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.

Microbial interactions shape cheese flavour formation.

Cheese fermentation and flavour formation are the result of complex biochemical reactions driven by the activity of multiple microorganisms. Here, we studied the roles of microbial interactions in flavour formation in a year-long Cheddar cheese making process, using a commercial starter culture containing Streptococcus thermophilus and Lactococcus strains. By using an experimental strategy whereby certain strains were left out from the starter culture, we show that S. thermophilus has a crucial role in boosting Lactococcus growth and shaping flavour compound profile. Controlled milk fermentations with systematic exclusion of single Lactococcus strains, combined with genomics, genome-scale metabolic modelling, and metatranscriptomics, indicated that S. thermophilus proteolytic activity relieves nitrogen limitation for Lactococcus and boosts de novo nucleotide biosynthesis. While S. thermophilus had large contribution to the flavour profile, Lactococcus cremoris also played a role by limiting diacetyl and acetoin formation, which otherwise results in an off-flavour when in excess. This off-flavour control could be attributed to the metabolic re-routing of citrate by L. cremoris from diacetyl and acetoin towards α-ketoglutarate. Further, closely related Lactococcus lactis strains exhibited different interaction patterns with S. thermophilus, highlighting the significance of strain specificity in cheese making. Our results highlight the crucial roles of competitive and cooperative microbial interactions in shaping cheese flavour profile.

Efficient production of acetoin from lactate by engineered Escherichia coli whole-cell biocatalyst.

Acetoin, an important and high-value added bio-based platform chemical, has been widely applied in fields of foods, cosmetics, chemical synthesis, and agriculture. Lactate is a significant intermediate short-chain carboxylate in the anaerobic breakdown of carbohydrates that comprise ~ 18% and ~ 70% in municipal wastewaters and some food processing wastewaters, respectively. In this work, a series of engineered Escherichia coli strains were constructed for efficient production of acetoin from cheaper and abundant lactate through heterogenous co-expression of fusion protein (α-acetolactate synthetase and α-acetolactate decarboxylase), lactate dehydrogenase and NADH oxidase, and blocking acetate synthesis pathways. After optimization of whole-cell bioconversion conditions, the engineered strain BL-11 produced 251.97 mM (22.20 g/L) acetoin with a yield of 0.434 mol/mol in shake flasks. Moreover, a titer of 648.97mM (57.18 g/L) acetoin was obtained in 30 h with a yield of 0.484 mol/mol lactic acid in a 1-L bioreactor. To the best of our knowledge, this is the first report on the production of acetoin from renewable lactate through whole-cell bioconversion with both high titer and yield, which demonstrates the economy and efficiency of acetoin production from lactate. Key Points • The lactate dehydrogenases from different organisms were expressed, purified, and assayed. • It is the first time that acetoin was produced from lactate by whole-cell biocatalysis. • The highest titer of 57.18 g/L acetoin was obtained with high theoretical yield in a 1-L bioreactor.

Improving acetoin production through construction of a genome-scale metabolic model.

Acetoin was widely used in food, medicine, and other industries, because of its unique fragrance. Bacillus amyloliquefaciens was recognized as a safe strain and a promising acetoin producer in fermentation. However, due to the complexity of its metabolic network, it had not been fully utilized. Therefore, a genome-scale metabolic network model (iJYQ746) of B. amyloliquefaciens was constructed in this study, containing 746 genes, 1736 reactions, and 1611 metabolites. The results showed that Mg2+, Mn2+, and Fe2+ have inhibitory effects on acetoin. When the stirring speed was 400 rpm, the maximum titer was 49.8 g L-1. Minimization of metabolic adjustments (MOMA) was used to identify potential metabolic modification targets 2-oxoglutarate aminotransferase (serC, EC 2.6.1.52) and glucose-6-phosphate isomerase (pgi, EC 5.3.1.9). These targets could effectively accumulate acetoin by increasing pyruvate content, and the acetoin synthesis rate was increased by 610% and 10%, respectively. This provides a theoretical basis for metabolic engineering to reasonably transform B. amyloliquefaciens and produce acetoin.

Metabolic Engineering of Microorganisms to Produce Pyruvate and Derived Compounds.

