Chemical imitation of yeast fermentation by the drosophilid-pollinated deceptive trap-flower Aristolochia baetica (Aristolochiaceae).

Deceptive flowers, unlike in mutualistic pollination systems, mislead their pollinators by advertising rewards which ultimately are not provided. Although our understanding of deceptive pollination systems increased in recent years, the attractive signals and deceptive strategies in the majority of species remain unknown. This is also true for the genus Aristolochia, famous for its deceptive and fly-pollinated trap flowers. Representatives of this genus were generally assumed to be oviposition-site mimics, imitating vertebrate carrion or mushrooms. However, recent studies found a broader spectrum of strategies, including kleptomyiophily and imitation of invertebrate carrion. A different deceptive strategy is presented here for the western Mediterranean Aristolochia baetica L. We found that this species is mostly pollinated by drosophilid flies (Drosophilidae, mostly Drosophila spp.), which typically feed on fermenting fruit infested by yeasts. The flowers of A. baetica emitted mostly typical yeast volatiles, predominantly the aliphatic compounds acetoin and 2,3-butandiol, and derived acetates, as well as the aromatic compound 2-phenylethanol. Analyses of the absolute configurations of the chiral volatiles revealed weakly (acetoin, 2,3-butanediol) to strongly (mono- and diacetates) biased stereoisomer-ratios. Electrophysiological (GC-EAD) experiments and lab bioassays demonstrated that most of the floral volatiles, although not all stereoisomers of chiral compounds, were physiologically active and attractive in drosophilid pollinators; a synthetic mixture thereof successfully attracted them in field and lab bioassays. We conclude that A. baetica chemically mimics yeast fermentation to deceive its pollinators. This deceptive strategy (scent chemistry, pollinators, trapping function) is also known from more distantly related plants, such as Arum palaestinum Boiss. (Araceae) and Ceropegia spp. (Apocynaceae), suggesting convergent evolution. In contrast to other studies working on floral scents in plants imitating breeding sites, the present study considered the absolute configuration of chiral compounds.

Role of Volatile Organic Compounds Produced by Kosakonia cowanii Cp1 during Competitive Colonization Interaction against Pectobacterium aroidearum SM2.

The competitive colonization of bacteria on similar ecological niches has a significant impact during their establishment. The synthesis speeds of different chemical classes of molecules during early competitive colonization can reduce the number of competitors through metabolic effects. In this work, we demonstrate for the first time that Kosakonia cowanii Cp1 previously isolated from the seeds of Capsicum pubescens R. P. produced volatile organic compounds (VOCs) during competitive colonization against Pectobacterium aroidearum SM2, affecting soft rot symptoms in serrano chili (Capsicum annuum L.). The pathogen P. aroidearum SM2 was isolated from the fruits of C. annuum var. Serrano with soft rot symptoms. The genome of the SM2 strain carries a 5,037,920 bp chromosome with 51.46% G + C content and 4925 predicted protein-coding genes. It presents 12 genes encoding plant-cell-wall-degrading enzymes (PCDEWs), 139 genes involved in five types of secretion systems, and 16 genes related to invasion motility. Pathogenic essays showed soft rot symptoms in the fruits of C. annuum L., Solanum lycopersicum, and Physalis philadelphica and the tubers of Solanum tuberosum. During the growth phases of K. cowanii Cp1, a mix of VOCs was identified by means of HS-SPME-GC-MS. Of these compounds, 2,5-dimethyl-pyrazine showed bactericidal effects and synergy with acetoin during the competitive colonization of K. cowanii Cp1 to completely reduce soft rot symptoms. This work provides novel evidence grounding a better understanding of bacterial interactions during competitive colonization on plant tissue, where VOC synthesis is essential and has a high potential capacity to control pathogenic microorganisms in agricultural systems.

Genetic engineering of a thermophilic acetogen, Moorella thermoacetica Y72, to enable acetoin production.

