Determination of 2,3-butanediol in high and low acetoin producers of Saccharomyces cerevisiae wine yeasts by automated multiple development (AMD)

P. ROMANO, G. SUZZI, V. BRANDOLINI, E. MENZIANI AND P. DOMIZIO. 1996. High performance thin layer chromatography with automated multiple development was used to determine 2,3-butanediol levels in wine produced by high and low acetoin-forming strains of Saccharomyces cerevisiae . An inverse correlation between acetoin and 2,3-butanediol content was found suggesting a leaky mutation in acetoin reductase of the low 2,3-butanediol producing strains.     

Acetoin production in Saccharomyces cerevisiae wine yeasts

Abstract One hundred strains of Saccharomyces cerevisiae were examined for the capacity to produce acetoin in synthetic medium and in grape must. The low production of acetoin was found to be the more common pattern in this species. Most strains exhibited a similar distribution in both media, production ranging from non-detectable amounts to 12 mg 1⁻¹. Only four strains produced high quantities of acetoin, up to 29.5 mg l⁻¹ in synthetic medium and up to 194.6 mg l⁻¹ in grape must. This biometric study showed the existence of two phenotypes, "low and high acetoin production", that could be selected for conferring a desirable flavour of the final product.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Metabolic characterization of Kloeckera apiculata strains from star fruit fermentation

A total of 37 strains of Kloeckera apiculata was isolated during the spontaneous fermentation of star fruit must. Each strain was differentiated from the others on the basis of its capacity to produce acetaldehyde, ethyl acetate, higher alcohols, acetoin and acetic acid. All the strains were characterized by the low production of higher alcohols and the high production of ethyl acetate, whereas consistent differences in the production of acetaldehyde, acetoin and acetic acid served to differentiate star fruit apiculate strains into six different phenotypes, present at different stages of the fermentation process. The metabolic strain diversity found can be interpreted as a natural consequence of environmental conditions, which influenced the frequency and selection of specific apiculate strains. From the biotechnological point of view the different metabolic biotypes represent an important source of strains for potential use as starter cultures for star fruit fermentation.     

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.     

白酒酒曲中高产四甲基吡嗪菌株的筛选和鉴定

为探索白酒酿造过程中四甲基吡嗪的合成机制,从白酒高温曲中筛选获得具有高产四甲基吡嗪的菌株。本研究应用Voges．Proskauer（V．P）反应和光度法对获得菌株的发酵液中的四甲基吡嗪含量进行测定从而获得高产菌株,综合菌落生长形态,16SrDNA序列分析及飞行时间质谱分析,对高产四甲基吡嗪菌株进行鉴定。结果表明,从曲中筛选获得的16株产乙偶姻菌株,其中S12、B1、S1001及B3产TTMP含量较高,分别为67．4mg／kg、50．7mg／kg、48．9mg／kg和43．2mg／kg。S12经进一步鉴定为Bacillussp．。     

Higher alcohol and acetoin production by Zygosaccharomyces wine yeasts

Seventy strains of Zygosaccharomyces isolated from grape musts were investigated for their ability to produce higher alcohols and acetoin in synthetic medium and grape must. The Zygosaccharomyces strains produced generally low amounts of higher alcohols. Within this genus, Z. fermentati behaved differently from Z. bailii producing less isobutanol in synthetic medium and more amyl alcohols and isobutanol in grape must. Zygosaccharomyces fermentati did not form detectable amounts of acetoin in any conditions whereas Z. bailii produced it both in synthetic medium and in grape must. These strains were found to contribute to aroma and taste of wine.     

Biotechnological suitability of Saccharomycodes ludwigii for fermented beverages

Nineteen Saccharomycodes ludwigii strains were tested for the production of secondary products in grape must fermentation. A predominant metabolic pattern characterized by high production of isobutyl alcohol, acetoin and ethyl acetate was obtained. The occurrence of some strains producing enhanced amounts of these compounds suggests a potential utilization of this species for industrial applications. Feijoa juice was inoculated with a selected S'codes ludwigii strain in comparison to a control strain of S. cerevisiae and evaluation of the fermented products was carried out by 30 consumers with respect to the odour, flavour and taste. The sample fermented by S'codes ludwigii was characterized by a fresh odour with a fruity flavour, identified as flavour of apple and kiwi-fruit. This product was compared to apple juice, with a more acid taste. Despite the high concentrations of acetic acid, this beverage might be considered a potential summer refreshing drink, addressed to a target of consumers who prefer fruit drinks that leave a slightly acid and little sugary taste in the mouth.     

Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.     

