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Lactic acid bacteria are commonly used in plant-based fermentation to reduce off-flavor and improve sensory characteristics. However, there have been few studies on Latilactobacillus sakei for plant-based yogurt fermentation and, particularly, its metabolic features at the genomic level remain unclear. This study aims to analyze the fermentation characteristics of the L. sakei DCF0720 strain and compare genetics and metabolic relations. For this, DCF0720 was used to ferment the black soybean milk and conduct the physicochemical analysis and sensory test. The genomic and metabolic analyses were performed by complete genome sequencing and 500 MHz 1 H NMR, respectively. As a result, DCF0720 exhibited enhanced fermentation performance and sensory evaluations at 37 °C compared to 30 °C, which is generally recognized as the optimal growth temperature for most L. sakei strains. It also produced flavor enhancing volatile compounds such as acetoin and hydroxyacetone, possessing all three key genes for acetoin biosynthesis. DCF0720 lacks 2,3-butanediol dehydrogenase, which leads to the inhibition of acetoin production. DCF0720 possesses a complete pathway to utilize primary black soybean carbon sources such as sucrose, raffinose, and stachyose. DCF0720 also possesses genes for the GH28 family, including the key enzymes in the hydrolysis of pectin substances, which means eliminating the main soybean nonstarch polysaccharides. This study demonstrates that DCF0720 is a suitable starter for plant-based yogurt fermentation, providing a better understanding of fermentation conditions with genetic and metabolic features for black soybean yogurt. Various carbon source utilization abilities with depth metabolic pathway analysis provide that DCF0720 can be employed to develop enhanced starter cultures for black soybean yogurt and diverse plant-based yogurts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ropy bread spoilage caused by aerobic spore-forming bacteria (ASF) is characterized by discoloration, sticky and stringy crumb degradation, and a fruity odor due to the release of volatile organic compounds (VOCs). Previous studies employing model experiments have demonstrated strain-specific spoilage potential. However, to gain a deeper understanding, it is essential to study rope spoilage within baked bread. This study aimed to objectively evaluate the effect of different ASF strains on bread quality. 82 strains were subjected to a comprehensive evaluation, including proteotypic and genotypic fingerprinting, and 13 were selected for further analysis, where ASF endospore-spiked dough samples were baked and incubated. Physical changes in crumb texture, discoloration, and VOC production were quantified using texture analysis, color measurements, and headspace solid-phase microextraction gas chromatography-mass spectrometry. The high intraspecies diversity was reflected in strain-specific variations in rope spoilage profiles. A significant reduction in crumb firmness and increased discoloration, as well as the development of high levels of relative acetoin, were associated with rope spoilage. While color measurements could not detect early-stage spoilage, changes in crumb texture and VOC development were detectable 24 h after baking. Even low levels of contamination (10 1  spores/g) could cause potential spoilage detectable by texture analysis, although not visible to the naked eye. This study provides an unbiased framework for future research and may help develop early detection tools for improved bread spoilage management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bio-refining lignocellulosic resource offers a renewable and sustainable approach for producing biofuels and biochemicals. However, the conversion efficiency of lignocellulosic resource is still challenging due to the intrinsic inefficiency in co-utilization of xylose and glucose. In this study, the industrial bacterium Bacillus licheniformis was engineered for biorefining lignocellulosic resource to produce acetoin. First, adaptive evolution was conducted to improve acetoin tolerance, leading to a 19.6 % increase in acetoin production. Then, ARTP mutagenesis and 60 Co-γ irradiation was carried out to enhance the production of acetoin, obtaining 73.0 g/L acetoin from glucose. Further, xylose uptake and xylose utilization pathway were rewired to facilitate the co-utilization of xylose and glucose, enabling the production of 60.6 g/L acetoin from glucose and xylose mixtures. Finally, this efficient cell factory was utilized for acetoin production from lignocellulosic hydrolysates with the highest titer of 68.3 g/L in fed-batch fermentation. This strategy described here holds great applied potential in the biorefinery of lignocellulose for the efficient synthesis of high-value chemicals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Some microbial volatile organic compounds (mVOCs) can act as antagonistic weapons against plant pathogens, but little information is available on the contribution of individual mVOC to biocontrol and how they interact with plant pathogens. In this study, the Bacillus subtilis strain N-18 isolated from the rhizosphere of healthy plants grown in areas where Fusarium crown and root rot (FCRR) of tomato occurs could reduce the 30% of the incidence of FCRR. Moreover, the volatile organic compounds (VOCs) produced by N-18 had inhibitory effects on Fusarium oxysporum f. sp. radicis-lycopersici (FORL). The identification of VOCs of N-18 was analyzed by the solid-phase microextraction coupled to gas chromatography-mass spectrometry. Meanwhile, we conducted sensitivity tests with these potential active ingredients and found that the volatile substances acetoin and 2-heptanol can reduce the 41.33% and 35% of the incidence of FCRR in tomato plants. In addition, the potential target protein of acetoin, found in the cheminformatics and bioinformatics database, was F. oxysporum of hypothetical protein AU210_012600 (FUSOX). Molecular docking results further predicted that acetoin interacts with FUSOX protein. These results reveal the VOCs of N-18 and their active ingredients in response to FORL and provide a basis for further research on regulating and controlling FCRR., (© 2022 Wiley Periodicals LLC.)

