Acetoin Production Research Articles

Metabolic engineering of Escherichia coli for improved cofactor regeneration in lactate to acetoin via whole-cell conversion

Background: Acetoin is a crucial intermediate in asymmetric syntheses of high-value chemicals and pharmaceuticals. However, its production still relies on traditional fossil-based processes. Developing efficient microbial cell factories for green and low-cost acetoin production is urgently needed. Methods: Acetoin was produced from inexpensive and shortcut lactate substrate using whole-cell Escherichia coli through overexpression of highly active α-acetolactate synthetase and decarboxylase from Bacillus subtilis (annotated as SD). Precise stepwise optimization of pathway and enzymatic reaction was executed by (1) harboring the most efficient cofactor-regenerating system, (2) tuning expression design, (3) disrupting byproduct pathway, and (4) optimizing a series of biotransformation parameters. Significant Findings: The recombinant E. coli successfully produced acetoin. The titer was gradually increased by expressing a pyruvate-producing gene from NAD+ dependent or independent system and its cofactor regeneration systems. Co-expressing lactate oxidase (lox) and catalase (cat) achieved a conversion efficiency of 50% and eliminated NAD+ usage. The conversion efficiency was further pulled by knocking out acetate-generating genes (pta and poxB), thus boosting acetoin conversion to 92.4%. Under optimized whole-cell biotransformation parameters, the highest acetoin titer reached 20.6 g/L within 30 h. This work provides an economical biomanufacturing process for acetoin from lactate via whole-cell bioconversion with remarkable yield.

Artificial cell-free system for the sustainable production of acetoin from bioethanol.

The present work introduces and validates an artificial cell free system for the synthesis of acetoin from ethanol, representing a greener alternative to conventional chemical synthesis. The one pot multi-enzymatic system, which employs pyruvate decarboxylase from Zymobacter palmae (ZpPDC), alcohol dehydrogenase from Saccharomyces cerevisiae (ScADH), and NADH oxidase from Streptococcus pyogenes (SpNOX), achieves nearly 100 % substrate conversion and reaction yield within 6 h under optimal conditions (pH 7.5, enzyme activities: ZpPDC 100 U·mL-1, ScADH 50 U·mL-1, SpNOX 127 U·mL-1, and 1 mM NAD+). Using air for oxygen supply mitigates enzyme inactivation while effectively accelerating the regeneration of NAD+. The use of bioethanol as a substrate demonstrates the robustness and sustainability of the bioprocess, enabling the production of natural acetoin from renewable resources. This environmentally friendly approach offers significant advantages for industrial applications, aligning with green chemistry principles.

Xenorhabdus bharatensis sp. nov., Xenorhabdus entomophaga sp. nov., Xenorhabdus siamensis sp. nov., and Xenorhabdus thailandensis sp. nov. Isolated from Steinernema Entomopathogenic Nematodes

