•A total of 510 ester compounds have been identified in traditional fermented baijiu.•Nine ethyl esters make important contributions to the baijiu flavor.•In the fermentation process of strong-aroma ...baijiu, 508 species have been identified.•Microbial fatty acid and amino acid metabolism contribute greatly to ester synthesis.•The metabolic processes of a minimum set of microbes help to stabilize baijiu flavor.
Chinese traditional fermented baijiu is a famous alcoholic beverage with unique flavor. Despite its consumption for millennia, the flavor mystery behind baijiu is still unclear. Studies indicate that esters are the most important flavor substances, and bring health benefits. However, the aroma contribution and formation mechanism of esters still need to be clarified to reveal the flavor profile of baijiu. This review systematically summarizes all the 510 esters and finds 9 ethyl esters contribute greatly to the flavor of baijiu. The 508 different microbial species that have been identified affect the synthesis of esters through fatty acid and amino acid metabolism. The determination of minimum functional microbial groups and the analysis of their metabolic characteristics are crucial to reveal the mechanism of formation of baijiu flavor, and ensure the reproducible formation of flavor substances.
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•Lactobacillus and Issatchenkia are dominant genera in the fermentation process.•Ester compounds and p-cresol contribute to different flavors of fermented products.•Correlation ...analysis indicates the relationships of flavor chemicals with microbes.•Bacterial and fungal synthetic pathways of p-cresol are firstly proposed.
Sauce-flavor baijiu is a representative of Chinese traditional fermented baijiu using grains as the raw materials through the co-fermentation of microorganisms. The whole manufacturing process includes 7 times of distillation and generates 7 kinds of base baijius. The final product is a mixture of the 7 kinds of base baijius. Thus the base baijius greatly affect the quality of the final product. The quality of the base baijiu obtained by the sixth distillation is obviously poorer than that of the fifth one. However, the reason is still unclear and limits the quality control of baijiu fermentation. In this study, the flavor substances and microbiota in the up, middle and bottom layers of fermented grains in the fifth and sixth rounds were compared. Some flavor esters showed obviously decreased concentrations in the sixth round, including ethyl benzoneacetic acid, ethyl hexanoic acid, ethyl dodecanoic acid, diethyl butanedioic acid, and ethyl 2-hydroxyl-propanoic acid. Meanwhile, an off-flavor p-cresol was detected in the sixth round. Correlation analysis of flavor chemicals and microbiota indicated that fungi in the fifth round played an important role for ester synthesis. Some bacterial and fungal species were both positively correlated with p-cresol synthesis, and the related p-cresol metabolic pathways were proposed for the first time. These results revealed flavor divergences of fermented grains between the fifth and sixth rounds, and will ultimately help to improve baijiu quality.
Petro-plastic wastes cause serious environmental contamination that require effective solutions. Developing alternatives to petro-plastics and exploring feasible degrading methods are two solving ...routes. Bio-plastics like polyhydroxyalkanoates (PHAs), polylactic acid (PLA), polycaprolactone (PCL), poly (butylene succinate) (PBS), poly (ethylene furanoate) s (PEFs) and poly (ethylene succinate) (PES) have emerged as promising alternatives. Meanwhile, biodegradation plays important roles in recycling plastics (e.g., bio-plastics PHAs, PLA, PCL, PBS, PEFs and PES) and petro-plastics poly (ethylene terephthalate) (PET) and plasticizers in plastics (e.g., phthalate esters, PAEs). All these bio- and petro-materials show structure similarity by connecting monomers through ester bond. Thus, this review focused on bio-plastics and summarized the sequences and structures of the microbial enzymes catalyzing ester-bond synthesis. Most of these synthetic enzymes belonged to α/β-hydrolases with conserved serine catalytic active site and catalyzed the polymerization of monomers by forming ester bond. For enzymatic plastic degradation, enzymes about PHAs, PBS, PCL, PEFs, PES and PET were discussed, and most of the enzymes also belonged to the α/β hydrolases with a catalytic active residue serine, and nucleophilically attacked the ester bond of substrate to generate the cleavage of plastic backbone. Enzymes hydrolysis of the representative plasticizer PAEs were divided into three types (I, II, and III). Type I enzymes hydrolyzed only one ester-bond of PAEs, type II enzymes catalyzed the ester-bond of mono-ester phthalates, and type III enzymes hydrolyzed di-ester bonds of PAEs. Divergences of catalytic mechanisms among these enzymes were still unclear. This review provided references for producing bio-plastics, and degrading or recycling of bio- and petro-plastics from an enzymatic point of view.