Traditional cheesemaking processes often involve backslopping practice. However, over successive inoculations, acidification deficiencies may arise. In such cases, adding a starter is recommended to restore the ecosystem stability. This study examines the impact of an autochthonous starter composed of three Lactococcus lactis strains on a raw goat milk microbial community during their evolution. Bacterial composition and technological features (acidification and aroma) were analyzed during communities' evolution over 800 generations. 16S rRNA gene metabarcoding showed that Lactococcus lactis strains predominated. The raw goat milk community acidification capacities varied early in the evolution and then remained stable. Adding the L. lactis starter to this community stabilized the ecosystem from the beginning of the evolution. The acetoin production was associated with the starter presence, consistent with the establishment of the diacetylatis biovar strain from the starter in the raw goat milk community throughout the evolution. Increased or decreased production of some volatile organic compounds when the starter was added revealed a specific aroma footprint due to interactions between the two communities. This study showed that adding a starter could help to achieve the maximum acidification rate from the early inoculation cycles and could significantly modify the aroma profile during long-term backslopping., Competing Interests: Declaration of competing interest The authors of the manuscript “Interaction between a Lactococcus lactis autochthonous starter and a raw goat milk microbial community during long-term backslopping” submitted at Food Microbiol. have no conflict of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plasmid-mediated antibiotic-free fermentation holds significant industrial potential. However, the requirements for host elements and energy during plasmid inheritance often cause cell burden, leading to plasmid loss and reduced production. The stable maintenance of plasmids is primarily achieved through a complex mechanism, making it challenging to rationally design plasmid-stabilizing strains and characterize the associated genetic factors. In this study, we introduced a fluorescence-based high-throughput method and successfully screened plasmid-stabilizing strains from the genomic fragment-deletion strains of Escherichia coli MG1655 and Bacillus subtilis 168. The application of Ec Δ50 in antibiotic-free fermentation increased the alanine titer 2.9 times. The enhanced plasmid stability in Ec Δ50 was attributed to the coordinated deletion of genes involved in plasmid segregation and replication control, leading to improved plasmid maintenance and increased plasmid copy number. The increased plasmid stability of Bs Δ 44 was due to the deletion of the phage SPP1 surface receptor gene yueB , resulting in minimized sporulation, improved plasmid segregational stability and host adaptation. Antibiotic-free fermentation results showed that strain Bs Δ yueB exhibited a 61.99% higher acetoin titer compared to strain Bs 168, reaching 3.96 g/L. When used for the fermentation of the downstream product, 2,3-butanediol, strain Bs Δ yueB achieved an 80.63% higher titer than Bs 168, reaching 14.94 g/L using rich carbon and nitrogen feedstocks. Overall, our work provided a plasmid-stabilizing chassis for E. coli and B. subtilis , highlighting their potential for antibiotic-free fermentation of valuable products and metabolic engineering applications.