Pyruvate is a hub of various endogenous metabolic pathways, including glycolysis, TCA cycle, amino acid, and fatty acid biosynthesis. It has also been used as a precursor for pyruvate-derived compounds such as acetoin, 2,3-butanediol (2,3-BD), butanol, butyrate, and L-alanine biosynthesis. Pyruvate and derivatives are widely utilized in food, pharmaceuticals, pesticides, feed additives, and bioenergy industries. However, compounds such as pyruvate, acetoin, and butanol are often chemically synthesized from fossil feedstocks, resulting in declining fossil fuels and increasing environmental pollution. Metabolic engineering is a powerful tool for producing eco-friendly chemicals from renewable biomass resources through microbial fermentation. Here, we review and systematically summarize recent advances in the biosynthesis pathways, regulatory mechanisms, and metabolic engineering strategies for pyruvate and derivatives. Furthermore, the establishment of sustainable industrial synthesis platforms based on alternative substrates and new tools to produce these compounds is elaborated. Finally, we discuss the potential difficulties in the current metabolic engineering of pyruvate and derivatives and promising strategies for constructing efficient producers.

Structural and enzymatic characterization of Bacillus subtilis R,R-2,3-butanediol dehydrogenase.

2,3-butanediol dehydrogenase (BDH, EC 1.1.1.76) also known as acetoin reductase (AR, EC 1.1.1.4) is the key enzyme converting acetoin (AC) into 2,3-butanediol (BD) and undertaking the irreversible conversion of diacetyl to acetoin in various microorganisms. The existence of three BDHs (R,R-, meso-, and S,S-BDH) product different BD isomers. Catalyzing mechanisms of meso- and S,S-BDH have been understood with the assistance of their X-ray crystal structures. However, the lack of structural data for R,R-BDH restricts the integral understanding of the catalytic mechanism of BDHs. In this study, we successfully crystallized and solved the X-ray crystal structure of Bacillus subtilis R,R-BDH. A zinc ion was found locating in the catalytic center and coordinated by Cys37, His70 and Glu152, helping to stabilize the chiral substrates observed in the predicted molecular docking model. The interaction patterns of different chiral substrates in the molecular docking model explained the react priority measured by the enzyme activity assay of R,R-BDH. Site-directed mutation experiments determined that the amino acids Cys37, Thr244, Ile268 and Lys340 are important in the catalytically active center. The structural information of R,R-BDH presented in this study accomplished the understanding of BDHs catalytic mechanism and more importantly provides useful guidance for the directional engineering of R,R-BDH to obtain high-purity monochiral BD and AC.

NADH dehydrogenases drive inward electron transfer in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a promising chassis organism for microbial electrosynthesis because it has a well-defined biochemical pathway (the Mtr pathway) that can connect extracellular electrodes to respiratory electron carriers inside the cell. We previously found that the Mtr pathway can be used to transfer electrons from a cathode to intracellular electron carriers and drive reduction reactions. In this work, we hypothesized that native NADH dehydrogenases form an essential link between the Mtr pathway and NADH in the cytoplasm. To test this hypothesis, we compared the ability of various mutant strains to accept electrons from a cathode and transfer them to an NADH-dependent reaction in the cytoplasm, reduction of acetoin to 2,3-butanediol. We found that deletion of genes encoding NADH dehydrogenases from the genome blocked electron transfer from a cathode to NADH in the cytoplasm, preventing the conversion of acetoin to 2,3-butanediol. However, electron transfer to fumarate was not blocked by the gene deletions, indicating that NADH dehydrogenase deletion specifically impacted NADH generation and did not cause a general defect in extracellular electron transfer. Proton motive force (PMF) is linked to the function of the NADH dehydrogenases. We added a protonophore to collapse PMF and observed that it blocked inward electron transfer to acetoin but not fumarate. Together these results indicate a link between the Mtr pathway and intracellular NADH. Future work to optimize microbial electrosynthesis in S. oneidensis MR-1 should focus on optimizing flux through NADH dehydrogenases.

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 PTSNtr 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.

Enzymes and pathways in microbial production of 2,3-butanediol and 3-acetoin isomers.

2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.

Characteristic Analysis of Soil-Isolated Bacillus velezensis HY-3479 and Its Antifungal Activity Against Phytopathogens.

