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The present study investigates the natural ability of Bacillus velezensis R22 to produce 2,3-BD from two inulin-rich substrates - insoluble and soluble chicory flour. After complex optimization of the media content and process parameters by consecutive application of Plackett-Burman design and response surface methodology, the strain R22 was capable of producing 71.2 g/L (95.5 % R,R-2,3-BD) and 96.4 g/L 2,3-BD (86.2 % R,R-2,3-BD) from insoluble and soluble chicory flour without preliminary hydrolysis. The optimal conditions were: K 2 HPO 4 concentration of 3.43 g/L, pH 6.68, and agitation speed of 363 rpm. The total inulinase activity of R22 ranged between 85 and 94 U/mL and was provided by the synergistic action of two enzymes: cytoplasmic SacC and membrane-located LevB, which were purified and characterized. The ability to directly convert inulin, its non-pathogenic nature, and the formation of predominantly R,R-2,3-BD singled out B. velezensis R22 as a new promising industrial producer of 2,3-BD from inulin-rich plant biomass., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kaloyan Petrov reports financial support was provided by the Republic of Bulgaria Ministry of Education and Science. Other 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 © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Within the context of the circular economy, the transformation of agri-food waste or by-products into valuable products is essential to promoting a transition towards more sustainable and efficient utilisation of resources. Whey is a very abundant by-product of dairy manufacturing. Apart from partial reutilisation in animal feed or some food supplements, the sustainable management and disposal of whey still represent significant environmental challenges. In this work, whey is considered a valuable resource for producing high-value products, specifically 2,3-butanediol (2,3-BDO), which was produced through fermentation using the bacterial strain Lactococcus lactis 43103. The described process yielded a >90% purity of 2,3-BDO, which was evaluated as a potential chain extender in the synthesis of bio-based waterborne polyurethane dispersions (PUDs). The incorporation of whey-derived 2,3-BDO led to the development of PUDs with up to 90% bio-based content without detrimental effects on the process or liquid-phase properties. The combination of 100% bio-based polyether polyols with partially renewable L-lysine ethyl ester diisocyanate and whey-derived 2,3-BDO as a chain extender generated totally stable, low-particle-size water dispersions of amorphous polymers characterised by similar structure and molecular weight compared to those of alternative petroleum-based PUDs. These results open up the possibility of incorporating fermentation-derived 2,3-BDO as a totally renewable component in bio-based PUDs as potential sustainable resinous systems for further formulation of water-based coatings or adhesives., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Germán-Ayuso, Cobos, Lorenzo, Río, Prieto-Fernández, Roncal and Cuevas.)

In order to establish an efficient microbial transformation platform based on seaweed feedstocks, experiments were performed to isolate a salt-tolerant strain capable of producing alginate lyase and 2,3-butanediol (2,3-BDO). Its physiological and biochemical characteristics, carbon source utilization, and product synthesis capabilities were investigated, and then the process for co-producing alginate lyase and 2,3-BDO from Laminaria japonica was optimized. Results showed that the isolated strain was identified as Vibrio alginolyticus, which was capable of utilizing multiple carbon sources to produce alginate lyase and 2,3-BDO even in the presence of 5 % NaCl. The highest reducing sugar yield was achieved as the Laminaria japonica pretreated with 1 % (v/v) sulfuric acid at 120 °C for 18 min. The enzymatic hydrolysis was boosted by devising a novel tween 80-assisted enzyme complex containing 60 FPU/g of cellulase, 15 U/g of pectinase, 20 U/g of alginate lyase, and 90 mg/g of tween 80. After establishing a semi-simultaneous saccharification and fermentation (S-SSF) strategy, the sugars could be fully utilized, yielding 14.83 g/L 2,3-BDO and 11.02 kU/L alginate lyase, respectively. Mass balance calculations indicating that up to 140 g of 2,3-butanediol and 120 kU of alginate lyase can be obtained from per kg of Laminaria japonica via this integrated approach., 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.)