Upgrading biomass-derived bioethanol to higher-order alcohols using conventional biotechnological approaches is challenging. Herein, a novel, magnetic metal-organic-framework-based cofactor regeneration system was developed using ethanol dehydrogenase (EtDH:D46G), NADH oxidase (NOX), formolase (FLS:L482S), and nicotinamide adenine dinucleotide (NAD + ) for converting rice straw-derived bioethanol to acetoin. A magnetic zeolitic imidazolate framework-8@Fe 3 O 4 /NAD + (ZIF-8@Fe 3 O 4 /NAD + ) regeneration system for cell-free cascade reactions was introduced and used to encapsulate EtDH:D46G, NOX, and FLS:L482S (ENF). ZIF-8@Fe 3 O 4 /NAD + ENF created an efficient microenvironment for three-step enzyme cascades. Under the optimized conditions, the yield of acetoin from 100 mM bioethanol using ZIF-8@Fe 3 O 4 /NAD + ENF was 90.4 %. The regeneration system showed 97.1 % thermostability at 50 °C. The free enzymes retained only 16.3 % residual conversion, compared with 91.2 % for ZIF-8@Fe 3 O 4 /NAD + ENF after ten cycles. The magnetic metal-organic-framework-based cofactor regeneration system is suitable for enzymatic cascade biotransformations and can be extended to other cascade systems for potential biotechnological applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare no competing interests. The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The synergistic fermentation of milk by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus is one of the key factors that determines the quality of yogurt. In this study, the mechanism whereby yogurt flavor compounds are produced by a mixture of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B were investigated by examining the flavor production, growth, and gene transcription of these strains. The results showed that yogurt produced by a 10:1 mixture of the aforementioned strains had the highest abundance of acetoin, whereas yogurt produced by a 1:1 mixture had the highest abundance of diacetyl and acetaldehyde. In addition, the growth of S. thermophilus SIT-20.S was enhanced in the 10:1 mixture. Transcriptomic analysis revealed differentially expressed genes in the flavor-compound-related pathways of S. thermophilus SIT-20.S and L. delbrueckii ssp. bulgaricus SIT-17.B in yogurts produced by 10:1 and 1:1 mixtures compared with those produced by either strain alone. Mixed fermentations regulated the expression of genes related to glycolysis, resulting in an increase of pyruvate, which is an important precursor for diacetyl and acetoin synthesis. The gene encoding the acetoin reductase (SIT-20S_orf01454) was decreased in S. thermophilus SIT-20.S, which ensured the accumulation of acetoin. In addition, the gene encoding the acetaldehyde dehydrogenase (SIT-20S_orf00949) was upregulated in S. thermophilus SIT-20.S, and the expression of alcohol dehydrogenase (SIT-20S_orf01479; SIT-17B_orf00943) was downregulated in both strains, maintaining the abundance of acetaldehyde. In addition, the gene encoding the NADH oxidase (SIT-17B_orf00860) in L. delbrueckii ssp. bulgaricus SIT-17.B were upregulated, which promoted the accumulation of diacetyl and acetoin. Overall, we characterized the mechanism by which S. thermophilus and L. delbrueckii ssp. bulgaricus synergistically generated yogurt flavor compounds during their production of yogurt and highlighted the importance of appropriate proportions of fermentation starters for improving the flavor of yogurts., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

BACKGROUND This study aimed to detect the volatile organic compound (VOC), 3-hydroxy-2-butanone (acetoin) using gas chromatography-ion mobility spectrometry (GC-IMS) in antimicrobial-resistant Klebsiella pneumoniae (K. pneumoniae) carbapenemase (KPC)-producing bacteria. MATERIAL AND METHODS Using stromal fluid of blood culture bottles (BacT/ALERT® SA) as the medium, 3-hydroxy-2-butanone (acetoin) released by K. pneumoniae during growth was detected using GC-IMS. The impact of imipenem (IPM) and carbapenemase inhibitors [avibactam sodium or pyridine-2,6-dicarboxylic acid (DPA)] on the emission of 3-hydroxy-2-butanone (acetoin) from various carbapenemase-producing K. pneumoniae was further investigated. Subsequently, VOCal software was used to generate a pseudo-3D plot of 3-hydroxy-2-butanone (acetoin), and the relative peak volumes were exported for data analysis. Standard strains served as references, and the findings were validated with clinical isolates. RESULTS The pattern of temporal changes in the 3-hydroxy-2-butanone (acetoin) release from K. pneumoniae in the absence of IPM was consistent with the growth curve. After the IPM addition, carbapenemase-positive strains released significantly higher contents of 3-hydroxy-2-butanone (acetoin) than carbapenemase-negative strains at the late exponential growth phase (T2). Notably, adding avibactam sodium significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class A carbapenemase-producing strains as compared to the absence of the carbapenemase inhibitor. Conversely, adding DPA significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class B carbapenemase-producing strains (both standard and clinical strains, all P<0.05). CONCLUSIONS This study demonstrated the potential of 3-hydroxy-2-butanone (acetoin) as a VOC biomarker for detecting carbapenemase-producing K. pneumoniae, as revealed by GC-IMS analysis.