Four Gram-stain-negative bacterial strains, CS20T, AUT15.5T, XENO-11T, and CCN3.3T, isolated from Steinernema entomopathogenic nematodes, were found to represent novel species within the genus Xenorhabdus (Gammaproteobacteria, Morganellaceae). In this study, we described these new species using whole-genome phylogenomic reconstructions, sequence identity values from core genome sequences, and phenotypic characterization. Phylogenetic reconstructions based on 16S rRNA gene sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T, (ii) strain AUT15.5T is closely related to X. budapestensis DSM 16342T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The 16S rRNA gene sequence similarity value between strain CS20T and X. stockiae DSM 17904T is 97.8%, between strain AUT15.5T and X. budapestensis DSM 16342T is 98.1%, between strain XENO-11T and X. khoisanae DSM 25463T is 97.8%, and between strain CCN3.3T and X. griffiniae DSM 17911T is 98.6%. Phylogenomic reconstructions using whole-genome sequences showed that: (i) strain CS20T is closely related to X. stockiae DSM 17904T and X. innexi DSM 16336T, (ii) strain AUT15.5T is closely related to X. indica DSM 17382T, (iii) strain XENO-11T is closely related to X. khoisanae DSM 25463T, and (iv) strain CCN3.3T is closely related to X. griffiniae DSM 17911T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains CS20T, AUT15.5T, XENO-11T, and CCN3.3T and the type strains of their more closely related species are below the 70% and the 95–96% divergence thresholds, respectively, used for prokaryotic species delineation. Hence, we propose the following four new species: Xenorhabdus bharatensis sp. nov. (the type strain is CS20T=CCM 9320T=CCOS 2070T), X. entomophaga sp. nov. (the type strain is XENO-11T=CCM 9389T=CCOS 2111T), X. siamensis sp. nov. (the type strain is AUT15.5T=CCM 9405T=CCOS 2116T), and X. thailandensis sp. nov. (the type strain is CCN3.3T=CCM 9406T=CCOS 2115T). The following biochemical tests may be useful for differentiating the novel species from their more closely related taxa: acetoin production, arginine dihydrolase, citrate utilization, gelatinase, glucose oxidation, indole production, and tryptophan deaminase. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of entomopathogenic bacteria associated with insect parasitic nematodes.

Interaction between a Lactococcus lactis autochthonous starter and a raw goat milk microbial community during long-term backslopping

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.

Investigation of Acetoin Biosynthesis by Bacillus subtilis ACA-DC 1176 Growing on Crude Glycerol in Flask and Bioreactor Trials

Acetoin biosynthesis by two Bacillus subtilis strains valorising crude glycerol was thoroughly explored within a pre-defined range of culture conditions and systems. B. subtilis ACA-DC 1176 stood out for its higher efficiency in acetoin production, prompting an investigation into the potential for enhanced productivity through the evaluation of diverse culture conditions and media compositions. The primary by-products of the biodiesel and corn industries, namely crude glycerol and corn steep liquor, respectively, were successfully employed as the principal carbon and nitrogen sources of the newly developed low-cost culture medium. Furthermore, the results of the various feeding strategies that were tested indicated that the conversion of 2,3-butanediol (BDO) to acetoin occurred exclusively when the concentration of glycerol was below approximately 5 g/L. This seemed to be necessary for the production of NADH, which is essential for maintaining cellular processes. Following the complete depletion of glycerol, acetic acid increased and became the predominant metabolite, while both acetoin and BDO decreased, presumably resulting in ATP generation. This is likely a mechanism employed by the cell to generate energy in the absence of a carbon source. In the fed-batch bioreactor culture, the kinetics of metabolites differed, as there was no conversion of BDO to acetoin at the final depletion of glycerol. At volumetric mass transfer coefficient (kLa) levels exceeding approximately 70 1/h, the production of acetoin was favoured over that of BDO, with the highest observed acetoin/BDO ratio reaching 4.29 g/g. Conversely, at kLa values below approximately 60 1/h, the titres of acetoin and BDO were found to be nearly equal. The final concentrations of acetoin and BDO reached 36.0 g/L and 25.5 g/L, respectively, resulting in a total yield of both (acetoin + BDO) per glycerol consumption of 0.40 g/g. To the best of our knowledge, this is the first study to focus on acetoin production from crude glycerol fermentative valorisation. The study presents new findings regarding the parameters influencing the level of BDO conversion to acetoin. However, further research is required in order to gain a comprehensive understanding of the underlying phenomena and metabolic pathways involved.

Microbiomics and metabolomics insights into the microbial regulation on the formation of flavor components in the traditional fermentation process of Chinese Hongqu aged vinegar

