. Taken together, this study contributes to the understanding of the genomic basis of P. freudenreichii and sheds light on its activities during cheese ripening.

Sugaring-out extraction of acetoin from fermentation broth by coupling with fermentation.

Acetoin is a natural flavor and an important bio-based chemical which could be separated from fermentation broth by solvent extraction, salting-out extraction or recovered in the form of derivatives. In this work, a novel method named as sugaring-out extraction coupled with fermentation was tried in the acetoin production by Bacillus subtilis DL01. The effects of six solvents on bacterial growth and the distribution of acetoin and glucose in different solvent-glucose systems were explored. The operation parameters such as standing time, glucose concentration, and volume ratio of ethyl acetate to fermentation broth were determined. In a system composed of fermentation broth, glucose (100%, m/v) and two-fold volume of ethyl acetate, nearly 100% glucose was distributed into bottom phase, and 61.2% acetoin into top phase without coloring matters and organic acids. The top phase was treated by vacuum distillation to remove solvent and purify acetoin, while the bottom phase was used as carbon source to produce acetoin in the next batch of fermentation.

Engineering of Bacillus subtilis for the Production of 2,3-Butanediol from Sugarcane Molasses.

2,3-butanediol is known to be a platform chemical with several potential industrial applications. Sustainable industrial scale production can be attained by using a sugarcane molasses based fermentation process using Bacillus subtilis. However, the accumulation of acetoin needs to be reduced to improve process efficiency. In this work, B. subtilis was genetically modified in order to increase the yield of 2,3-butanediol. Metabolic engineering strategies such as cofactor engineering and overexpression of the key enzyme butanediol dehydrogenase were attempted. Both the strategies individually led to a statistically significant increase in the 2,3-butanediol yields for sugarcane molasses based fermentation. Cofactor engineering led to a 26 % increase in 2,3-butanediol yield and overexpression of bdhA led to a 11 % increase. However, the combination of the two strategies did not lead to a synergistic increase in 2,3-butanediol yield.

Direct Analysis of Free and Sulfite-Bound Carbonyl Compounds in Wine by Two-Dimensional Quantitative Proton and Carbon Nuclear Magnetic Resonance Spectroscopy.

Recent developments that have accelerated 2D NMR methods and improved quantitation have made these methods accessible analytical procedures, and the large signal dispersion allows for the analysis of complex samples. Few natural samples are as complex as wine, so the application to challenges in wine analysis look promising. The analysis of carbonyl compounds in wine, key oxidation products, is complicated by a multitude of kinetically reversible adducts, such as acetals and sulfonates, so that sample preparation steps can generate complex interferences. These challenges could be overcome if the compounds could be quantified in situ. Here, two-dimensional ((1)H-(1)H) homonuclear and heteronuclear ((13)C-(1)H) single quantum correlations (correlation spectroscopy, COSY, and heteronuclear single quantum coherence, HSQC) nuclear magnetic resonance spectra of undiluted wine samples were observed at natural abundance. These techniques achieve simultaneous direct identification and quantitation of acetaldehyde, pyruvic acid, acetoin, methylglyoxal, and α-ketoglutaric acid in wine with only a small addition of D2O. It was also possible to observe and sometimes quantify the sulfite, hydrate, and acetal forms of the carbonyl compounds. The accuracy of the method was tested in wine samples by spiking with a mixture of all analytes at different concentrations. The method was applied to 15 wine samples of various vintages and grape varieties. The application of this method could provide a powerful tool to better understand the development, evolution, and perception of wine oxidation and insight into the impact of these sulfite bound carbonyls on antimicrobial and antioxidant action by SO2.

How sotolon can impart a Madeira off-flavor to aged beers.

4,5-Dimethyl-3-hydroxy-2(5H)-furanone or sotolon is known to impart powerful Madeira-oxidized-curry-walnut notes to various alcoholic beverages. It has been much studied in oxidized Jura flor-sherry wines, aged Roussillon sweet wines, and old Port wines, in which it contributes to the characteristic "Madeira-oxidized" aroma of these beverages. No scientific paper describes how sotolon might be involved in the Madeira off-flavor found in aged beers. The specific extraction procedure applied here allowed us to quantify this lactone in 7 special beers, at levels sometimes well above its threshold (from 5 to 42 µg/L after 6, 12, 18, and 24 months of natural aging, while unquantifiable in fresh beer). Investigation of spiked beers led us to highlight the key role of pro-oxidants and acetaldehyde. Addition of ascorbic acid without sulfites should be avoided by brewers, as the former would intensify sotolon synthesis. Acetoin, a beer fermentation byproduct, also emerged as possible precursor in beer when combined with serine.