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 Glf L445I , 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-glf L445I alsSalsD, 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., (© 2024 International Union of Biochemistry and Molecular Biology, Inc.)

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., (© 2024. The Author(s).)

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., (© 2023. Chr. Hansen.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 Mg 2+ , Mn 2+ , and Fe 2+ 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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest All authors declare that we have no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

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., (© 2022 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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.

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.

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., (© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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.

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 β-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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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-1β, 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., (© 2022. The Author(s).)

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 P sod 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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

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, pdc 1 and pdc 5 double knockout strains of S. cerevisiae H14-02 ( MAT a type) and S. cerevisiae H5-02 ( MAT α type) were obtained by the Cre/loxP technique. The effects of the deletion of pdc 1 and pdc 5 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.

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol ( t=2.7-6.4 h) and on ethanol ( t=15-50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth ( t=3-9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme. [ABSTRACT FROM AUTHOR]

Acetoin, 3-hydroxyl,2-butanone, is extensively used as a flavor additive in food products. This volatile compound is produced by the dairy bacterium Lactococcus lactis when aerobic respiration is activated by haem addition, and comprises ∼70% of carbohydrate degradation products. Here we investigate the targets of acetoin toxicity, and determine how acetoin impacts L. lactis physiology and survival. Acetoin caused damage to DNA and proteins, which related to reactivity of its keto group. Acetoin stress was reflected in proteome profiles, which revealed changes in lipid metabolic proteins. Acetoin provoked marked changes in fatty acid composition, with massive accumulation of cycC19:0 cyclopropane fatty acid at the expense of its unsaturated C18:1 fatty acid precursor. Deletion of the cfa gene, encoding the cycC19:0 synthase, sensitized cells to acetoin stress. Acetoin-resistant transposon mutagenesis revealed a hot spot in the high affinity phosphate transporter operon pstABCDEF , which is known to increase resistance to multiple stresses. This work reveals the causes and consequences of acetoin stress on L. lactis, and may facilitate control of lactic acid bacteria production in technological processes. IMPORTANCE Acetoin, 3-hydroxyl,2-butanone, has diverse uses in chemical industry, agriculture, and dairy industries as a volatile compound that generates aromas. In bacteria, it can be produced in high amount by Lactococcus lactis when it grows under aerobic respiration. However, acetoin production can be toxic and detrimental for growth and/or survival. Our results showed that it damages DNA and proteins via its keto group. We also showed that acetoin modifies membrane fatty acid composition with the production of cyclopropane C19:0 fatty acid at the expense of an unsaturated C18:1. We isolated mutants more resistant to acetoin than the wild-type strain. All of them mapped to a single locus pstABCDEF operon, suggesting a simple means to limit acetoin toxicity in dairy bacteria and to improve its production.

2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2R,3R)-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2R,3R)-2,3-BD or meso-2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3R/3S)-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2R,3R)-2,3-BD (K m  = 0.16 mM, k cat /K m  = 673 s -1 · mM -1 ) and meso-BD (K m  = 0.72 mM, k cat /K m  = 165 s -1 · mM -1 ). Moreover, it could also reduce (3R/3S)-acetoin with a K m of 0.14 mM and a k cat /K m of 885 s -1 · mM -1 . The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.

Objectives: The aim of this work was to study the changes of bacterial cell growth, acetion formation and glucose consumption with fermentation time during batch cultivation., Results: A mathematical model of cell growth, product synthesis, and substrate consumption changes with time during the batch cultivation of acetion was established. By analyzing the fitting curve of the kinetic model, it is found that the calculated value of the model fits well with the experimental value, and the fitting model R2 is greater than 0.98., Conclusions: The kinetic model established in this experiment can better reflect the batch cultivation process of acetion.