This study aimed to investigate microbial succession and metabolic dynamics during the traditional fermentation of Hongqu aged vinegar, and explore the core functional microbes closely related to the formation of flavor components. Microbiome analysis demonstrated that Lactobacillus, Acetobacter, Bacillus, Enterobacter, Lactococcus, Leuconostoc and Weissella were the predominant bacterial genera, while Aspergillus piperis, Aspergillus oryzae, Monascus purpureus, Candida athensensis, C. xylopsoci, Penicillium ochrosalmoneum and Simplicillium aogashimaense were the predominant fungal species. Correlation analysis revealed that Acetobacter was positively correlated with the production of tetramethylpyrazine, acetoin and acetic acid, Lactococcus showed positive correlation with the production of 2-nonanone, 2-heptanone, ethyl caprylate, ethyl caprate, 1-hexanol, 1-octanol and 1-octen-3-ol, C. xylopsoci and C. rugosa were positively associated with the production of diethyl malonate, 2,3-butanediyl diacetate, acetoin, benzaldehyde and tetramethylpyrazine. Correspondingly, non-volatile metabolites were also detected through ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. A variety of amino acids and functional dipeptides were identified during the traditional brewing of Hongqu aged vinegar. Correlation analysis revealed that Lactobacillus was significantly associated with DL-lactate, indolelactic acid, D-(+)-3-phenyllactic acid, pimelic acid, pregabalin and 3-aminobutanoic acid. This study is useful for understanding flavor formation mechanism and developing effective strategies for the suitable strains selection to improve the flavor quality of Hongqu aged vinegar.

Genomic and metabolomic analysis of Latilactobacillus sakei DCF0720 for black soybean yogurt fermentation

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 1H 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.

Multilevel systemic engineering of Bacillus licheniformis for efficient production of acetoin from lignocellulosic hydrolysates

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 60Co-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.

Strain and model development for auto- and heterotrophic 2,3-butanediol production using Cupriavidus necator H16

The production of platform chemicals from renewable energy sources is a crucial step towards a post-fossil economy. This study reports on the production of acetoin and 2,3-butanediol heterotrophically with fructose as substrate and autotrophically from CO2 as carbon source, H2 as electron donor and O2 as electron acceptor with Cupriavidus necator. In a previous study, the strain was developed for the production of acetoin with high carbon efficiency. Acetoin can serve as a precursor for the synthesis of 2,3-butanediol by the integration of a butanediol dehydrogenase. In this study, different plasmid backbones and butanediol dehydrogenases were evaluated regarding efficiency for CO2-based 2,3-butanediol production. The developed strain utilizes the pBBR1 plasmid bearing a 2,3-butanediol dehydrogenase from Enterobacter cloacae and is characterized by 2,3-butanediol as the main product and a heterotrophic total product yield of 88.11%, an autotrophic volumetric productivity of 39.45 mg L−1 h−1, a total product carbon yield of 81.6%, an H2 efficiency of 33.46%, and a specific productivity of 0.016 g product per gram of biomass per hour. In addition, a mathematical model was developed to simulate the processes under these conditions. With this model, it was possible to calculate productivities and substrate usage at distinct time points of the production processes and calculate productivities and substrate usage with high resolution which will be useful in future applications.

Photorhabdus africana sp. nov. isolated from Heterorhabditis entomopathogenic nematodes

One Gram-negative, rod-shaped bacterial strain, isolated from an undescribed Heterorhabditis entomopathogenic nematode species was characterized to determine its taxonomic position. The 16S rRNA gene sequences indicate that it belongs to the class Gammaproteobacteria, to the family Morganellaceae, to the genus Photorhabdus, and likely represents a novel bacterial species. This strain, designated here as CRI-LCT, was therefore molecularly, biochemically, and morphologically characterized to describe the novel bacterial species. Phylogenetic reconstructions using 16S rRNA gene sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. The 16rRNA gene sequences between CRI-LCT and P. laumondii subsp. laumondii TT01T are 99.1% identical, and between CRI-LCT and P. laumondii subsp. clarkei BOJ-47T are 99.2% identical. Phylogenetic reconstructions using whole genome sequences show that CRI-LCT is closely related to P. laumondii subsp. laumondii TT01T and to P. laumondii subsp. clarkei BOJ-47T. Moreover, digital DNA-DNA hybridization (dDDH) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 65% and 63%, respectively. In addition, we observed that average nucleotide identity (ANI) values between CRI-LCT and its two relative species P. laumondii subsp. laumondii TT01T and P. laumondii subsp. clarkei BOJ-47T are 95.8% and 95.5%, respectively. These values are below the 70% dDDH and the 95–96% ANI divergence thresholds that delimits prokaryotic species. Based on these genomic divergence values, and the phylogenomic separation, we conclude that CRI-LCT represents a novel bacterial species, for which we propose the name Photorhabdus africana sp. nov. with CRI-LCT (= CCM 9390T = CCOS 2112T) as the type strain. The following biochemical tests allow to differentiate P. africana sp. nov. CRI-LCT from other species of the genus, including its more closely related taxa: β-Galactosidase, citrate utilization, urease and tryptophan deaminase activities, indole and acetoin production, and glucose and inositol oxidation. Our study contributes to a better understanding of the taxonomy and biodiversity of this important bacterial group with great biotechnological and agricultural potential.