The production of biofuels by recombinant Escherichia coli is restricted by the toxicity of the products. 2,3-Butanediol (2,3-BD), a platform and fuel bio-chemical with low toxicity to microbes, ...could be a promising alternative for biofuel production. However, the yield and productivity of 2,3-BD produced by recombinant E. coli strains are not sufficient for industrial scale fermentation. In this work, the production of 2,3-BD by recombinant E. coli strains was optimized by applying a systematic approach. 2,3-BD biosynthesis gene clusters were cloned from several native 2,3-BD producers, including Bacillus subtilis, Bacillus licheniformis, Klebsiella pneumoniae, Serratia marcescens, and Enterobacter cloacae, inserted into the expression vector pET28a, and compared for 2,3-BD synthesis. The recombinant strain E. coli BL21/pETPT7-EcABC, carrying the 2,3-BD pathway gene cluster from Enterobacter cloacae, showed the best ability to synthesize 2,3-BD. Thereafter, expression of the most efficient gene cluster was optimized by using different promoters, including PT7, Ptac, Pc, and Pabc. E. coli BL21/pET-RABC with Pabc as promoter was superior in 2,3-BD synthesis. On the basis of the results of biomass and extracellular metabolite profiling analyses, fermentation conditions, including pH, agitation speed, and aeration rate, were optimized for the efficient production of 2,3-BD. After fed-batch fermentation under the optimized conditions, 73.8g/L of 2,3-BD was produced by using E. coli BL21/pET-RABC within 62h. The values of both yield and productivity of 2,3-BD obtained with the optimized biological system are the highest ever achieved with an engineered E. coli strain. In addition to the 2,3-BD production, the systematic approach might also be used in the production of other important chemicals through recombinant E. coli strains.
•2,3-BD was selected as the target biofuel produced through recombinant E. coli.•Gene clusters in several native strains were compared for 2,3-BD production.•Expression of the most efficient gene cluster was optimized using different promoters.•Fermentative conditions were optimized by extracellular metabolite profile analysis.•2,3-BD production was highly acquired by systematically engineered E. coli.
Biotechnological production of biofuels is restricted by toxicity of the products such as ethanol and butanol. As its low toxicity to microbes, 2,3-butanediol (2,3-BD), a fuel and platform ...bio-chemical, could be a promising alternative for biofuel production from renewable bioresources. In addition, no bacterial strains have been reported to produce enantiopure 2,3-BD using lignocellulosic hydrolysates. In this study, Enterobacter cloacae strain SDM was systematically and metabolically engineered to construct an efficient biocatalyst for production of the fuel and enantiopure bio-chemical—(2R,3R)-2,3-BD. First, the various (2R,3R)-2,3-BD dehydrogenase encoding genes were expressed in a meso-2,3-BD dehydrogenase encoding gene disrupted E. cloacae strain under native promoter Pb of the 2,3-BD biosynthetic gene cluster of E. cloacae. Then, carbon catabolite repression was eliminated via inactivation of the glucose transporter encoding gene ptsG and overexpression of a galactose permease encoding gene galP. The resultant strain could utilize glucose and xylose simultaneously. To improve the efficiency of (2R,3R)-2,3-BD production, the byproduct-producing genes (ldh and frdA) were knocked out, thereby enhancing the yield of (2R,3R)-2,3-BD by 16.5% in 500-mL Erlenmeyer flasks. By using fed-batch fermentation in a 5-L bioreactor, 152.0g/L (2R,3R)-2,3-BD (purity>97.5%) was produced within 44h with a specific productivity of 3.5g/Lh and a yield of 97.7% from a mixture of glucose and xylose, two major carbohydrate components in lignocellulosic hydrolysates. In addition, when a lignocellulosic hydrolysate was used as the substrate, 119.4g/L (2R,3R)-2,3-BD (purity>96.0%) was produced within 51h with a productivity of 2.3g/Lh and a yield of 95.0%. These results show that the highest records have been acquired for enantiopure (2R,3R)-2,3-BD production by a native or engineered strain from biomass-derived sugars. In addition to producing the 2,3-BD, our systematic approach might also be used in the production of other important chemicals by using lignocellulose-derived sugars.
•(2R,3R)-2,3-BD producers were constructed from a native 2,3-BD producer.•The engineered strain enabled efficient co-utilization of glucose and xylose for BD production.•The highest records were acquired for the BD production by an engineered strain.
Fatty acid ethyl esters are important flavor chemicals in strong-flavor baijiu. Microorganisms are the main contributors to ester synthesis during baijiu manufacture. However, the ester synthesis was ...unstable between batches. This was owing to a limited knowledge of the mechanisms for ester synthesis by microorganisms. In this work, a fatty acid ethyl ester synthesizing
strain CGMCC (China General Microbiological Culture Collection) 3.4309 was identified. The conversion ratios of ethyl valerate, ethyl caproate, ethyl caprylate, and ethyl caprate were 7.87, 29.20, 94.80, and 85.20%, respectively, under the optimized conditions. A comparison of transcriptomes under the initial and optimized ester synthetic conditions indicated that 23 genes were upregulated in transcription level and encoded enzymes with potential abilities for ester synthesis. Eleven of the enzymes were expressed, and three of them, numbered An605, An1097, and An3131, showed the ability to catalyze fatty acid ethyl ester synthesis under aqueous phase, with capric acid as the preferred substrate. The possible enzymatic catalytic mechanism was proposed based on homology modeling and molecular docking. This study reported for the first time that
showed the ability to efficiently catalyze the synthesis of short- and medium-chain fatty acid ethyl esters in aqueous phase, identified the key enzymes, and analyzed the basic enzymatic properties. This is helpful to promote the application of related microorganisms and enzyme resources in the baijiu industry.