Acetogens are among the key microorganisms involved in the bioproduction of commodity chemicals from diverse carbon resources, such as biomass and waste gas. Thermophilic acetogens are particularly attractive because fermentation at higher temperatures offers multiple advantages. However, the main target product is acetic acid. Therefore, it is necessary to reshape metabolism using genetic engineering to produce the desired chemicals with varied carbon lengths. Although such metabolic engineering has been hampered by the difficulty involved in genetic modification, a model thermophilic acetogen, M. thermoacetica ATCC 39073, is the case with a few successful cases of C2 and C3 compound production, other than acetate. This brief report attempts to expand the product spectrum to include C4 compounds by using strain Y72 of Moorella thermoacetica. Strain Y72 is a strain related to the type strain ATCC 39073 and has been reported to have a less stringent restriction-modification system, which could alleviate the cumbersome transformation process. A simplified procedure successfully introduced a key enzyme for acetoin (a C4 chemical) production, and the resulting strains produced acetoin from sugars and gaseous substrates. The culture profile revealed varied acetoin yields depending on the type of substrate and culture conditions, implying the need for further engineering in the future. Thus, the use of a user-friendly chassis could benefit the genetic engineering of M. thermoacetica.

Floral scent of eastern skunk cabbage (Symplocarpus foetidus: Araceae).

Symplocarpus foetidus (L.) Salisb. (eastern skunk cabbage) occurs across a broad geographic range of northeastern North America, blooming in winter between December and March. The inflorescences are well-known for their thermogenic and thermoregulatory metabolic capabilities. The perceptual qualities of their fetid floral aroma have been described widely in the literature, but to date the floral volatile composition remained largely unknown. Here we present a detailed study of the floral scent produced by S. foetidus collected from intact female- and male-stage inflorescences and from dissected floral parts. Our results show a large range of biosynthetically diverse volatiles including nitrogen- and sulfur-containing compounds, monoterpenes, benzenoids, and aliphatic esters and alcohols. We document high inter-individual variation with some organ-specific volatile trends but no clear strong variation based on sexual stage. Multivariate data analysis revealed two distinct chemotypes from our study populations that are not defined by sexual stage or population origin. The chemotype differences may explain the bimodal perceptual descriptions in earlier work which vary between highly unpleasant/fetid and pleasant/apple-like. We discuss the results in ecological contexts including potential for floral mimicry, taking into account existing pollination studies for the species. We also discuss the results in evolutionary contexts, comparing our scent data to published scent data from the close sister species Symplocarpus renifolius. Future work should more closely examine the chemotype occurrence and frequency within these and other populations, and the impact these chemotypes may have on pollinator attraction and reproductive success.

Design of a synthetic enzyme cascade for the in vitro fixation of formaldehyde to acetoin.

Formaldehyde (FALD) has gained prominence as an essential C1 building block in the synthesis of valuable chemicals. However, there are still challenges in converting FALD into commodities. Recently, cell-free biocatalysis has emerged as a popular approach for producing such commodities. Acetoin, also known as 3-hydroxy-2-butanone, has been widely used in food, cosmetic, agricultural and the chemical industry. It is valuable to develop a process to produce acetoin from FALD. In this study, a cell-free multi-enzyme catalytic system for the production of acetoin using FALD as the substrate was designed and constructed. It included three scales: FALD utilization pathway, glycolysis pathway and acetoin synthesis pathway. After the optimization of the reaction system, 20.17 mM acetoin was produced from 122 mM FALD, with a yield of 0.165 mol/mol, reaching 99.0% of the theoretical yield. The pathway provides a new approach for high-yield acetoin production from FALD, which consolidates the foundation for the production of high value-added chemicals using cheap one-carbon compounds.

L-Ascorbic Acid Restricts Vibrio cholerae Survival in Various Growth Conditions.

Cholera, a deadly diarrheal disease, continues to ravage various parts of the world. It is caused by Vibrio cholerae, an important member of the gamma-proteobacteria. Based on certain genetic and phenotypic tests, the organism is classified into two major biotypes, namely classical and El Tor. The El Tor and its variants are majorly responsible for the ongoing seventh pandemic across the globe. Previously, we have shown that cross-feeding of glucose metabolic acidic by-products of gut commensals can severely affect the viability of the biotypes. In this work, we examined the effect of L-ascorbic acid on the survival of Vibrio cholerae strains belonging to both biotypes and different serotypes. We observed that L-ascorbic acid effectively restricts the growth of all strains under various conditions including strains adapted to acid stress. In addition, L-ascorbic acid is also effective in decreasing bile-induced biofilms of Vibrio cholerae.

Untargeted metabolomics unveiled the role of butanoate metabolism in the development of Pseudomonas aeruginosa hypoxic biofilm.