Engineered Serratia marcescens for efficient (3R)-acetoin and (2R,3R)-2,3-butanediol production.

(3R)-Acetoin and (2R,3R)-2,3-butanediol are important pharmaceutical intermediates. However, until now, the quantity of natural microorganisms with the ability to produce single configuration of optically pure (3R)-acetoin and (2R,3R)-2,3-butanediol is rare. In this study, a meso-2,3-butanediol dehydrogenase encoded by the slaC gene from Serratia marcescens MG1 was identified for meso-2,3-butanediol and (2S,3S)-2,3-butanediol biosynthesis. Inactivation of the slaC gene could significantly decrease meso-2,3-butanediol and (2S,3S)-2,3-butanediol and result in a large quantity of (3R)-acetoin accumulation. Furthermore, a (2R,3R)-2,3-butanediol dehydrogenase encoded by the bdhA gene from Bacillus subtilis 168 was introduced into the slaC mutant strain of Serratia marcescens MG1. Excess (2R,3R)-2,3-butanediol dehydrogenase could accelerate the reaction from (3R)-acetoin to (2R,3R)-2,3-butanediol and lead to (2R,3R)-2,3-butanediol accumulation. In fed-batch fermentation, the excess (2R,3R)-2,3-butanediol dehydrogenase expression strain could produce 89.81 g/l (2R,3R)-2,3-butanediol with a productivity of 1.91 g/l/h at 48 h. These results provided potential applications for (3R)-acetoin and (2R,3R)-2,3-butanediol production.

A rapid, one step preparation for measuring selected free plus SO2-bound wine carbonyls by HPLC-DAD/MS.

Carbonyl compounds are produced during fermentation and chemical oxidation during wine making and aging, and they are important to wine flavor and color stability. Since wine also contains these compounds as α-hydroxysulfonates as a result of their reaction with sulfur dioxide, an alkaline pre-treatment requiring oxygen exclusion has been used to release these bound carbonyls for analysis. By modifying the method to hydrolyze the hydroxysulfonates with heating and acid in the presence of 2,4-dinitrophenylhydrazine (DNPH), the carbonyl compounds are simultaneously and quickly released and derivatized, resulting in a simpler and more rapid method. In addition, the method avoids air exclusion complications during hydrolysis by the addition of sulfur dioxide. The method was optimized for temperature, reaction time, and the concentrations of DNPH, sulfur dioxide and acid. The hydrazones were shown to be stable for 10 h, adequate time for chromatographic analysis by HPLC-DAD/MS. This method is demonstrated for 2-ketoglutaric acid, pyruvic acid, acetoin and acetaldehyde, wine carbonyls of very different reactivities, and it offers good specificity, high recovery and low limits of detection. This new rapid, simple method is demonstrated for the measurement of carbonyl compounds in a range of wines of different ages and grape varieties.

Identification and characterization of a mycobacterial NAD⁺-dependent alcohol dehydrogenase with superior reduction of diacetyl to (S)-acetoin.

An enzyme capable of reducing acetoin in the presence of NADH was purified from Mycobacterium sp. B-009, a non-clinical bacterial strain of soil origin. The enzyme is a homotetramer and can be classified as a medium-chain alcohol dehydrogenase/reductase based on the molecular weight of the monomer. Identification of the structural gene revealed a limited distribution of homologous genes only among actinomycetes. In addition to its activity as a reductase specific for (S)-acetoin (EC 1.1.1.76), the enzyme showed both diacetyl reductase (EC 1.1.1.304) and NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1) activities. (S)-Acetoin and diacetyl reductases belong to a group of short-chain alcohol dehydrogenase/reductases but do not have superior abilities to dehydrogenate monoalcohols. Thus, the purified enzyme can be readily distinguished from other enzymes. We used the dual functionality of the enzyme to effectively reduce diacetyl to (S)-acetoin, coupled with the oxidation of 1-butanol.

Vitamin B1-catalyzed acetoin formation from acetaldehyde: a key step for upgrading bioethanol to bulk C4 chemicals.