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools and is routinely used to study individual enzymatic reactions but is not generally used to study systems-level properties of metabolism. Here we demonstrate a new approach that we call "cell-free systems biology", where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a three-enzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO 2 from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum , an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

Bacillus licheniformis 24 (BL24) is an efficient, non-pathogenic producer of 2,3-butanediol (2,3-BD). However, during inulin fermentation, the strain produces large amounts of exopolysaccharides (EPS), which interfere with the process' performance. The present study aims to investigate the effect that inactivation of the sacB gene, encoding levansucrase in BL24, has on 2,3-BD production efficiency. Knockout of the sacB gene was accomplished via insertional inactivation. The sacB -knockout variant formed 0.57 g/L EPS from sucrose and 0.7-0.8 g/L EPS from glucose and fructose, a 15- and 2.5-fold reduction relative to the wild type, respectively. Likewise, during batch fermentation with soluble inulin Frutafit ® CLR, the mutant BLΔ sacB produced significantly less EPS than the wild type, allowing the maintenance of pH at values favoring 2,3-BD synthesis. At pH 6.50, BLΔ sacB reached a record titer of 128.7 g/L 2,3-BD, with productivity of 1.65 g/L/h, and a yield of 85.8% of the theoretical maximum. The obtained concentration of 2,3-BD is two-fold higher compared to that of the wild type. Subsequent RT-qPCR assays confirmed a successful sacB knockout. Three of the genes involved in inulin hydrolysis ( sacA , sacC , and fruA ) maintained their expression levels compared to the wild type, while that of levB increased. Although total EPS accumulation could not be completely eliminated via sacB gene knockout alone, the overall reduction in EPS content has enabled the highest yield of 2,3-BD from inulin to date, a promising result for the industrial production from inulin-rich substrates.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

This study explores the synthesis and performance of novel copolyesters containing 2,3-butanediol (2,3-BDO) as a biobased secondary diol. This presents an opportunity for improving their thermal properties and reducing crystallinity, while also being more sustainable. It is, however, a challenge to synthesize copolyesters of sufficient molecular weight that also have high 2,3-BDO content, due to the reduced reactivity of secondary diols compared to primary diols. Terephthalate-based polyesters were synthesized in combination with different ratios of 2,3-BDO and ethylene glycol (EG). With a 2,3-BDO to EG ratio of 28:72, an M n of 31.5 kDa was reached with a T g of 88 °C. The M n dropped with increasing 2,3-BDO content to 18.1 kDa for a 2,3-BDO to EG ratio of 78:22 (T g = 104 °C) and further to 9.8 kDa (T g = 104 °C) for the homopolyester of 2,3-BDO and terephthalate. The water and oxygen permeability both increased significantly with increasing 2,3-BDO content and even the lowest content of 2,3-BDO (28% of total diol) performed significantly worse than PET. The incorporation of 2,3-BDO had little effect on the tensile properties of the polyesters, which were similar to PET. The results suggest that 2,3-BDO can be potentially applied for polyesters requiring higher T g and lower crystallinity than existing materials (mainly PET).

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 CO 2 as carbon source, H 2 as electron donor and O 2 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 CO 2 -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 H 2 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., (© 2024. The Author(s).)