During the investigation of beneficial agricultural microorganisms, a novel Bacillus strain was isolated. To isolate an effective microorganism that has antifungal activity, soil samples were collected from an agricultural field in the southern area of Pohang, Korea. One strain that had specificity on plant pathogens was analyzed. According to 16S rRNA sequencing, the isolated bacterium was identified as Bacillus velezensis and was designated as HY-3479. Few assays were taken to analyze the characteristics of the HY-3479 strain. In agar plate assay, HY-3479 showed antifungal effects on Colletotrichum acutatum, Cylindrocarpon destructans, Rhizoctonia solani, and Sclerotinia sclerotiorum. The strain also had various enzymatic activities including protease, amylase, and ß-1,3-glucanase, which were relatively higher than control strains. Metabolites study of strain HY-3479 was conducted by GC-MS analysis and the bacterium contained many plant growth promoters like 3-methyl-1-butanol, (R, R)-2,3-butanediol, acetoin, and benzoic acid which were not found in untreated TSB medium. In gene expression analysis, antifungal lipopeptide genes like srfc (surfactin) and ituD (iturin A) were highly produced in the HY-3479 strain compared to the control strain KCTC 13417. B. velezensis strain HY-3479 may be the candidate to be an effective microorganism in agriculture and become a beneficial biocontrol agent with plant growth-promoting activities.

Harnessing diversity of Lactococcus lactis from raw goat milk: Design of an indigenous starter for the production of Rocamadour, a French PDO cheese.

Twenty-four strains of Lactococcus lactis isolated from raw goat milk collected in the Rocamadour PDO area were analysed by MLST typing and phenotypic characterisation. The strains were combined to design an indigenous starter for the production of Rocamadour PDO cheese. The strains were divided into three classes based on their technological properties: acidifying and proteolytic strains in class I (12/24 strains), slightly acidifying and non-proteolytic strains in class II (2/24 strains), and non-acidifying and non-proteolytic strains in class III (10/24 strains). Interestingly, all but three strains (21/24) produced diacetyl/acetoin despite not having citrate metabolism genes, as would classically be expected for the production of these aroma compounds. Three strains (EIP07A, EIP13D, and EIP20B) were selected for the indigenous starter based on the following inclusion/exclusion criteria: (i) no negative interactions between included strains, (ii) ability to metabolize lactose and at least one strain with the prtP gene and/or capable of producing diacetyl/acetoin, and (iii) selected strains derived from different farms to maximise genetic and phenotypic diversity. Despite consisting exclusively of L. lactis strains, the designed indigenous starter allowed reproducible cheese production with performances similar to those obtained with an industrial starter and with the sensory qualities expected of Rocamadour PDO cheese.

A host-vector toolbox for improved secretory protein overproduction in Bacillus subtilis.

Target proteins in biotechnological applications are highly diverse. Therefore, versatile flexible expression systems for their functional overproduction are required. In order to find the right heterologous gene expression strategy, suitable host-vector systems, which combine different genetic circuits, are useful. In this study, we designed a novel Bacillus subtilis expression toolbox, which allows the overproduction and secretion of potentially toxic enzymes. This toolbox comprises a set of 60 expression vectors, which combine two promoter variants, four strong secretion signals, a translation-enhancing downstream box, and three plasmid backbones. This B. subtilis toolbox is based on a tailor-made, clean deletion mutant strain, which is protease and sporulation deficient and exhibits reduced autolysis and secondary metabolism. The appropriateness of this alternative expression platform was tested for the overproduction of two difficult-to-produce eukaryotic model proteins. These included the sulfhydryl oxidase Sox from Saccharomyces cerevisiae, which forms reactive hydrogen peroxide and undesired cross-linking of functional proteins, and the human interleukin-1ß, a pro-inflammatory cytokine. For the best performing Sox and interleukin, overproducing and secreting variants of these new B. subtilis toolbox fermentation strategies were developed and tested. This study demonstrates the suitability of the prokaryotic B. subtilis host-vector system for the extracellular production of two eukaryotic proteins with biotechnological relevance. KEY POINTS: • Construction of a versatile Bacillus subtilis gene expression toolbox. • Verification of the toolbox by the secretory overproduction of two difficult-to-express proteins. • Fermentation strategy for an acetoin-controlled overproduction of heterologous proteins.

Metabolic engineering of Corynebacterium glutamicum for efficient production of optically pure (2R,3R)-2,3-butanediol.