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

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

Effect of crude glycerol, 2,3-butanediol, and acetoin initial concentrations on Enterobacter aerogenes ATCC 13048 growth in batch runs

The 2,3-butanediol (BD) and acetoin production from several carbon sources and microorganisms has been extensively studied, mainly due to the possibility of replacing petroleum derivatives by the diol. Crude glycerol is highlighted among the substrates that can be used to produce 2,3-BD. However, this carbon source contains impurities that can affect microbial metabolism. Furthermore, there are scarce studies about the inhibitory effect of fermentation products, such as 2,3-BD and acetoin, on the process. The aim of this study was to evaluate the effect of crude glycerol, 2,3-BD and acetoin initial concentrations on Enterobacter aerogenes growth and metabolism. For this purpose, batch runs were performed using crude glycerol concentrations varying from 20 to 80 g.L-1. To evaluate the inhibitory effect of 2,3-BD plus acetoin, batch runs were performed using product addition from 0 to 40 g.L-1. During batch runs performed using increasing crude glycerol concentrations, the values of maximum specific growth rates were decreasing, evidencing the metabolism inhibition possibly due to the osmotic pressure and substrate composition. In all batch runs, acetic and lactic acids, and ethanol were identified as co-products. In the case of fermentation products, the 2,3-BD plus acetoin initial concentration of 20 g.L-1 was found inhibitory to E. aerogenes growth by a reduction of 45% in maximum specific growth rate. The data also proved the major influence of 2,3-BD rather than acetoin on the overall rate of fermentation, since acetoin concentrations of up to 3.5 g.L-1 did not influence the E. aerogenes growth. Furthermore, the strain ability to adapt to the 2,3-BD and acetoin initial concentrations was demonstrated, opening possibilities for new processes strategies.

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

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

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

Serratia marcescens is utilized as a significant enterobacteria in the production of various high-value secondary metabolites. Acetoin serves as a crucial foundational compound of development and finds application in a broad range of fields. Furthermore, S. marcescens HBQA-7 is capable of utilizing xylose as its exclusive carbon source for acetoin production. The objective of this study was to utilize a constitutive promoter screening strategy to enhance both xylose utilization and acetoin production in S. marcescens HBQA-7. By utilizing RNA-seq, we identified the endogenous constitutive promoter P6 that is the most robust, which facilitated the overexpression of the sugar transporter protein GlfL445I , α-acetyl lactate synthase, and α-acetyl lactate decarboxylase, respectively. The resultant recombinant strains exhibited enhanced xylose utilization rates and acetoin yields. Subsequently, a recombinant plasmid, denoted as pBBR1MCS-P6-glfL445I 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 7.14g/L acetoin in the final fermentation medium. Subsequently, in a 5 L fermenter setup, the acetoin yield reached 48.75g/L, corresponding to a xylose-to-acetoin conversion yield of 0.375g/g.