Influence of temperature on flavour compound production from citrate by Lactobacillus rhamnosus ATCC 7469

The citrate utilization by Lactobacillus rhamnosus ATCC 7469 was found to be temperature-dependent. The maximum citrate utilization and incorporation of [1,5-14C]citrate rate were observed at 37 degreesC. At this temperature, maximum citrate lyase activity and specific diacetyl and acetoin production (Y(DA%)) were observed. The high levels of alpha-acetolactate synthase and low levels of diacetyl reductase, acetoin reductase and L-lactate dehydrogenase found at 37 degreesC led to an accumulation of diacetyl and acetoin. Optimum lactic acid production was observed at 45 degreesC, according to the high lactate dehydrogenase activity. The NADH oxidase activity increased with increasing culture temperature from 22 degreesC to 37 degreesC. Thus there are greater quantities of pyruvate available for the production of alpha-acetolactate, diacetyl and aceotin, and less diacetyl and acetoin are reduced.     

Evaluation of gene modification strategies for the development of low-alcohol-wine yeasts

Saccharomyces cerevisiae has evolved a highly efficient strategy for energy generation which maximizes ATP energy production from sugar. This adaptation enables efficient energy generation under anaerobic conditions and limits competition from other microorganisms by producing toxic metabolites, such as ethanol and CO(2). Yeast fermentative and flavor capacity forms the biotechnological basis of a wide range of alcohol-containing beverages. Largely as a result of consumer demand for improved flavor, the alcohol content of some beverages like wine has increased. However, a global trend has recently emerged toward lowering the ethanol content of alcoholic beverages. One option for decreasing ethanol concentration is to use yeast strains able to divert some carbon away from ethanol production. In the case of wine, we have generated and evaluated a large number of gene modifications that were predicted, or known, to impact ethanol formation. Using the same yeast genetic background, 41 modifications were assessed. Enhancing glycerol production by increasing expression of the glyceraldehyde-3-phosphate dehydrogenase gene, GPD1, was the most efficient strategy to lower ethanol concentration. However, additional modifications were needed to avoid negatively affecting wine quality. Two strains carrying several stable, chromosomally integrated modifications showed significantly lower ethanol production in fermenting grape juice. Strain AWRI2531 was able to decrease ethanol concentrations from 15.6% (vol/vol) to 13.2% (vol/vol), whereas AWRI2532 lowered ethanol content from 15.6% (vol/vol) to 12% (vol/vol) in both Chardonnay and Cabernet Sauvignon juices. Both strains, however, produced high concentrations of acetaldehyde and acetoin, which negatively affect wine flavor. Further modifications of these strains allowed reduction of these metabolites.     

The production of 2,3-butanediol as a differentiating character in wine yeasts

The capacity to produce 2,3-butanediol by 90 strains of four different species of wine yeasts (Kloeckera apiculata, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Zygosaccharomyces bailii) was tested in grape must by automated multiple development HPTLC. The total amount of 2,3-butanediol produced varied between 23mg l–1 and 857.7mg l–1 according to the yeast species. S. cerevisiae and Z. bailii behaved similarly, producing elevated amounts of 2,3-butanediol. K. apiculata and Sc. ludwigii, in contrast, were low producers. When considerable amounts of 2,3-butanediol were found, little acetoin was present; the amounts of butanediol and acetoin were characteristic of the individual species.     

Fermentative production of chiral acetoin by wild-type Bacillus strains

Abstract

In recent years, there have been many studies on producing acetoin by microbial fermentation, while only a few studies have focused on chiral acetoin biosynthesis. The weight assignment method was first applied to balance the chiral purity (expressed as the enantiomeric excess value) and the titer of acetoin. Bacillus sp. H-18W, a thermophile, was selected from seven Bacillus strains for chiral acetoin production. To lower the cost of the fermentation medium, soybean meal was used as a feedstock. Four kinds of frequently used commercial proteinases with different active sites were tested for the hydrolyzation of the soybean meal, and the combination of the acidic proteinase and the neutral proteinase showed the best results. In a fermentation medium containing 100?g L^?1 glucose and 200?g L^?1 hydrolysate, Bacillus sp. H-18W produced 21.84?g L^?1 acetoin with an ee value of 96.25% at 60?h. This is the first report of using a thermophilic strain to produce chiral acetoin by microbial fermentation. Thermophilic fermentation can reduce the risk of bacterial contamination and can save cooling water. Using soybean meal hydrolysate and glucose as feedstocks, this work provides an economical and alternative method for the production of chiral pure acetoin.     

