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.

Acetoin is a common food flavor additive. This volatile compound widely exists in nature. Some microorganisms, higher plants, insects, and higher animals have the ability to synthesize acetoin using different enzymes and pathways under certain circumstances. As a very active molecule, acetoin acts as a precursor of dozens of compounds. Therefore, acetoin and its derivatives are frequently detected in the component analysis of a variety of foods using gas chromatography-mass spectrometry. Because of the increasing importance of these compounds, this paper reviews the origins and natural existence of these substances, physiological roles, the biological synthesis pathways, nonenzymatic spontaneous reactions, and the common determination methods in foods. This work is the first review on dietary natural acetoin.

Production of (3S)-acetoin ((3S)-AC), an important platform chemical, is desirable but difficult to perform. An NADPH-dependent carbonyl reductase (Gox0644) from Gluconobacter oxydans DSM 2003 was confirmed to have a good ability to reduce diacetyl (DA) to produce (3S)-AC. In this work, the NADPH-dependent carbonyl reductase was expressed and purified. Glucose dehydrogenase from Bacillus subtilis 168 was coupled with the NADPH-dependent carbonyl reductase to produce (3S)-AC from DA. Under the optimal conditions, 12.2gl611 (3S)-AC was produced from 14.3gl611 DA in 75min. Because DA can be biotechnological produced, the two-enzymes coupling system might be a promising alternative for the (3S)-AC production.

This report identifies twelve building block chemicals that can be produced from sugars via biological or chemical conversions. The twelve building blocks can be subsequently converted to a number of high-value bio-based chemicals or materials. Building block chemicals as considered for this analysis are molecules with multiple functional groups that possess the potential to be transformed into new families of useful molecules. The twelve sugar-based building blocks are 1,4-diacids (succinic fumaric and malic), 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sotitol, and xylitol/arabinitol.

Upgrading ethanol to higher order alcohols is desired but difficult using current biotechnological methods. In this study, we designed a completely artificial reaction pathway for upgrading ethanol to acetoin, 2,3-butanediol, and 2-butanol in a cell-free bio-system composed of ethanol dehydrogenase, formolase, 2,3-butanediol dehydrogenase, diol dehydratase, and NADH oxidase. Under optimized conditions, acetoin, 2,3-butanediol, and 2-butanol were produced at 88.78%, 88.28%, and 27.25% of the theoretical yield from 100 mM ethanol, respectively. These results demonstrate that this artificial synthetic pathway is an environmentally-friendly novel approach for upgrading bio-ethanol to acetoin, 2,3-butanediol, and 2-butanol.

乙偶姻,化学名为3-羟基-2-丁酮,是国际上常用的香料品种。本文综述了由丁二酮和丁二醇两种原料合成乙偶姻的研究现状,分析了乙偶姻合成研究的发展趋势。

The nutritional requirements for acetoin production by Bacillus subtilis CICC 10025 were optimized statistically in shake flask experiments using indigenous agroindustrial by-products. The medium components considered for initial screening in a Plackett–Burman design comprised a-molasses (molasses submitted to acidification pretreatment), soybean meal hydrolysate (SMH), KH 2 PO 4 ·3H 2 O, sodium acetate, MgSO 4 ·7H 2 O, FeCl 2 , and MnCl 2 , in which the first two were identified as significantly (at the 99% significant level) influencing acetoin production. Response surface methodology was applied to determine the mutual interactions between these two components and optimal levels for acetoin production. In flask fermentations, 37.902g l 611 acetoin was repeatedly achieved using the optimized concentrations of a-molasses and SMH [22.0% (v/v) and 27.8% (v/v), respectively]. a-Molasses and SMH were demonstrated to be more productive than pure sucrose and yeast extract plus peptone, respectively, in acetoin fermentation. In a 5-l fermenter, 35.402g l 611 of acetoin could be obtained after 56.402h of cultivation. To our knowledge, these results, i.e., acetoin yields in flask or fermenter fermentations, were new records on acetoin fermentation by B. subtilis .

以木糖为唯一碳源筛选了10株乙偶姻生产菌株,对乙偶姻产量最高菌株进行16SrDNA鉴定,测序结果表明该菌株为多粘芽孢杆菌,命名为LY107。经单因素实验优化培养条件为:培养温度37℃、pH值7.0、装液量15mL/250mL、接种量5%(体积比)。采用葡萄糖-木糖(2∶1,质量比,下同)为碳源模拟木质纤维素水解液发酵生产乙偶姻,经Plackett-Burman(PB)实验和RSM优化培养基组分(g.L-1)为:葡萄糖-木糖(2∶1)60、蛋白胨5、酵母粉16.5、硫酸锰0.05、硫酸亚铁0.005、磷酸二氢钾0.1、乙酸钠2.47。在优化培养条件和培养基组分下,乙偶姻最高产量达23.9g.L-1,葡萄糖-木糖转化率为79.9%。