Pseudomonas aeruginosa is a versatile opportunistic pathogen which causes a variety of acute and chronic human infections, some of which are associated with the biofilm phenotype of the pathogen. We hypothesize that defining the intracellular metabolome of biofilm cells, compared to that of planktonic cells, will elucidate the metabolic pathways and biomarkers indicative of biofilm inception. Disc-shaped stainless-steel coupons (12.7 mm diameter) were employed as a surface for static biofilm establishment. Each disc was immersed in a well, of a 24-well microtiter plate, containing a 1-mL Lysogeny broth (LB) suspension of P. aeruginosa ATCC 9027, a strain known for its biofilm prolificacy. This setup underwent oxygen-depleted incubation at 37°C for 24 hours to yield hypoxic biofilms and the co-existing static planktonic cells. In parallel, another planktonic phenotype of ATCC 9027 was produced in LB under shaking (200 rpm) incubation at 37°C for 24 hours. Planktonic and biofilm cells were harvested, and the intracellular metabolites were subjected to global untargeted metabolomic analysis using LC-MS technology, where small metabolites (below 1.5 kDa) were selected. Data analysis showed the presence of 324 metabolites that differed (p < 0.05) in abundance between planktonic and biofilm cells, whereas 70 metabolites did not vary between these phenotypes (p > 0.05). Correlation, principal components, and partial least square discriminant analyses proved that the biofilm metabolome is distinctly clustered away from that of the two planktonic phenotypes. Based on the functional enrichment analysis, arginine and proline metabolism were enriched in planktonic cells, but butanoate metabolism was enriched in biofilm cells. Key differential metabolites within the butanoate pathway included acetoacetate, 2,3-butandiol, diacetyl, and acetoin, which were highly upregulated in the biofilm compared to the planktonic cells. Exogenous supplementation of acetoin (2 mM), a critical metabolite in butanoate metabolism, augmented biofilm mass, increased the structural integrity and thickness of the biofilm, and maintained the intracellular redox potential by balancing NADH/NAD+ ratio. In conclusion, P. aeruginosa hypoxic biofilm has a specialized metabolic landscape, and butanoate pathway is a metabolic preference and possibly required for promoting planktonic cells to the biofilm state. The butanoate pathway metabolites, particularly acetoin, could serve as markers for biofilm development.

Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches.

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Effect of different isolation sources of Lactococcus lactis subsp. lactis on volatile metabolites in fermented milk.

Lactococcus lactis subsp. lactis (L. lactis subsp. lactis) is a commonly used starter cultures in fermented dairy products, contributing distinct flavor and texture characteristics with high application value. However, the strains from different isolates have different contributions to milk fermentation. Therefore, this study aimed to investigate the influence of L. lactis subsp. lactis isolated from various sources on the volatile metabolites present in fermented milk. In this study, L. lactis subsp. lactis from different isolation sources (yogurt, koumiss and goat yogurt) was utilized as a starter culture for fermentation. The volatile metabolites of fermented milk were subsequently analyzed by headspace solid phase microextraction gas chromatography-mass spectrography (HS-SPME-GC-MS). The results indicated significant differences in the structure and abundance of volatile metabolites in fermented milk produced with different isolates (R2Y = 0.96, Q2 = 0.88). Notably, the strains isolated from goat yogurt appeared to enhance the accumulation of ketones (goat yogurt vs yogurt milk: 50 %; goat yogur vs koumiss: 27.3 %)and aldehydes (goat yogurt vs yogurt milk: 21.4 %; goat yogurt vs koumiss: 54.5 %) in fermented milk than strains isolated from koumiss and yogurt milk. It significantly promoted the production of 8 flavor substances (1 substance with OAV ≥ 1 and 6 substances with OAV > 0.1) and enhanced the biosynthesis of valine, leucine, and isoleucine. This study provides valuable insights for the application of Lactococcus lactis subsp. lactis isolated from different sources in fermented dairy production and screening of potential starter cultures.

Comparative key aroma compounds and sensory correlations of aromatic coconut water varieties: Insights from GC × GC-O-TOF-MS, E-nose, and sensory analysis.

