乙偶姻生物合成代谢调控及其应用

许多微生物在糖酵解过程中能够将糖类转化为乙偶姻来避免过度酸化。乙偶姻能够调节NAD＋/NADH的比率并存储碳源。此外,乙偶姻作为一种具有特殊奶油香气的食用香料,广泛应用于食品,烟草、酒类和化妆品行业。近些年研究发现许多植物根际促生菌能够通过产生乙偶姻激活植物对外界环境压力的抗性,激活植物系统抗性,抵御病原菌的侵袭。乙偶姻还可以促进植物生长,提高产量。另外,乙偶姻还是调节根际促生菌与宿主植物相互作用的信号分子。简述乙偶姻的生物合成路径及其调控,并介绍乙偶姻在食品、医药、化工、化妆品、植物保护、生物燃料等方面的应用。     

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.     

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.     

Diacetyl and Acetoin Production by Lactobacillus casei

Agitation of broth cultures of Lactobacillus casei retarded cellular dry weight accumulation but enhanced production of both diacetyl and acetoin. Addition of pyruvate overcame this retardation, but addition of sulfhydryl-protecting reagents did not. Both pyruvate and citrate enhanced accumulated dry weight of L. casei incubated without agitation, but only pyruvate increased diacetyl accumulation. Both actively dividing cells and cells suspended in buffer converted pyruvate to diacetyl and acetoin. Maximum production of diacetyl and acetoin occurred during the late logarithmic or early stationary phases. Cells isolated from pyruvate- or citrate-containing cultures showed the greatest ability to convert pyruvate to diacetyl and acetoin. The optimum pH for diacetyl and acetoin formation by whole cells was in the range of 4.5 to 5.5. The presence of citrate or acetate enhanced diacetyl and acetoin formation by L. casei cells in buffer suspension.     

Exocellular products from Bifidobacterium adolescentis as immunomodifiers in the lymphoproliferative responses of mouse splenocytes

Abstract Pelobacter carbinolicus strain GraBd1 fermented methylacetoin, which is a good carbon source for growth ( μ = 0.16 h⁻¹) of this strict anaerobic bacterium, to acetone, acetate and ethanol (main products), acetoin, 2,3-butanediol and methylbutanediol (minor products). During growth on 2,3-butanediol, acetoin and methyl-acetoin the formation of a protein exhibiting acetoin: DCPIP oxidoreductase activity is induced. This enzyme amounts to a substantial portion of the soluble proteins. In vitro, it cleaves acetoin into acetate and acetaldehyde but reacts also with diacetyl or methylacetoin. We discussed four different models for the degradation of acetoin in the cells and came to the conclusion that in vivo the oxidative-thiolytic acetoin cleavage model is most probably realized in P. carbinolicus .     

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.     

乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响

【目的】研究乙酸合成途径阻断及NADH氧化酶表达对于谷氨酸棒杆菌生产乙偶姻的影响。【方法】在谷氨酸棒杆菌CGF2中异源表达als SD操纵子构建乙偶姻生产菌株CGT1,考察敲除乙酸生成途径cat和pqo对乙偶姻的影响。然后引入短乳杆菌的NADH氧化酶,在优化的溶氧条件下研究其对乙偶姻产量的影响。【结果】CGT1在摇瓶发酵中可积累6.27 g/L乙偶姻,敲除cat使乙偶姻产量显著提高30.94%,达到8.21 g/L;双敲除cat和pqo没有进一步提高产量。通过优化发酵的溶氧水平,乙偶姻产量达到10.06 g/L。在高溶氧水平下引入NADH氧化酶导致菌株的生长和糖代谢速率提高,但乙偶姻产量略有降低。在分批补料发酵中,重组菌株乙偶姻产量达到40.51 g/L,产率为0.51 g/(L?h)。【结论】在谷氨酸棒杆菌中阻断乙酸合成途径cat能够有效提高乙偶姻产量,NADH氧化酶在高溶氧水平下表达不利于乙偶姻的合成,需要进一步调节表达水平以确定其效果。     

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.     

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.     

Metabolic engineering of Bacillus subtilis for the co-production of uridine and acetoin

In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.

     

Effect of substrates on acetoin production by Torulopsis colliculosa and Enterobacter species.

Under optimal conditions, Torulopsis colliculosa NRRL 172 and Enterobacter B-87 (ATCC 27613) produced 50 to 500 mg of acetoin per g of substrate. Whereas cane molasses, gur, glucose, and sucrose were suitable substrates for acetoin production, lactose and mannitol supported very good growth but yielded little or no acetoin. Production of acetoin increased with increases in the concentration of glucose, yeast extract, and peptone. Combination of substrates and intermittent feeding of substrate failed to increase the yields.     

枯草芽孢杆菌(Bacillus subtilis)发酵生产乙偶姻的pH调控策略

【目的】为了提高Bacillus subtilis CCTCC M 208157发酵生产乙偶姻的效率。【方法】在7 L发酵罐水平上考察不同pH条件对菌株生长及乙偶姻合成的影响。【结果】pH对菌株合成乙偶姻有显著影响,pH 4.5有利于细胞合成乙偶姻,但是延迟期较长;pH 5.5时菌株生长较快,但乙偶姻的产量偏低。因此提出了两阶段pH控制策略:发酵前期(0 16 h),控制pH 5.5;发酵中后期(16 72 h),控制pH 4.5。【结论】通过此策略,菌株合成乙偶姻的能力得到进一步提高,乙偶姻的产量、产率和生产强度分别为32.7 g/L、0.41 g/g和0.91 g/(L.h),分别比初始发酵条件下提高了41%、42%和69%。     