Klebsiella pneumoniae can use glucose or glycerol as carbon sources to produce 1,3-propanediol or 2,3-butanediol, respectively. In the metabolism of Klebsiella pneumoniae, hydrogenase-3 is responsible for H 2 production from formic acid, but it is not directly related to the synthesis pathways for 1,3-propanediol and 2,3-butanediol. In the first part of this research, hycEFG, which encodes subunits of the enzyme hydrogenase-3, was knocked out, so K. pneumoniae ΔhycEFG lost the ability to produce H 2 during cultivation using glycerol as a carbon source. As a consequence, the concentration of 1,3-propanediol increased and the substrate (glycerol) conversion ratio reached 0.587 mol/mol. Then, K. pneumoniae ΔldhAΔhycEFG was constructed to erase lactic acid synthesis which led to the further increase of 1,3-propanediol concentration. A substrate (glycerol) conversion ratio of 0.628 mol/mol in batch conditions was achieved, which was higher compared to the wild type strain (0.545 mol/mol). Furthermore, since adhE encodes an alcohol dehydrogenase that catalyzes ethanol production from acetaldehyde, K. pneumoniae ΔldhAΔadhEΔhycEFG was constructed to prevent ethanol production. Contrary to expectations, this did not lead to a further increase, but to a decrease in 1,3-propanediol production. In the second part of this research, glucose was used as the carbon source to produce 2,3-butanediol. Knocking out hycEFG had distinct positive effect on 2,3-butanediol production. Especially in K. pneumoniae ΔldhAΔadhEΔhycEFG, a substrate (glucose) conversion ratio of 0.730 mol/mol was reached, which is higher compared to wild type strain (0.504 mol/mol). This work suggests that the inactivation of hydrogenase-3 may have a global effect on the metabolic regulation of K. pneumoniae, leading to the improvement of the production of two industrially important bulk chemicals, 1,3-propanediol and 2,3-butanediol., Competing Interests: Conflict of Interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024. Published by Elsevier Inc.)

The Expert Panel for Cosmetic Ingredient Safety (Panel) assessed the safety of 10 alkane diol ingredients as used in cosmetics. The alkane diols are structurally related to each other as small diols, and most are reported to function in cosmetics as solvents. The Panel reviewed the relevant data for these ingredients, and concluded that seven alkane diols are safe in cosmetics in the present practices of use and concentration described in this safety assessment, but that the available data are insufficient to make a determination of safety for three ingredients, namely 1,4-Butanediol, 2,3-Butanediol, and Octanediol., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The articles in this supplement were sponsored by the Cosmetic Ingredient Review.

Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h -1 in the reference to 0.175 h -1 in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h -1 with a glucose consumption rate of 29 mmol (g CDW) -1 h -1 , which was indeed higher than reference (23 mmol (g CDW) -1 h -1 ) and one of the highest reported for S. cerevisiae. The accompanying 2,3-butanediol- (15.8 mmol (g CDW) -1 h -1 ) and glycerol (19.6 mmol (g CDW) -1 h -1 ) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

L-Valine, a branched-chain amino acid with diversified applications, is biosynthesized with α-acetolactate as the key precursor. In this study, the metabolic flux in Klebsiella oxytoca PDL-K5, a Risk Group 1 organism producing 2,3-butanediol as the major fermentation product, was rearranged to L-valine production by introducing exogenous L-valine biosynthesis pathway and blocking endogenous 2,3-butanediol generation at the metabolic branch point α-acetolactate. After further enhancing L-valine efflux, strengthening pyruvate polymerization and selecting of key enzymes for L-valine synthesis, a plasmid-free K. oxytoca strain VKO-9 was obtained. Fed-batch fermentation with K. oxytoca VKO-9 in a 7.5 L fermenter generated 122 g/L L-valine with a yield of 0.587 g/g in 56 h. In addition, repeated fed-batch fermentation was conducted to prevent precipitation of L-valine due to oversaturation. The average concentration, yield, and productivity of produced L-valine in three cycles of repeated fed-batch fermentation were 81.3 g/L, 0.599 g/g, and 3.39 g/L/h, respectively., 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.)