Ligustrazine is an important active alkaloid in medicine and in the food industry. Here, we developed a combined biological-chemical approach to produce ligustrazine from acetaldehyde. First, we constructed a whole-cell biocatalytic system to produce the precursor acetoin from acetaldehyde by overexpressing formolase (FLS). Second, a two-step strategy was developed to enhance protein expression of FLS by codon usage optimization at the first 14 codons and the introduction of an overlapping gene before the start codon. Through expression optimization and directed evolution of FLS, we improved the titer of acetoin about 40 fold when the concentration of acetaldehyde was 1.5 M. Finally, after reaction conditions optimization, the titer of acetoin and ligustrazine reached 222 g L -1 and 94 g L -1 , with a 86.5% and 48% conversion rate from acetaldehyde, respectively. The developed one-pot synthesis for acetoin and ligustrazine is expected to be applied to industrial production in the future with the advantages of a green process, high efficiency, and low cost.

Lactic acid bacteria (LAB) are commonly used in soymilk fermentation to improve health-related functionality, but their contribution to sensory qualities is less valued. We characterized Lactobacillus harbinensis M1, Lactobacillus mucosae M2, Lactobacillus fermentum M4, Lactobacillus casei M8 and Lactobacillus rhamnosus C1 from naturally-fermented tofu whey, along with Streptococcus thermophilus ST3 from kefir XPL-1 fermented soymilk, to investigate their potential as starter cultures of fermented soymilk. They were characterized for antibiotic susceptibility, probiotic potential and their performance as starter cultures. All the LABs showed sensitivity to the tested antibiotics. L. casei M8 had strongest tolerance to synthetic gastrointestinal juice (<1.0 log CFU/mL loss), as well as antagonistic effects towards five food-borne pathogens. GC/MS analysis showed that L. harbinensis M1 produced significantly higher abundance (P < 0.05) of 2,3-butanedione (2.45 ppm) and acetoin (44.30 ppm), thus improving the overall sensory acceptability of fermented soymilk. The coding genes for the synthesis of 2,3-butanedione/acetoin (alsS, alsD, butA) were predicted from the whole-genome. A co-culture of L. harbinensis M1 and L. casei M8 produced a fermented soymilk product with both markedly improved flavor and good probiotic potential. It appears that L. harbinensis M1 has much potential for improving the organoleptic properties of fermented soymilk., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Microbial contamination, especially in large-scale processes, is partly a life-or-death issue for industrial fermentation. Therefore, the aim of this research was to create an antimicrobial contamination system in Bacillus subtilis 168 (an ideal acetoin producer for its safety and acetoin synthesis potential). First, introduction of the formamidase (FmdA) from Helicobacter pylori and the phosphite dehydrogenase (PtxD) from Pseudomonas stutzeri enabled the engineered Bacillus subtilis to simultaneously assimilate formamide and phosphite as nitrogen (N) and phosphorus (P) sources. Thus, the engineered B. subtilis became the dominant population in a potentially contaminated system, while contaminated microbes were starved of key nutrients. Second, stepwise metabolic engineering via chromosome-based overexpression of the relevant glycolysis and acetoin biosynthesis genes led to a 1.12-fold increment in acetoin titer compared with the starting host. Finally, with our best acetoin producer, 25.56 g/L acetoin was synthesized in the fed-batch fermentation, with a productivity of 0.33 g/L/h and a yield of 0.37 g/g under a nonsterilized and antibiotic-free system. More importantly, our work fulfills many key criteria of sustainable chemistry since sterilization is abolished, contributing to the simplified fermentation operation with lower energy consumption and cost.

The continuous and sole dependence on imidazolinone (IMI) herbicides for weedy rice control has led to the evolution of herbicide resistance in weedy rice populations across various countries growing IMI herbicide-resistant rice (IMI-rice), including Malaysia. A comprehensive study was conducted to elucidate occurrence, level, and mechanisms endowing resistance to IMI herbicides in putative resistant (R) weedy rice populations collected from three local Malaysian IMI-rice fields. Seed bioassay and whole-plant dose-response experiments were conducted using commercial IMI herbicides. Based on the resistance index (RI) quantification in both experiments, the cross-resistance pattern of R and susceptible (S) weedy rice populations and control rice varieties (IMI-rice variety MR220CL2 and non-IMI-rice variety MR219) to imazapic and imazapyr was determined. A molecular investigation was carried out by comparing the acetohydroxyacid synthase (AHAS) gene sequences of the R and S populations and the MR220CL2 and MR219 varieties. The AHAS gene sequences of R weedy rice were identical to those of MR220CL2, exhibiting a Ser-653-Asn substitution, which was absent in MR219 and S plants. In vitro assays were conducted using analytical grade IMI herbicides of imazapic (99.3%) and imazapyr (99.6%) at seven different concentrations. The results demonstrated that the AHAS enzyme extracted from the R populations and MR220CL2 was less sensitive to IMI herbicides than that from S and MR219, further supporting that IMI herbicide resistance was conferred by target-site mutation. In conclusion, IMI resistance in the selected populations of Malaysian weedy rice could be attributed to a Ser-653-Asn mutation that reduced the sensitivity of the target site to IMI herbicides. To our knowledge, this study is the first to show the resistance mechanism in weedy rice from Malaysian rice fields., Competing Interests: The authors have declared that no competing interests exist.