乙偶姻又称3-羟基-2-丁酮，天然存在于可可、干酪、香蕉、葡萄、玉米等食品中，具有奶油香、脂香的特征，因此多用于食品、乳品及饮料中香料的配制；此外，乙偶姻作为一种平台化合物，还广泛的应用于化工、医药等行业。目前，乙偶姻的合成方法主要有化学法和生物法。生物法以其原料来源广泛，产品绿色天然，越来越受到人们的喜爱。生物法合成乙偶姻主要以微生物发酵法为主，而有关研究多集中于优良菌种的筛选及培养基的优化方面，对于发酵过程的研究及相关的调控手段报道较少。 本文在实验室前期研究的基础上，得到了一株高产乙偶姻的枯草芽孢杆菌（Bacillussubtilis CCTCC M208157），对其发酵乙偶姻的过程进行了研究。首先对菌株发酵乙偶姻的培养基进行了优化，并通过代谢流量分析的方法，明确了该菌株乙偶姻发酵代谢流的分布情况；之后通过添加表明活性剂、pH分段调控和补料分批发酵等手段，提高菌株发酵乙偶姻的产量；最后，对发酵液中乙偶姻的提取条件做了初步探索，为菌株发酵生产乙偶姻的进一步放大奠定了基础。 主要研究结果如下： (1)首先对B. subtilis CCTCC M208157发酵生产乙偶姻使用的培养基进行了优化，得到培养基组成为：葡萄糖80g/L、酵母膏20g/L、（NH_4）_2SO_410g/L、K_2HPO_410g/L、MgSO_41g/L、MnSO_40.05g/L。通过对培养基的优化，乙偶姻产量可达23.6g/L，相比对照提高了18.3%。 (2)采用代谢流量分析的方法对B. subtilis CCTCC M208157进行了研究，通过构建菌株的乙偶姻代谢网络模型，计算得到菌株乙偶姻发酵过程的代谢流量分布情况，菌株的葡萄糖代谢流量有86.6%用于乙偶姻的合成，说明菌株具有较强的乙偶姻合成能力。 (3)考察了添加不同表面活性剂对B. subtilis CCTCC M208157发酵生产乙偶姻的影响；其中添加二甲亚砜（DMSO）可提高菌株利用底物能力，当添加3.0g/L DMSO时乙偶姻产量可达26.8g/L，发酵结束后无底物残留，相比对照提高了16.3%。 (4)考察不同pH条件下B. subtilis CCTCC M208157乙偶姻发酵过程，通过发酵动力学参数的分析，提出了相应的pH调控策略如下：0-16h，pH5.5；16-72h，pH4.5。通过两段pH控制策略，菌株发酵乙偶姻的产量、产率、生产强度分别为32.7g/L、0.41g/g、0.91g/(L·h)，使得其相比初始发酵条件下分别提高了41%、42%、69%。 (5)提出了补料分批发酵策略：初始葡萄糖浓度为80g/L，发酵24h，开始以4g/(L·h)的流加速度向发酵体系中补加500g/L的葡萄糖溶液，共补加100g/L的葡萄糖。通过补料分批发酵，乙偶姻产量达到58.7g/L，为目前野生菌种报道的最高值。 (6)对发酵液中乙偶姻的提取做了初步探索，提取流程如下：首先在-0.098MPa，45℃条件下蒸馏，收集馏分得到乙偶姻水溶液，之后添加等体积的乙酸乙酯萃取，重复6次萃取合并各次萃取有机相，再于-0.098MPa，30℃蒸馏，除去乙酸乙酯，得到乙偶姻产品，经气相色谱检测纯度可达95%。

Abstract BACKGROUND Optically pure acetoin is an important potential pharmaceutical intermediate. It has also been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites. Recombinant Escherichia coli was developed for efficient (3 R )-acetoin production. Culture medium optimization and process control were carried out to improve (3 R )-acetoin yield by the engineered strain. RESULTS A synthetic pathway involved the budRAB genes from Serratia marcescens and NADH oxidase gene from Lactobacillus brevis in E. coli was developed for efficient (3 R )-acetoin production. Batch culture showed that 23.4 g L611 of (3 R )-acetoin could be obtained from 60 g L611 glucose by the engineered strain. Chiral-column GC analysis indicated that the stereoisomeric purity of (3 R )-acetoin produced was 97.3%. Further, the medium composition was optimized in shake flasks by an orthogonal design method. Under optimal conditions, (3 R )-acetoin concentration reached 38.3 g L611 in flask fermentation. Fed-batch fermentation based on a suitable agitation speed was carried out in a 5 L bioreactor, and maximum (3 R )-acetoin concentration of 60.3 g L611 was achieved with a productivity of 1.55 g L611 h611 and yield 86.3%. CONCLUSION An engineering E. coli for efficient (3 R )-acetoin production was constructed. The optimization of fermentation variables and fed-batch culture resulted in a maximum (3 R )-acetoin concentration of 60.3 g L611. 08 2013 Society of Chemical Industry