Aroma is a key criterion in evaluating aromatic coconut water. A comparison regarding key aroma compounds and sensory correlations was made between Thailand Aromatic Green Dwarf (THD) and Cocos nucifera L. cv. Wenye No. 4 coconut water using E-nose and GC × GC-O-TOF-MS combined with chemometrics. Twenty-one volatile components of coconut water were identified by GC × GC-O-TOF-MS, and 5 key aroma compounds were analyzed by relative odor activity value and aroma extract dilution analysis. Moreover, the combination of the E-nose with orthogonal partial least squares was highly effective in discriminating between the two coconut water samples and screened the key sensors responsible for this differentiation. Additionally, the correlation between volatile compounds and sensory properties was established using partial least squares. The key aroma compounds of coconut water exhibited positive correlations with the corresponding sensory properties.

Construction and characterization of a promoter library with varying strengths to enhance acetoin production from xylose in Serratia marcescens.

Serratia marcescens is utilized as a significant enterobacteria in the production of various high-value secondary metabolites. Acetoin serves as a crucial foundational compound of development and finds application in a broad range of fields. Furthermore, S. marcescens HBQA-7 is capable of utilizing xylose as its exclusive carbon source for acetoin production. The objective of this study was to utilize a constitutive promoter screening strategy to enhance both xylose utilization and acetoin production in S. marcescens HBQA-7. By utilizing RNA-seq, we identified the endogenous constitutive promoter P6 that is the most robust, which facilitated the overexpression of the sugar transporter protein GlfL445I, α-acetyl lactate synthase, and α-acetyl lactate decarboxylase, respectively. The resultant recombinant strains exhibited enhanced xylose utilization rates and acetoin yields. Subsequently, a recombinant plasmid, denoted as pBBR1MCS-P6-glfL445IalsSalsD, was constructed, simultaneously expressing the aforementioned three genes. The resulting recombinant strain, designated as S3, demonstrated a 1.89-fold boost in xylose consumption rate compared with the original strain during shake flask fermentation. resulting in the accumulation of 7.14 g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75 g/L, corresponding to a xylose-to-acetoin conversion yield of 0.375 g/g.

Limiting acetoin generation during 2,3-butanediol fermentation with Paenibacillus polymyxa using lignocellulosic hydrolysates.

Recent decarbonization efforts have led to interests in producing more bio-based chemicals. One attractive compound produced biochemically is the platform chemical known as 2,3-butanediol (2,3-BDO). In this work a mild alkaline pretreatment using sodium carbonate was performed on corn stover (CS) and switchgrass (SG) to generate hydrolysates for fermentation with the 2,3-BDO producer bacteria strain Paenibacillius polymyxa. Enzymatic hydrolysis performed on the pretreated CS and SG produced theoretical sugar yields of 80 % and 95 % for glucose and xylose, respectively. Fermentations with P. polymxya conducted in anaerobic bottles produced 2,3-BDO reaching concentrations ranging from 14 to 18 g/L with negligible conversion into acetoin. Bioreactor fermentations using the hydrolysate media generated up to 43 g/L and 34 g/L of 2,3-BDO from pretreated CS and SG, respectively, within 24 h of fermentation. However, 2,3-BDO product output was reduced by 40-50 % over the remainder of the fermentation due to conversion into acetoin caused by glucose depletion.

The volatile organic compound acetoin enhances the colonization of Azorhizobium caulinodans ORS571 on Sesbania rostrata.

Chemoreceptors play a crucial role in assisting bacterial sensing and response to environmental stimuli. Genome analysis of Azorhizobium caulinodans ORS571 revealed the presence of 43 putative chemoreceptors, but their biological functions remain largely unknown. In this study, we identified the chemoreceptor AmaP (methyl-accepting protein of A. caulinodans), characterized by the presence of the CHASE3 domain and exhibited a notable response to acetoin. Thus, we investigated the effect of acetoin sensing on its symbiotic association with the host. Our findings uncovered a compelling role for acetoin as a key player in enhancing various facets of A. caulinodans ORS571's performance including biofilm formation, colonization, and nodulation abilities. Notably, acetoin bolstered A. caulinodans ORS571's efficacy in promoting the growth of S. rostrata, even under moderate salt stress conditions. This study not only broadens our understanding of the AmaP protein with its distinctive CHASE3 domain but also highlights the promising potential of acetoin in fortifying the symbiotic relationship between A. caulinodans and Sesbania rostrata.

Metabolic engineering of Caldicellulosiruptor bescii for 2,3-butanediol production from unpretreated lignocellulosic biomass and metabolic strategies for improving yields and titers.