The solid-state fermentation of Baijiu is complicated by the co-fermentation of many microorganisms. The instability of the composition and abundance of the microorganisms in the fermentation process ...leads to fluctuations of product quality, which is one of the bottleneck problems faced by the Strong-flavor Baijiu industry. In this study, we established a combination of functional microorganisms for the stable fermentation of the main flavor compounds of Baijiu, including medium and long-chain fatty acid ethyl esters such as hexanoic acid, ethyl ester; butanoic acid, ethyl ester; octanoic acid, ethyl ester; acetic acid, ethyl ester; 9,12-octadecadienoic acid, ethyl ester; and decanoic acid, ethyl ester in the fermented grains. Our study investigated the effects of microbial combinations on the fermentation from three aspects: microbial composition, microbial interactions, and microbial association with flavor compounds. The results showed that the added functional microorganisms (
,
,
,
, and
) became the dominant species in the fermentation system and formed positive interactions with other microorganisms, while the negative interactions between microorganisms were significantly reduced in the fermentation systems that contained both Daqu and functional microorganisms. The redundancy analysis showed that the functional microorganisms (
,
,
,
,
,
, and
) showed strong positive correlations with the main flavor compounds (hexanoic acid, ethyl ester; lactic acid, ethyl ester; butanoic acid, ethyl ester; acetic acid, ethyl ester; and octanoic acid, ethyl ester). These results indicated that it was feasible to produce Baijiu with a functional microbial combination, and that this could promote stable Baijiu production.
Fatty acid ethyl esters are important flavor chemicals in strong-flavor Baijiu.
YJX-8 is recognized as an important microorganism for ester synthesis in the fermentation process. Enzyme LIP05 from ...YJX-8 can efficiently catalyze the synthesis of fatty acid ethyl esters under aqueous phase, but the key catalytic sites affecting esterification were unclear. The present work combined homology modeling, molecular dynamics simulation, molecular docking and site-directed mutation to analyze the catalytic mechanism of LIP05. Protein structure modeling indicated LIP05 belonged to α/β fold hydrolase, contained a lid domain and a core catalytic pocket with conserved catalytic triad Ser150-His215-Asp202, and the oxyanion hole composed of Gly73 and Thr74. Ile30 and Leu37 of the lid domain were found to affect substrate specificity. The π-bond stacking between Tyr116 and Tyr149 played an important role in stabilizing the catalytic active center of LIP05. Tyr116 and Ile204 determined the substrate spectrum by composing the substrate-entrance channel. Residues Leu83, Ile204, Ile211 and Leu216 were involved in forming the hydrophobic substrate-binding pocket through steric hindrance and hydrophobic interaction. The catalytic mechanism for esterification in aqueous phase of LIP05 was proposed and provided a reference for clarifying the synthesis of fatty acid ethyl esters during the fermentation process of strong-flavor Baijiu.
Sauce-flavor Baijiu is representative of solid-state fermented Baijiu. It is significant to deeply reveal the dynamic changes of microorganisms in the manufacturing process and their impact on the ...formation of flavor chemicals correlated with the quality of Baijiu. Sauce-flavor Baijiu manufacturing process can be divided into seven rounds, from which seven kinds of base Baijius are produced. The quality of base Baijiu in the third round is significantly better than that in the first and second rounds, but the mystery behind the phenomenon has not yet been revealed. Based on high-throughput sequencing and flavor analysis of fermented grains, and correlation analysis, the concentrations of flavor chemicals in the third round of fermented grains were enhanced, including esters hexanoic acid, ethyl ester; octanoic acid, ethyl ester; decanoic acid, ethyl ester; dodecanoic acid, ethyl ester; phenylacetic acid, ethyl ester; 3-(methylthio)-propionic acid ethyl ester; acetic acid, phenylethyl ester; hexanoic acid, butyl ester, and other flavor chemicals closely related to the flavor of sauce-flavor Baijiu, such as tetramethylpyrazine. The changes in flavor chemicals should be an important reason for the quality improvement of the third round of base Baijiu. Correlation analysis showed that ester synthesis was promoted by the bacteria genus
and many low abundances of fungal genera, and these low abundances of fungal genera also had important contributions to the production of tetramethylpyrazine. Meanwhile, the degrading metabolic pathway of tetramethylpyrazine was investigated, and the possible microorganisms were correlated. These results clarified the base Baijiu quality improvement of the third round and helped to provide a theoretical basis for improving base Baijiu quality.
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