Effects of corn steep liquor on production of 2,3-butanediol and acetoin by Bacillus subtilis

The initial concentration of corn steep liquor (CSL) have remarkable effects on not only 2,3-butanediol (2,3-BD) and acetoin (metabolic precursor) production, but also on the ratio of 2,3-BD to acetoin. When a high concentration of CSL was supplemented, cell growth was improved, acetoin reductase (ACR) was stimulated, the concentration of 2,3-BD increased by 78.6%, acetoin decreased by 61.9%, and the ratio of 2,3-BD to acetoin increased by 3.69-fold. The acr gene, encoding ACR, was over-expressed in Bacillus subtilis. Compared to the control (parent strain), low levels of CSL in the engineered strain increased 2,3-BD concentration and the ratio 2,3-BD to acetoin by 13.9% and 39.5%, respectively, and decreased acetoin titer by 18.3%. Acetoin became a major product under low levels of CSL. Also, a knockout strain carrying an acr::cat insertion mutation was constructed. As expected, the loss of ACR activity led to an accumulation of acetoin in the supernatants of acr:: cat mutant cultures. Additionally, the productivity of acetoin was improved by high concentration of CSL. The results above demonstrate the feasibility of using B. subtilis for the production of not only 2,3-BD but also acetoin as a major product.     

Biosynthesis of Diacetyl in Bacteria and Yeast

Both diacetyl and acetoin were produced by cell-free extracts and cultures of Pseudomonas fluorescens, Aerobacter aerogenes, Lactobacillus brevis, and Saccharomyces cerevisiae 299, whereas only acetoin was produced by cell-free extracts and cultures of Streptococcus lactis, Serratia marcescens, Escherichia coli, and S. cerevisiae strains 513 and 522. Cell-free extracts that produced diacetyl did not produce it from acetoin; they produced it from pyruvate, but only if acetyl-coenzyme A was was added to the reaction mixtures. Production of diacetyl by S. cerevisiae 299 was prevented by valine, inhibited by sodium arsenite, and stimulated by pantothenic acid. Valine did not prevent the production of acetoin. E. coli and the three strains of S. cerevisiae did not decarboxylate alpha-acetolactate but did use acetaldehyde in the production of acetoin from pyruvate. The other organisms produced acetoin from pyruvate via alpha-acetolactate.     

Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Citric acid metabolism in hetero- and homofermentative lactic acid bacteria.

The effect of citrate on production of diacetyl and acetoin by four strains each of heterofermentative and homofermentative lactic acid bacteria capable of utilizing citrate was studied. Acetoin was quantitatively the more important compound. The heterofermentative bacteria produced no acetoin or diacetyl in the absence of citrate, and two strains produced traces of acetoin in its presence. Citrate stimulated the growth rate of the heterofermentative lactobacilli. Acidification of all heterofermentative cultures with citric acid resulted in acetoin production. Destruction of accumulated acetoin appeared to coincide with the disappearance of citrate. All homofermentative bacteria produced more acetoin and diacetyl in the presence of citrate than in its absence. Citrate utilization was begun immediately by the streptococci but was delayed until at least the middle of the exponential phase in the case of the lactobacilli.     

Enhanced 2,3-Butanediol Production by Optimizing Fermentation Conditions and Engineering Klebsiella oxytoca M1 through Overexpression of Acetoin Reductase

Microbial production of 2,3-butanediol (2,3-BDO) has been attracting increasing interest because of its high value and various industrial applications. In this study, high production of 2,3-BDO using a previously isolated bacterium Klebsiella oxytoca M1 was carried out by optimizing fermentation conditions and overexpressing acetoin reductase (AR). Supplying complex nitrogen sources and using NaOH as a neutralizing agent were found to enhance specific production and yield of 2,3-BDO. In fed-batch fermentations, 2,3-BDO production increased with the agitation speed (109.6 g/L at 300 rpm vs. 118.5 g/L at 400 rpm) along with significantly reduced formation of by-product, but the yield at 400 rpm was lower than that at 300 rpm (0.40 g/g vs. 0.34 g/g) due to acetoin accumulation at 400 rpm. Because AR catalyzing both acetoin reduction and 2,3-BDO oxidation in K. oxytoca M1 revealed more than 8-fold higher reduction activity than oxidation activity, the engineered K. oxytoca M1 overexpressing the budC encoding AR was used in fed-batch fermentation. Finally, acetoin accumulation was significantly reduced by 43% and enhancement of 2,3-BDO concentration (142.5 g/L), yield (0.42 g/g) and productivity (1.47 g/L/h) was achieved compared to performance with the parent strain. This is by far the highest titer of 2,3-BDO achieved by K. oxytoca strains. This notable result could be obtained by finding favorable fermentation conditions for 2,3-BDO production as well as by utilizing the distinct characteristic of AR in K. oxytoca M1 revealing the nature of reductase.