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii, an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C5 and C6 sugars. In addition, C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii.IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications.

The effective of bacterial community dynamics driven by different starter cultures on the flavor development of Chinese fermented sausages.

This study aimed to understand the community successions driven by different starters and their effects on the flavor development of Chinese fermented sausages. The results showed that the bacterial genus (67.6%) and pH (32.4%) were the key factors influencing the volatile profile. Inoculated the starters composed of Pediococcus and staphylococci maintained the stable community succession patterns dominated by staphylococci (samples T and S). Although the highly acidic environment (pH < 5.2) caused the community to exhibit a fluctuation in succession pattern, the inoculation of Latilactobacillus paracasei (sample Y) maintained microbial diversity and was conducive to the accumulation of aldehydes and esters. In sample P, inoculated the starter with Latilactobacillus and Staphylococcus also maintained microbial diversity, the moderately acidic environment (pH > 5.4) resulted in a stable succession pattern of the microbial community, and it was not conducive to the accumulation of aldehydes, alcohols and esters.

Utilization of plant derived lactic acid bacteria for efficient bioconversion of brewers' spent grain into acetoin.

Brewers' spent grain (BSG) is a major side-stream from the beer industry, with an annual estimated production of 39 million tons worldwide. Due to its high nutritional value, high abundance and low price, it has been proposed as an ingredient in human food. Here we investigated the ability of different lactic acid bacteria to produce the flavor molecule acetoin in liquid BSG extract, in order to broaden the possibilities of utilization of BSG in human food. All the investigated lactic acid bacteria (LAB) covering the Leuconostoc, Lactobacillus and Lactoccocus species were able to convert the fermentable sugars in liquid BSG into acetoin. Production levels varied significantly between the different LAB species, with Leuconostoc pseudomesenteroides species reaching the highest titers of acetoin with only acetate as the main byproduct, while also being the fastest consumer of the fermentable sugars present in liquid BSG. Surprisingly, the currently best investigated LAB for acetoin production, L. lactis, was unable to consume the maltose fraction of liquid BSG and was therefore deemed unfit for full conversion of the sugars in BSG into acetoin. The production of acetoin in Leu. pseudomesenteroides was pH dependent as previously observed in other LAB, and the conversion of BSG into acetoin was scalable from shake flasks to 1 L bioreactors. While all investigated LAB species produced acetoin under aerobic conditions, Leu. pseudomesenteroides was found to be an efficient and scalable organism for bioconversion of liquid BSG into a safe acetoin rich food additive.

Superior anodic electro-fermentation by enhancing capacity for extracellular electron transfer.

Anodic electro-fermentation (AEF), where an anode replaces the terminal electron acceptor, shows great promise. Recently a Lactococcus lactis strain blocked in NAD+ regeneration was demonstrated to use ferricyanide as an alternative electron acceptor to support fast growth, but the need for high concentrations of this non-regenerated electron acceptor limits practical applications. To address this, growth of this L. lactis strain, and an adaptively evolved (ALE) mutant with enhanced ferricyanide respiration capacity were investigated using an anode as electron acceptor in a bioelectrochemical system (BES) setup. Both strains grew well, however, the ALE mutant significantly faster. The ALE mutant almost exclusively generated 2,3-butanediol, whereas its parent strain mainly produced acetoin. The ALE mutant interacted efficiently with the anode, achieving a record high current density of 0.81 ± 0.05 mA/cm2. It is surprising that a Lactic Acid Bacterium, with fermentative metabolism, interacts so well with an anode, which demonstrates the potential of AEF.

Adaptation to pollination by fungus gnats underlies the evolution of pollination syndrome in the genus Euonymus.