Diacetyl Biosynthesis in Streptococcus diacetilactis and Leuconostoc citrovorum

Pyruvate was shown to be the precursor of diacetyl and acetoin in Streptococcus diacetilactis, but dialyzed cell-free extracts of S. diacetilactis and Leuconostoc citrovorum that had been treated with anion-exchange resin to remove coenzyme A (CoA) formed only acetoin from pyruvate in the presence of thiamine pyrophosphate (TPP) and Mg(++) or Mn(++) ions. The ability to produce diacetyl was restored by the addition of acetyl-CoA. Acetyl-phosphate did not replace the acetyl-CoA. Neither diacetyl nor acetoin was formed when the otherwise complete reaction system was modified by using boiled extract or by omitting the extract, pyruvate, TPP, or the metal ions. Free acetaldehyde was not involved in the biosynthesis of diacetyl or acetoin from pyruvate, dialyzed cell-free extracts of the bacteria produced only acetoin (besides CO(2)) from alpha-acetolactate, and acetoin was not involved in the biosynthesis of diacetyl. Only one of the optical isomers present in racemic alpha-acetolactate was attacked by the extracts, and there was no appreciable spontaneous decarboxylation of the alpha-acetolactate at the pH (4.5) used in experiments.     

Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824

Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin. The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824. The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long. Expression of the K. pneumoniae acetoin reductase gene in E. coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The K. pneumoniae acetoin reductase gene was cloned into a clostridial/E. coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E. coli and C. acetobutylicum. While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C. acetobutylicum, 2,3-butanediol is not. Analysis of culture supernatants by gas chromatography revealed that introduction of the K. pneumoniae acetoin reductase gene into C. acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin. 2,3-Butanediol was produced by cultures of C. acetobutylicum containing the gene only when commercial acetoin was added. Journal of Industrial Microbiology & Biotechnology (2001) 27, 220–227. Received 12 September 2000/ Accepted in revised form 26 June 2001     

Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis

The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent K_m values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited α-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.     

Diacetyl, Acetoin, and Acetaldehyde Production by Mixed-Species Lactic Starter Cultures

Citrate utilization and acetoin, diacetyl, acetaldehyde, and lactic acid production in milk at 21 C by five different mixed-strain starters, containing Streptococcus diacetilactis (D type), Leuconostoc (B type), and S. diacetilactis and Leuconostoc (BD type), were measured. BD and D cultures utilized citrate more rapidly and produced more diacetyl, acetoin, and acetaldehyde than B types. All cultures produced much more acetoin than diacetyl, with the BD and D cultures producing four to five times larger amounts of acetoin than the B cultures. Reduction of diacetyl and acetoin toward the end of the normal incubation period was characteristic of BD and D cultures, whereas a similar reduction of acetaldehyde was characteristic of BD and especially of B cultures. Continued incubation of B cultures beyond 17 h also resulted in reduction of diacetyl and acetoin. Addition of citrate to the milk retarded diacetyl and acetoin reduction. Mn²⁺ had no effect on diacetyl production by a BD culture but increased citrate utilization and, as a consequence, caused greater diacetyl destruction in one of the B cultures.     

The acetoin assimilation pathway of Pseudomonas putida KT2440 is regulated by overlapping global regulatory elements that respond to nutritional cues

Many microorganisms produce and excrete acetoin (3-hydroxy-2-butanone) when growing in environments that contain glucose or other fermentable carbon sources. This excreted compound can then be assimilated by other bacterial species such as pseudomonads. This work shows that acetoin is not a preferred carbon source of Pseudomonas putida, and that the induction of genes required for its assimilation is down-modulated by different, independent, global regulatory systems when succinate, glucose or components of the LB medium are also present. The expression of the acetoin degradation genes was found to rely on the RpoN alternative sigma factor and to be modulated by the Crc/Hfq, Cyo and PTS^Ntr regulatory elements, with the impact of the latter three varying according to the carbon source present in addition to acetoin. Pyruvate, a poor carbon source for P. putida, did not repress acetoin assimilation. Indeed, the presence of acetoin significantly improved growth on pyruvate, revealing these compounds to have a synergistic effect. This would provide a clear competitive advantage to P. putida when growing in environments in which all the preferred carbon sources have been depleted and pyruvate and acetoin remain as leftovers from the fermentation of sugars by other microorganisms.     

Evidence for oxidative thiolytic cleavage of acetoin in Pelobacter carbinolicus analogous to aerobic oxidative decarboxylation of pyruvate

Dihydrolipoamide dehydrogenase and dihydrolipoamide acetyltransferase were formed when Pelobacter carbinolicus strain GraBd1 was grown on acetoin. The specific activities of these enzymes amounted to 0.50 and 28.7 U/mg protein, respectively. The crude extract catalyzed the CoASH- and NAD+-dependent formation of acetyl-CoA from acetoin and methylacetoin. From ethylene glycol-grown cells these activities were absent. Crude extracts also exhibited acetoin: methyl viologen and acetoin: metronidazole oxidoreductase activity. As shown by reconstitution experiments methylviologen reduction was dependent on the presence of a light-brownish protein (Mr 220,000 +/- 10,000); metronidazole reduction was in addition dependent on the presence of a dark-brownish protein (Mr 4,900 +/- 800), which is probably a ferredoxin. However, both components were synthesized constitutively. We discussed a model for oxidative-thiolytic cleavage of acetoin which is analogous to the reaction of the pyruvate dehydrogenase enzyme complex rather than to pyruvate: ferredoxin oxidoreductase.