Lactococcus lactis subsp. lactis biovar diacetylactis is widely used in dairy fermentations as it can form the butter aroma compounds acetoin and diacetyl from citrate in milk. Here, we explore the possibility of producing acetoin from the more abundant lactose. Starting from a dairy isolate of L. lactis biovar diacetylactis, we obtained a series of mutants with low lactate dehydrogenase ( ldh ) activity. One isolate, RD1M5, only had a single insertion mutation in the ldh gene compared to its parental strain as revealed by whole genome resequencing. We tested the ability of RD1M5 to produce acetoin in milk. With aeration, all the lactose could be consumed, and the only product was acetoin. In a simulated cheese fermentation, a 50% increase in acetoin concentration could be achieved. RD1M5 turned out to be an excellent cell factory for acetoin and was able to convert lactose in dairy waste into acetoin with high titer (41 g/L) and high yield (above 90% of the theoretical yield). Summing up, RD1M5 was found to be highly robust and to grow excellently in milk or dairy waste. Being natural in origin opens up for applications within dairies as well as for safe production of food-grade acetoin from low-cost substrates.

Background: Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses., Results: In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%., Conclusion: To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.

Cell division can perturb the metabolic performance of industrial microbes. The C period of cell division starts from the initiation to the termination of DNA replication, whereas the D period is the bacterial division process. Here, we first shorten the C and D periods of E. coli by controlling the expression of the ribonucleotide reductase NrdAB and division proteins FtsZA through blue light and near-infrared light activation, respectively. It increases the specific surface area to 3.7 μm -1 and acetoin titer to 67.2 g·L -1 . Next, we prolong the C and D periods of E. coli by regulating the expression of the ribonucleotide reductase NrdA and division protein inhibitor SulA through blue light activation-repression and near-infrared (NIR) light activation, respectively. It improves the cell volume to 52.6 μm 3 and poly(lactate-co-3-hydroxybutyrate) titer to 14.31 g·L -1 . Thus, the optogenetic-based cell division regulation strategy can improve the efficiency of microbial cell factories.

Acetoin (3-hydroxy-2-butanone) is an important physiological metabolic product in many microorganisms. Acetoin breakdown is catalyzed by the acetoin dehydrogenase enzyme system (AoDH ES), which is encoded by acoABCL operon. In this study, we analyzed transcription and regulation of the aco operon in Bacillus thuringiensis (Bt). RT-PCR analysis revealed that acoABCL forms one transcriptional unit. The Sigma 54 controlled consensus sequence was located 12 bp from the acoA transcriptional start site (TSS). β-galactosidase assay revealed that aco operon transcription is induced by acetoin, controlled by sigma 54, and positively regulated by AcoR. The HTH domain of AcoR recognized and specifically bound to a 13-bp inverted repeat region that participates in 30-bp fragment mapping 81 bp upstream of the acoA TSS. The GAF domain in AcoR represses enhancer transcriptional activity at the acoA promoter. Transcriptions of the aco operon and acoR were repressed by glucose via CcpA, and CcpA specifically bound to sequences within the acoR promoter fragment. In the acoABCL and acoR mutants, acetoin use was abolished, suggesting that the aco operon is essential for utilization of acetoin. The data presented here improve our understanding of the regulation of the aco gene cluster in bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Hemp seed flour represents a potential ingredient for protein enrichment of gluten-free bakery products, the nutritional value of which could be further increased by fermentation with sourdough or with beneficial lactic acid bacteria strains. In this study, a metabolomic approach was used to evaluate the effect of hemp seed flour addition and sourdough fermentation on the production of flavoring and health-related volatile organic compounds (VOCs) in a gluten-free bread. Multivariate analysis of VOCs provided an in-depth description of the effects of hemp seed flour addition and sourdough fermentation on flavoring and bioactive compounds. In particular, an increased concentration of antimicrobial compounds, a larger spectrum of bioactive VOCs and a typical flavoring profile was evidenced in comparison to standard products. Furthermore, an increase of fermentation metabolites was observed in comparison to a standard dough, relating to abundances of 2-butanone-3-hydroxy, acetic acid, ethanol, and 1,4-butanediol. This study provides new insights on the evolution of flavoring and bioactive hemp seed flour constituents during sourdough fermentation, evidencing their retention in baked goods, and describes a new approach that could guide the formulation of innovative, fermented food with enhanced nutritional value.