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

Food waste is a major issue that is increasingly affecting our environment. More than one-third of food is wasted, resulting in over $400 billion in losses to the U.S. economy. While composting and other small recycling practices are encouraged from person-to-person, it is not enough to balance the net loss of 80 million tons per year. Currently, one of the most promising routes for reducing food waste is through microbial fermentation, which can convert the waste into valuable bioproducts. Among the compounds produced from fermentation, 2,3-butanediol (2,3-BDO) has gained interest recently due to its molecular structure as a building block for many other derivatives used in perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, and pharmaceuticals. Waste feedstocks, such as food waste, are a potential source of renewable energy due to their lack of cost and availability. Food waste also possesses microbial requirements for growth such as carbohydrates, proteins, fats, and more. However, food waste is highly inconsistent and the variability in composition may hinder its ability to be a stable source for bioproducts such as 2,3-BDO. This current study focuses specifically on post-consumer food waste and how 2,3-BDO can be produced through a non-model organism, Bacillus licheniformis YNP5-TSU during non-sterile fermentation. From the dining hall at Tennessee State University, 13 food waste samples were collected over a 6-month period and the compositional analysis was performed. On average, these samples consisted of fat (19.7%), protein (18.7%), ash (4.8%), fiber (3.4%), starch (27.1%), and soluble sugars (20.9%) on a dry basis with an average moisture content of 34.7%. Food waste samples were also assessed for their potential production of 2,3-BDO during non-sterile thermophilic fermentation, resulting in a max titer of 12.12 g/L and a 33% g/g yield of 2,3-BDO/carbohydrates. These findings are promising and can lead to the better understanding of food waste as a defined feedstock for 2,3-BDO and other fermentation end-products.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis , this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O 2 ) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O 2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O 2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design., Competing Interests: The authors declare no conflict of interest.

The platform chemical 2,3-butanediol (2,3-BDO) is used to derive products, such as 1,3-butadiene and methyl ethyl ketone, for the chemical and fuel production industries. Efficient microbial 2,3-BDO production at industrial scales has not been achieved yet for various reasons, including product inhibition to host organisms, mixed stereospecificity in product formation, and dependence on expensive substrates (i.e., glucose). In this study, we explore engineering of a 2,3-BDO pathway in Caldicellulosiruptor bescii , an extremely thermophilic (optimal growth temperature = 78°C) and anaerobic bacterium that can break down crystalline cellulose and hemicellulose into fermentable C 5 and C 6 sugars. In addition , C. bescii grows on unpretreated plant biomass, such as switchgrass. Biosynthesis of 2,3-BDO involves three steps: two molecules of pyruvate are condensed into acetolactate; acetolactate is decarboxylated to acetoin, and finally, acetoin is reduced to 2,3-BDO. C. bescii natively produces acetoin; therefore, in order to complete the 2,3-BDO biosynthetic pathway, C. bescii was engineered to produce a secondary alcohol dehydrogenase (sADH) to catalyze the final step. Two previously characterized, thermostable sADH enzymes with high affinity for acetoin, one from a bacterium and one from an archaeon, were tested independently. When either sADH was present in C. bescii, the recombinant strains were able to produce up to 2.5-mM 2,3-BDO from crystalline cellulose and xylan and 0.2-mM 2,3-BDO directly from unpretreated switchgrass. This serves as the basis for higher yields and productivities, and to this end, limiting factors and potential genetic targets for further optimization were assessed using the genome-scale metabolic model of C. bescii .IMPORTANCELignocellulosic plant biomass as the substrate for microbial synthesis of 2,3-butanediol is one of the major keys toward cost-effective bio-based production of this chemical at an industrial scale. However, deconstruction of biomass to release the sugars for microbial growth currently requires expensive thermochemical and enzymatic pretreatments. In this study, the thermo-cellulolytic bacterium Caldicellulosiruptor bescii was successfully engineered to produce 2,3-butanediol from cellulose, xylan, and directly from unpretreated switchgrass. Genome-scale metabolic modeling of C. bescii was applied to adjust carbon and redox fluxes to maximize productivity of 2,3-butanediol, thereby revealing bottlenecks that require genetic modifications., Competing Interests: The authors declare no conflict of interest.