BACKGROUND AND AIMS: Dipteran insects are known pollinators of many angiosperms, but knowledge on how flies affect floral evolution is relatively scarce. Some plants pollinated by fungus gnats share a unique set of floral characters (dark red display, flat shape and short stamens), which differs from any known pollination syndromes. We tested whether this set of floral characters is a pollination syndrome associated with pollination by fungus gnats, using the genus Euonymus as a model. METHODS: The pollinator and floral colour, morphology and scent profile were investigated for ten Euonymus species and Tripterygium regelii as an outgroup. The flower colour was evaluated using bee and fly colour vision models. The evolutionary association between fungus gnat pollination and each plant character was tested using a phylogenetically independent contrast. The ancestral state reconstruction was performed on flower colour, which is associated with fungus gnat pollination, to infer the evolution of pollination in the genus Euonymus. KEY RESULTS: The red-flowered Euonymus species were pollinated predominantly by fungus gnats, whereas the white-flowered species were pollinated by bees, beetles and brachyceran flies. The colour vision analysis suggested that red and white flowers are perceived as different colours by both bees and flies. The floral scents of the fungus gnat-pollinated species were characterized by acetoin, which made up >90 % of the total scent in three species. Phylogenetically independent contrast showed that the evolution of fungus gnat pollination is associated with acquisition of red flowers, short stamens and acetoin emission. CONCLUSIONS: Our results suggest that the observed combination of floral characters is a pollination syndrome associated with the parallel evolution of pollination by fungus gnats. Although the role of the red floral display and acetoin in pollinator attraction remains to be elucidated, our finding underscores the importance of fungus gnats as potential contributors to floral diversification.

Mechanism of microbial production of acetoin and 2,3-butanediol optical isomers and substrate specificity of butanediol dehydrogenase.

3-Hydroxybutanone (Acetoin, AC) and 2,3-butanediol (BD) are two essential four-carbon platform compounds with numerous pharmaceutical and chemical synthesis applications. AC and BD have two and three stereoisomers, respectively, while the application of the single isomer product in chemical synthesis is superior. AC and BD are glucose overflow metabolites produced by biological fermentation from a variety of microorganisms. However, the AC or BD produced by microorganisms using glucose is typically a mixture of various stereoisomers. This was discovered to be due to the simultaneous presence of multiple butanediol dehydrogenases (BDHs) in microorganisms, and AC and BD can be interconverted under BDH catalysis. In this paper, beginning with the synthesis pathways of microbial AC and BD, we review in detail the studies on the formation mechanisms of different stereoisomers of AC and BD, summarize the properties of different types of BDH that have been tabulated, and analyze the structural characteristics and affinities of different types of BDH by comparing them using literature and biological database data. Using microorganisms, recent research on the production of optically pure AC or BD was also reviewed. Limiting factors and possible solutions for chiral AC and BD production are discussed.

One-pot synthesis of fuel precursor from acetoin fermentation broth using ionic liquid-based salting-out extraction system.

BACKGROUND: The development of biofuels, especially liquid hydrocarbon fuels, has been widely concerned due to the depletion of fossil resources. In order to obtain fuel precursors, the reaction of C-C bond formation is usually carried out with biomass derived ketones/aldehydes as reactants. Acetoin and 2,3-butanediol are two platform chemicals, which are co-existed in fermentation broth and traditionally separated by distillation, and then acetoin could be use as C4 building block to prepare hydrocarbon fuels. In order to mitigate the process complexity, direct aldol condensation reaction of acetoin in fermentation broth was studied in this work. RESULTS: A one-pot process of product separation and acetoin derivative synthesis was proposed based on salting-out extraction (SOE). Aldol condensation reaction of acetoin and 5-methyl furfural in different SOE systems was compared, and the results showed that the synthesis of C10 fuel precursors and separation of C10 products and 2,3-butanediol from fermentation broth were achieved in one-pot with ethanolammonium butyrate (EOAB) and K2HPO4 as SOE reagents and catalysts. The SOE and reaction conditions such as the concentrations of EOAB and K2HPO4, reaction temperature and time were optimized. When the system was composed of 6 wt% EOAB-44 wt% K2HPO4 and the mixture was stirred for 6 h at 200 rpm, 40 â„ƒ, the yield of C10 products was 80.7%, and 95.5% 2,3-butanediol was distributed to the top EOAB-rich phase. The exploration of reaction mechanism showed that an imine intermediate was rapidly formed and the subsequent C10 product formation was the key step for aldol condensation reaction. CONCLUSIONS: With EOAB and K2HPO4 as SOE reagents and catalysts, one-pot synthesis of fuel precursor from acetoin fermentation broth was achieved without prior purification. A yield of 80.7% for C10 products was obtained which was accumulated at the interface of two aqueous-phase, and 95.5% 2,3-BD was distributed to the top EOAB-rich phase. This work provides a new integration process of product separation and derivative synthesis from fermentation broth based on ionic liquid SOE.