The production of bulk chemicals and fuels from renewable biobased feedstocks is of significant importance for the sustainability of human society. The production of ethanol from biomass has dramatically increased and bioethanol also holds considerable potential as a versatile building block for the chemical industry. Herein, we report a highly selective process for the conversion of ethanol to C4 bulk chemicals, such as 2,3-butanediol and butene, via a vitamin B1 (thiamine)-derived N-heterocyclic carbene (NHC)-catalyzed acetoin condensation as the key step to assemble two C2 acetaldehydes into a C4 product. The environmentally benign and cheap natural catalyst vitamin B1 demonstrates high selectivity (99%), high efficiency (97% yield), and high tolerance toward ethanol and water impurities in the acetoin reaction. The results enable a novel and efficient process for ethanol upgrading.

Optimisation of ultrasound-assisted reverse micelles dispersive liquid-liquid micro-extraction by Box-Behnken design for determination of acetoin in butter followed by high performance liquid chromatography.

A novel approach, ultrasound-assisted reverse micelles dispersive liquid-liquid microextraction (USA-RM-DLLME) followed by high performance liquid chromatography (HPLC) was developed for selective determination of acetoin in butter. The melted butter sample was diluted and homogenised by n-hexane and Triton X-100, respectively. Subsequently, 400µL of distilled water was added and the microextraction was accelerated by 4min sonication. After 8.5min of centrifugation, sedimented phase (surfactant-rich phase) was withdrawn by microsyringe and injected into the HPLC system for analysis. The influence of effective variables was optimised using Box-Behnken design (BBD) combined with desirability function (DF). Under optimised experimental conditions, the calibration graph was linear over the range of 0.6-200mgL(-1). The detection limit of method was 0.2mgL(-1) and coefficient of determination was 0.9992. The relative standard deviations (RSDs) were less than 5% (n=5) while the recoveries were in the range of 93.9-107.8%.

Strategies for enhancing fermentative production of acetoin: a review.

Acetoin is a volatile compound widely used in foods, cigarettes, cosmetics, detergents, chemical synthesis, plant growth promoters and biological pest controls. It works largely as flavour and fragrance. Since some bacteria were found to be capable of vigorous acetoin biosynthesis from versatile renewable biomass, acetoin, like its reduced form 2,3-butanediol, was also classified as a promising bio-based platform chemical. In spite of several reviews on the biological production of 2,3-butanediol, little has concentrated on acetoin. The two analogous compounds are present in the same acetoin (or 2,3-butanediol) pathway, but their production processes including optimal strains, substrates, derivatives, process controls and product recovery methods are quite different. In this review, the usages of acetoin are reviewed firstly to demonstrate its importance. The biosynthesis pathway and molecular regulation mechanisms are then outlined to depict the principal network of functioning in typical species. A phylogenetic tree is constructed and the relationship between taxonomy and acetoin producing ability is revealed for the first time, which will serve as a useful guide for the screening of competitive acetoin producers. Genetic engineering, medium optimization, and process control are effective strategies to improve productivity as well. Currently, downstream processing is one of the main barriers in efficient and economical industrial acetoin fermentation. The future prospects of microbial acetoin production are discussed in light of the current progress, challenges, and trends in this field.

Sunlight-initiated chemistry of aqueous pyruvic acid: building complexity in the origin of life.

Coupling chemical reactions to an energy source is a necessary step in the origin of life. Here, we utilize UV photons provided by a simulated sun to activate aqueous pyruvic acid and subsequently prompt chemical reactions mimicking some of the functions of modern metabolism. Pyruvic acid is interesting in a prebiotic context due to its prevalence in modern metabolism and its abiotic availability on early Earth. Here, pyruvic acid (CH3COCOOH, a C3 molecule) photochemically reacts to produce more complex molecules containing four or more carbon atoms. Acetoin (CH3CHOHCOCH3), a C4 molecule and a modern bacterial metabolite, is produced in this chemistry as well as lactic acid (CH3CHOHCOOH), a molecule which, when coupled with other abiotic chemical reaction pathways, can provide a regeneration pathway for pyruvic acid. This chemistry is discussed in the context of plausible environments on early Earth such as near the ocean surface and atmospheric aerosol particles. These environments allow for combination and exchange of reactants and products of other reaction environments (such as shallow hydrothermal vents). The result could be a contribution to the steady increase in chemical complexity requisite in the origin of life.