Acetoin (3-hydroxy-2-butanone) is an important flavour compound and is applied in cosmetics, pharmacy and chemical synthesis. In contrast to chemical syntheses or fermentations an enzymatic route facilitates enantioselective acetoin production. The discovery of a (S)-selective alcohol dehydrogenase enables a novel production process of (R)-acetoin from meso-2,3-butanediol. It was shown that the regeneration of oxidised nicotinamide adenine dinucleotide is a key point in preparative application of dehydrogenases for the oxidative route. An electrochemical regeneration system was successful combined with the ADH catalysed reaction. Up to 48mM (R)-acetoin was produced in the reaction system while productivities up to 2mMh 1 were reached. The possibility to apply an electrochemical system in a semi-preparative synthesis will stimulate further research of electroenzymatic processes with oxidoreductases.

Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis.

Paenibacillus polymyxa DSM 365, an efficient producer of (R,R)-2,3-butanediol, is known to show the highest production titer and productivity reported to date. Here, the first draft genome sequence of this promising strain may provide the genetic basis for further insights into the molecular mechanisms underlying the production of (R,R)-2,3-butanediol with high optical purity and at a high titer. It will also facilitate the design of rational strategies for further strain improvements, as well as construction of artificial biosynthetic pathways through synthetic biology for asymmetric synthesis of chiral 2,3-butanediol or acetoin in common microbial hosts.

Fed-batch fermentations for the production of 2,3-butanediol (BDL) with Paenibacillus polymyxa DSM 365 were investigated in 2-L-fermenters. A suitable micro-aerobic set-up enabled high product selectivity of up to 98% R,R-BDL towards meso-BDL and acetoin. Up to 111gL611R,R-BDL within 54h could be achieved with sufficient supply of complex medium (yeast extract). To the best of the knowledge of the authors, this is the highest titer so far reported for P. polymyxa indicating its high potential as a non pathogenic BDL-producer. Fermentation in low nutritional medium (5gL611 yeast extract) yielded up to 72gL611 BDL+acetoin (79% R,R-BDL), yet was affected by formation of exopolysaccharides (EPS). In the range of 30–40°C EPS formation decreased with raising temperature although growth rate and BDL-production remained similar. Additionally, Tween8003 was found to be a good additive to reduce viscosity caused by EPS.

(2R,3R)-2,3-Butanediol has many industrial applications, such as it is used as an antifreezeagent and low freezing point fuel. In addition, it is particularly important to provide chiral groups indrugs. In recent years, this valuable bio-based chemical has attracted increasing attention, and significantprogress has been made in the development of microbial cell factories for (2R,3R)-2,3-butanediolproduction. This article reviews recent advances and challenges in microbial routes to (2R,3R)-2,3-butanediol production, and highlights the metabolic engineering and synthetic biological approachesused to improve titers, yields, productivities, and optical purities. Finally, a systematic and integrativestrategy for developing high-performance microbial cell factories is proposed

【目的】对多粘类芽孢杆菌Paenibacillus polymya的玉米粉同步糖化发酵工艺进行优化，以获得低成本、高效的（R，R）-2，3-丁二醇生产技术。【方法】研究玉米粉浓度、氮源种类和氮源浓度对菌体生长、耗糖能力以及（R，R）-2，3-丁二醇产量、产率、得率和转化率的影响，并在此基础上进一步考察培养基中其它成分对（R，R）-2，3-丁二醇发酵的影响。【结果】优化后的培养基组分为玉米干粉140g／L，酵母粉30g／L，Na。HP043g／L，KH2P043g／L，（NH4）zS042g／L，MgS040．8g／L，微量元素溶液2mL／L。使用优化后的培养基进行同步糖化发酵，发酵50h后（R，R）-2，3-丁二醇产量达到56．28g／L（光学纯度为98．3％），对葡萄糖的得率为0．44g／g，产率为1．13g／（L·h）。【结论】（R，R）-2，3-丁二醇生产技术低价高效，可为其工业化生产提供参考。

BackgroundThe high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.Methodology/Principal FindingsSimultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.Conclusions/SignificanceA recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis.

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 M NADP+. Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde. Biotechnol. Bioeng. 2010;106: 541 552. 2010 Wiley Periodicals, Inc.