Promoter plays the critical role in regulating gene transcription, and dual-promoter has received the widespread attentions due to its high efficiency and continuity, here, we want to construct an efficient dual-promoter for protein production and metabolic pathway enhancement. Firstly, our results indicated that P43 promoter efficiently transcribed at logarithmic period, while the σ B -type promoters (PylB, PgsiB, PykzA) were active at stationary phase. Then, several dual promoters were constructed by coupling these σ B -type promoters with P43, and the attained dual-promoter PykzA-P43 showed the best performance, which led to 1.72-, 3.46- and 1.85-fold increases of green fluorescence intensity, red fluorescence intensity and α-amylase activity, compared with those of the recognized strong promoter P43, respectively. Furthermore, α-amylase activity was further increased to 389.65 U/mL by 32.20 % via optimizing sigma factor binding sites (-10 and -35 boxes) of PykzA-P43, attaining the optimized dual promoter Pdual3. Finally, Pdual3 was applied in metabolic pathway enhancement, and the yields of Poly γ-glutamic acid, acetoin and 2, 3-butanediol were respectively improved by 82.01 %, 17.09 % and 99.39 %. Our results indicated that dual-promoter significantly enhanced gene expression, and this study provided an energetic dual-promoter Pdual3 for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lactococcus lactis subsp. lactis bv. diacetylactis strains are often used as starter cultures by the dairy industry due to their production of acetoin and diacetyl, important substances that add buttery flavor notes in dairy products. Twenty-three L. lactis subsp. lactis isolates were obtained from dairy products (milk and cheese) and dairy farms (silage), identified at a biovar level, fingerprinted by rep-PCR and characterized for some technological features. Fifteen isolates presented molecular and phenotypical (diacetyl and citrate) characteristics coherent with L. lactis subsp. lactis bv. diacetylactis and rep-PCR allowed the identification of 12 distinct profiles (minimum similarity of 90%). Based on technological features, only two isolates were not able to coagulate skim milk and 10 were able to produce proteases. All isolates were able to acidify skim milk: two isolates, in special, presented high acidifying ability due to their ability in reducing more than two pH units after 24 h. All isolates were also able to grow at different NaCl concentrations (0 to 10%, w/v), and isolates obtained from peanut and grass silages presented the highest NaCl tolerance (10%, w/v). These results indicate that the L. lactis subsp. lactis bv. diacetylactis isolates presented interesting technological features for potential application in fermented foods production. Despite presenting promising technological features, the isolates must be assessed according to their safety before being considered as starter cultures.

Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

2,3,5,6-Tetramethylpyrazine (TMP) has health care functions, especially for cardiovascular and cerebrovascular health. In this study, we found that Bacillus coagulans , a well-known probiotic, has the capability to produce acetoin, a precursor of TMP. The culture conditions and medium for the production of TMP by B. coagulans CICC 20138 were optimized. Then, a novel three-step process was successfully performed for the production of TMP from edible materials by B. coagulans . First, in the acetoin enrichment process, 12.61 ± 0.34 g/L acetoin was generated at 36 h. Second, in the spore enrichment process, various factors were optimized to make the bacteria produce more spores to improve the resistance to subsequent high-temperature reactions. Third, in the TMP enrichment process, the final concentration of TMP and B. coagulans spores contained in the product reached 2.54 ± 0.26 g/L and 8.81 × 10 8 CFU/mL at 46 h, respectively. This is the first report of using a probiotic bacterium to produce TMP. Using edible materials and the probiotic strain, this work provides a novel method for the production of a TMP food additive rich in B. coagulans spores.

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