A novel plant growth-promoting rhizobacterium, Rhizosphaericola mali gen. nov., sp. nov., isolated from healthy apple tree soil

The rhizosphere microbial community is closely associated with plant disease by regulating plant growth, agricultural production, nutrient availability, plant hormone and adaptation to environmental changes. Therefore, it is very important to identify the rhizosphere microbes around plant roots and understand their functions. While studying the differences between the rhizosphere microbiota of healthy and diseased apple trees to find the cause of apple tree disease, we isolated a novel strain, designated as B3-10T, from the rhizosphere soil of a healthy apple tree. The genome relatedness indices between strain B3-10T and other type species of family Chitinophagaceae were in the ranges of 62.4–67.0% for ANI, 18.6–32.1% for dDDH, and 39.0–56.6% for AAI, which were significantly below the cut‑off values for the species delineation, indicating that strain B3-10T could be considered to represent a novel genus in family Chitinophagaceae. Interestingly, the complete genome of strain B3-10T contained a number of genes encoding ACC-deaminase, siderophore production, and acetoin production contributing to plant-beneficial functions. Furthermore, strain B3-10T was found to significantly promote the growth of shoots and roots of the Nicotiana benthamiana, which is widely used as a good model for plant biology, demonstrating that strain B3-10T, a rhizosphere microbe of healthy apple trees, has the potential to promote growth and reduce disease. The phenotypic, chemotaxonomic, phylogenetic, genomic, and physiological properties of this plant growth-promoting (rhizo)bacterium, strain B3-10T supported the proposal of a novel genus in the family Chitinophagaceae, for which the name Rhizosphaericola mali gen. nov., sp. nov. (= KCTC 72123T = NBRC 114178T).

Metabolic reprogramming in the food-borne pathogen Listeria monocytogenes as a critical defence against acid stress.

The ability to sense and respond effectively to acidic stress is important for microorganisms to survive and proliferate in fluctuating environments. As specific metabolic activities can serve to buffer the cytoplasmic pH, microorganisms rewire their metabolism to favour these reactions and thereby mitigate acid stress. The orally acquired pathogen Listeria monocytogenes exploits alternative metabolic activities to overcome the acidic stress encountered in the human stomach or food products. In this minireview, we discuss the metabolic processes in L. monocytogenes that mitigate acid stress, with an emphasis on the proton-depleting reactions, including glutamate decarboxylation, arginine/agmatine deimination, and fermentative acetoin production. We also summarize the recent findings on regulatory mechanisms that control the expression of genes that are responsible for these metabolic activities, including the general stress response regulator SigB, arginine repressor ArgR, and the recently discovered RofA-like transcriptional regulatory GadR. We further discuss the importance of this metabolic reprogramming in the context of food products and within the host. Finally, we highlight some outstanding challenges in the field, including an understanding of acid-sensing mechanisms, the role of intraspecies heterogeneity in acid resistance, and how a fundamental understanding of acid stress response can be exploited for food formulation to improve food safety and reduce food waste.

Development of the Cre-Cas 2.0 system for evaluating acetoin production in Bacillus subtilis

BackgroundAcetoin is a vital platform compound widely used in the food, pharmaceutical, and chemical industries. Since 2004, the United States Department of Energy has listed it as one of the 30 platform compounds for priority development. Bacillus subtilis, a generally recognized safe microorganism, is an ideal platform microorganism for the engineered production of valuable bio-based chemicals. MethodsThe Cre-Cas 2.0 system derived from the Cre-Cas system was applied to inactive, enhance, or recover native metabolic genes. This system was designed to evaluate which native metabolic genes affect acetoin production in B. subtilis. Significant findingsUtilizing the Cre-Cas 2.0 system, acetoin reductase (bdhA), l-lactate dehydrogenase (ldh), and acetoin dehydrogenase (acoA) were inactivated, while the native acetoin synthesis genes, alsSD operon and alsR, were enhanced. This approach resulted in the creation of sixteen recombinant strains for the assessment of acetoin production. Finally, the acetoin production of strain ACN11 was 55 % higher than the original strain B. subtilis DB428. Moreover, employing the Cre-Cas 2.0 concept for editing the chromosomal genes of B. subtilis to enhance acetoin and other biochemical production could offer flexibility and applicability.