BACKGROUND: 2,3-butanediol is an important platform compound which has a wide range of applications, involving in medicine, chemical industry, food and other fields. Especially the optically pure (2R,3R)-2,3-butanediol can be employed as an antifreeze agent and as the precursor for producing chiral compounds. However, some (2R,3R)-2,3-butanediol overproducing strains are pathogenic such as Enterobacter cloacae and Klebsiella oxytoca. RESULTS: In this study, a (3R)-acetoin overproducing C. glutamicum strain, CGS9, was engineered to produce optically pure (2R,3R)-2,3-butanediol efficiently. Firstly, the gene bdhA from B. subtilis 168 was integrated into strain CGS9 and its expression level was further enhanced by using a strong promoter Psod and ribosome binding site (RBS) with high translation initiation rate, and the (2R,3R)-2,3-butanediol titer of the resulting strain was increased by 33.9%. Then the transhydrogenase gene udhA from E. coli was expressed to provide more NADH for 2,3-butanediol synthesis, which reduced the accumulation of the main byproduct acetoin by 57.2%. Next, a mutant atpG was integrated into strain CGK3, which increased the glucose consumption rate by 10.5% and the 2,3-butanediol productivity by 10.9% in shake-flask fermentation. Through fermentation engineering, the most promising strain CGK4 produced a titer of 144.9 g/L (2R,3R)-2,3-butanediol with a yield of 0.429 g/g glucose and a productivity of 1.10 g/L/h in fed-batch fermentation. The optical purity of the resulting (2R,3R)-2,3-butanediol surpassed 98%. CONCLUSIONS: To the best of our knowledge, this is the highest titer of optically pure (2R,3R)-2,3-butanediol achieved by GRAS strains, and the result has demonstrated that C. glutamicum is a competitive candidate for (2R,3R)-2,3-butanediol production.

Food web and metabolic interactions of the lung inhabitants Streptococcus pneumoniae and Pseudomonas aeruginosa.

Bacteria that successfully adapt to different substrates and environmental niches within the lung and overcome the immune defence can cause serious lung infections. Such infections are generally complex, and recognized as polymicrobial in nature. Both Pseudomonas aeruginosa and Streptococcus pneumoniae can cause chronic lung infections and were both detected in cystic fibrosis (CF) lung at different stages. In this study, single and dual species cultures of Pseudomonas aeruginosa and Streptococcus pneumoniae were studied under well-controlled planktonic growth conditions. Under pH-controlled conditions, both species apparently benefited from the presence of the other. In co-culture with P. aeruginosa, S. pneumoniae grew efficiently under aerobic conditions, whereas in pure S. pneumoniae culture, growth inhibition occurred in bioreactors with dissolved oxygen concentrations above the microaerobic range. Lactic acid and acetoin that are produced by S. pneumoniae were efficiently utilized by P. aeruginosa. In pH-uncontrolled co-cultures, the low pH triggered by S. pneumoniae assimilation of glucose and lactic acid production negatively affected the growth of both strains. Nevertheless, ammonia production improved significantly, and P. aeruginosa growth dominated at later growth stages. This study revealed unreported metabolic interactions of two important pathogenic microorganisms and shed new lights into pathophysiology of bacterial lung infection.

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens.

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.

Biotechnological production of chiral acetoin.

Acetoin (AC) is an important platform bulk chemical with versatile applications. It exists in two stereoisomeric forms: (3R)-AC and (3S)-AC. Both stereoisomers could be potentially applied in the pharmaceutical industry, agriculture, and in optically active α-hydroxyketone derivative synthesis. Chiral AC production has recently become a new research focus in biotechnology. Fermentative and biocatalytic routes that can produce (3R)-AC or (3S)-AC with high optical purity have been developed over the past several years. In this review we summarize recent advances in strain screening, metabolic engineering, and biocatalytic system construction aimed at improving the production of chiral AC. Limiting factors and possible solutions for chiral AC production are discussed.

Prospects on bio-based 2,3-butanediol and acetoin production: Recent progress and advances.

The bio-based platform chemicals 2,3-butanediol (BDO) and acetoin have various applications in chemical, cosmetics, food, agriculture, and pharmaceutical industries, whereas the derivatives of BDO could be used as fuel additives, polymer and synthetic rubber production. This review summarizes the novel technological developments in adapting genetic and metabolic engineering strategies for selection and construction of chassis strains for BDO and acetoin production. The valorization of renewable feedstocks and bioprocess development for the upstream and downstream stages of bio-based BDO and acetoin production are discussed. The techno-economic aspects evaluating the viability and industrial potential of bio-based BDO production are presented. The commercialization of bio-based BDO and acetoin production requires the utilization of crude renewable resources, the chassis strains with high fermentation production efficiencies and development of sustainable purification or conversion technologies.

High and Economical Nattokinase Production with Acetoin as a Useful Byproduct from Soybean Milk and Glucose.

Nattokinase (NK) is a potent fibrinolytic enzyme with wide pharmaceutical and nutraceutical applications. Safe and high NK-yielding strains are urgently needed. In this study, the best strain NDF was isolated from one of the 11 natto samples and then identified as Bacillus subtilis. The effects of carbon and nitrogen sources on NK production were investigated, and glucose and soybean milk were finally selected as the optimal carbon and nitrogen sources, respectively. Acetoin, a valuable compound with versatile usages, was detected as the main byproduct of carbon overflow. In a 6-L fermenter, NK and acetoin reached their peak concentrations simultaneously (10,220 IU/mL and 25.9 g/L, respectively) at 25 h in a culture medium containing 180 g/L of soybean milk and 105 g/L of glucose. The NK product was verified by sequencing of the aprN gene and SDS-PAGE analysis. Only very limited kinds of proteins were found in the supernatant of the fermentation broth, and NK was one of the main bands. This study has developed an economical and high NK production method with acetoin as a useful byproduct.

Effects of pyruvate decarboxylase (pdc1, pdc5) gene knockout on the production of metabolites in two haploid Saccharomyces cerevisiae strains.

Saccharomyces cerevisiae has good reproductive ability in both haploid and diploid forms, a pyruvate decarboxylase plays an important role in S. cerevisiae cell metabolism. In this study, pdc1 and pdc5 double knockout strains of S. cerevisiae H14-02 (MATa type) and S. cerevisiae H5-02 (MATα type) were obtained by the Cre/loxP technique. The effects of the deletion of pdc1 and pdc5 on the metabolites of the two haploid S. cerevisiae strains were consistent. In S. cerevisiae H14-02, the ethanol conversion decreased by 30.19%, the conversion of glycerol increased by 40.005%, the concentration of acetic acid decreased by 43.54%, the concentration of acetoin increased by 12.79 times, and the activity of pyruvate decarboxylase decreased by 40.91% compared to those in the original H14 strain. The original S. cerevisiae haploid strain H14 produced a small amount of acetoin but produced very little 2,3-butanediol. However, S. cerevisiae H14-02 produced 1.420 ± 0.063 g/L 2,3-BD. This study not only provides strain selection for obtaining haploid strains with a high yield of 2,3-BD but also lays a foundation for haploid S. cerevisiae to be used as a new tool for genetic research and breeding programs.

Advances in biological production of acetoin: a comprehensive overview.

Acetoin, a high-value-added bio-based platform chemical, is widely used in foods, cosmetics, agriculture, and the chemical industry. It is an important precursor for the synthesis of: 2,3-butanediol, liquid hydrocarbon fuels and heterocyclic compounds. Since the fossil resources are becoming increasingly scarce, biological production of acetoin has received increasing attention as an alternative to chemical synthesis. Although there are excellent reviews on the: application, catabolism and fermentative production of acetoin, little attention has been paid to acetoin production via: electrode-assisted fermentation, whole-cell biocatalysis, and in vitro/cell-free biocatalysis. In this review, acetoin biosynthesis pathways and relevant key enzymes are firstly reviewed. In addition, various strategies for biological acetoin production are summarized including: cell-free biocatalysis, whole-cell biocatalysis, microbial fermentation, and electrode-assisted fermentation. The advantages and disadvantages of the different approaches are discussed and weighed, illustrating the increasing progress toward economical, green and efficient production of acetoin. Additionally, recent advances in acetoin extraction and recovery in downstream processing are also briefly reviewed. Moreover, the current issues and future prospects of diverse strategies for biological acetoin production are discussed, with the hope of realizing the promises of industrial acetoin biomanufacturing in the near future.