Toxigenic characterization, spoilage potential, and antimicrobial susceptibility of coagulase-positive Staphylococcus species isolated from Minas Frescal cheese

This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for deterioration in psychrotrophic and mesophilic conditions, verify the toxigenic potential of Staphylococcus aureus, and determine the antimicrobial susceptibility profile of toxigenic S. aureus. Species determination was performed based on the detection of β-hemolysis in 5% ovine blood agar; fermentation of mannitol, maltose, and trehalose sugars; and production of acetoin. After species determination, DNA extraction and analysis was performed for S. aureus colonies for genes encoding staphylococcal toxins (eta, etb, tst, sea, seb, sec, sed, and see) using 2 multiplex PCR assays. Isolates identified as toxigenic S. aureus were tested for antimicrobial susceptibility to tetracycline, erythromycin, clindamycin, gentamicin, ciprofloxacin, sulfazotrim, trimethoprim, streptomycin, cefoxitin, vancomycin and enrofloxacin. Elevated CPS counts were observed with an average of >6 log cfu/g. Of the 355 isolates, 177 (49.86%) were identified as S. aureus. Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus coagulans were identified in 3 (0.84%), 2 (0.56%), 2 (0.56%), and 1 (0.28%) isolates, respectively. Of the total number of S. aureus, 25 (52.08%) were positive for the gene that encodes for toxic shock toxin (TSST-1). Another 16 (33.33%) were positive for the sea gene, and 4 isolates (8.33%) were positive for see and one isolate each was positive for seb (2.08%), sec (2.08%), and etb (2.08%) genes. All isolates demonstrated lipolytic activity under mesophilic and psychrotrophic conditions. S. intermedius and S. hyicus had the most prominent proteolytic potential. Multidrug resistance was observed in most of the potentially toxigenic isolates, with clindamycin having the lowest efficiency (40%), whereas the aminoglycosides (gentamicin and streptomycin) had the highest effectiveness demonstrating inhibition in all evaluated isolates. Methicillin-resistant S. aureus (MRSA) was detected. Minas Frescal cheeses, marketed in the north of Tocantins in the Brazilian Amazon region, do not comply with legal quality standards and pose a public health risk due to the enterotoxigenic potential of multiresistant isolates, in addition to low shelf life of the samples given the high spoilage potential of this microbiota.

Potential of Cheese-Associated Lactic Acid Bacteria to Metabolize Citrate and Produce Organic Acids and Acetoin.

Lactic acid bacteria (LAB) are pivotal in shaping the technological, sensory, and safety aspects of dairy products. The evaluation of proteolytic activity, citrate utilization, milk pH reduction, and the production of organic compounds, acetoin, and diacetyl by cheese associated LAB strains was carried out, followed by Principal Component Analysis (PCA). Citrate utilization was observed in all Leuconostoc (Le.) mesenteroides, Le. citreum, Lactococcus (Lc.) lactis, Lc. garvieae, and Limosilactobacillus (Lm.) fermentum strains, and in some Lacticaseibacillus (Lact.) casei strains. Most strains exhibited proteolytic activity, reduced pH, and generated organic compounds. Multivariate PCA revealed Le. mesenteroides as a prolific producer of acetic, lactic, formic, and pyruvic acids and acetoin at 30 °C. Enterococcus sp. was distinguished from Lact. casei based on acetic, formic, and pyruvic acid production, while Lact. casei primarily produced lactic acid at 37 °C. At 42 °C, Lactobacillus (L.) helveticus and some L. delbrueckii subsp. bulgaricus strains excelled in acetoin production, whereas L. delbrueckii subsp. bulgaricus and Streptococcus (S.) thermophilus strains primarily produced lactic acid. Lm. fermentum stood out with its production of acetic, formic, and pyruvic acids. Overall, cheese-associated LAB strains exhibited diverse metabolic capabilities which contribute to desirable aroma, flavor, and safety of dairy products.

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

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