2,3-Butanediol (2,3-BDO) is an important gateway molecule for many chemical derivatives. Currently, microbial production is gradually being recognized as a green and sustainable alternative to petrochemical synthesis, but the titer, yield, and productivity of microbial 2,3-BDO remain suboptimal. Here, we used systemic metabolic engineering strategies to debottleneck the 2,3-BDO production in Enterobacter aerogenes. Firstly, the pyruvate metabolic network was reconstructed by deleting genes for by-product synthesis to improve the flux toward 2,3-BDO synthesis, which resulted in a 90% increase of the product titer. Secondly, the 2,3-BDO productivity of the IAM1183-LPCT/D was increased by 55% due to the heterologous expression of DR1558 which boosted cell resistance to abiotic stress. Thirdly, carbon sources were optimized to further improve the yield of target products. The IAM1183-LPCT/D showed the highest titer of 2,3-BDO from sucrose, 20% higher than that from glucose, and the yield of 2,3-BDO reached 0.49 g/g. Finally, the titer of 2,3-BDO of IAM1183-LPCT/D in a 5-L fermenter reached 22.93 g/L, 85% higher than the wild-type strain, and the titer of by-products except ethanol was very low. KEY POINTS: Deletion of five key genes in E. aerogenes improved 2,3-BDO production The titer of 2,3-BDO was increased by 90% by regulating metabolic flux Response regulator DR1558 was expressed to increase 2,3-BDO productivity., (© 2024. The Author(s).)

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Anodic electro-fermentation (AEF), where an anode replaces the terminal electron acceptor, shows great promise. Recently a Lactococcus lactis strain blocked in NAD + regeneration was demonstrated to use ferricyanide as an alternative electron acceptor to support fast growth, but the need for high concentrations of this non-regenerated electron acceptor limits practical applications. To address this, growth of this L. lactis strain, and an adaptively evolved (ALE) mutant with enhanced ferricyanide respiration capacity were investigated using an anode as electron acceptor in a bioelectrochemical system (BES) setup. Both strains grew well, however, the ALE mutant significantly faster. The ALE mutant almost exclusively generated 2,3-butanediol, whereas its parent strain mainly produced acetoin. The ALE mutant interacted efficiently with the anode, achieving a record high current density of 0.81 ± 0.05 mA/cm 2 . It is surprising that a Lactic Acid Bacterium, with fermentative metabolism, interacts so well with an anode, which demonstrates the potential of AEF., 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Saccharomyces cerevisiae is a promising host for the bioproduction of higher alcohols, such as 2,3-butanediol (2,3-BDO). Metabolically engineered S. cerevisiae strains that produce 2,3-BDO via glycolysis have been constructed. However, the specific 2,3-BDO production rates of engineered strains must be improved. To identify approaches to improving the 2,3-BDO production rate, we investigated the factors contributing to higher ethanol production rates in certain industrial strains of S. cerevisiae compared to laboratory strains. Sequence analysis of 11 industrial strains revealed the accumulation of many nonsynonymous substitutions in RIM15 , a negative regulator of high fermentation capability. Comparative metabolome analysis suggested a positive correlation between the rate of ethanol production and the activity of the pyruvate-consuming pathway. Based on these findings, RIM15 was deleted, and the pyruvate-consuming pathway was activated in YHI030, a metabolically engineered S. cerevisiae strain that produces 2,3-BDO. The titer, specific production rate, and yield of 2,3-BDO in the test tube-scale culture using the YMS106 strain reached 66.4 ± 4.4 mM, 1.17 ± 0.017 mmol (g dry cell weight h) -1 , and 0.70 ± 0.03 mol (mol glucose consumed) -1 . These values were 2.14-, 2.92-, and 1.81-fold higher than those of the vector control, respectively. These results suggest that bioalcohol production via glycolysis can be enhanced in a metabolically engineered S. cerevisiae strain by deleting RIM15 and activating the pyruvate-consuming pathway.

Enterobacter hormaechei is part of the Enterobacter cloacae complex (ECC), which is widespread in nature. It is a facultative Gram-negative bacterium of medical and industrial importance. We assessed the metabolic and genetic repertoires of a new Enterobacter isolate. Here, we report the whole-genome sequence of a furfural- and 5-hydroxymethyl furfural (HMF)-tolerant strain of E. hormaechei (UW0SKVC1), which uses glucose, glycerol, xylose, lactose and arabinose as sole carbon sources. This strain exhibits high tolerance to furfural (IC 50 = 34.2 mM; ~3.3 g/L) relative to Escherichia coli DH5α (IC 50 = 26.0 mM; ~2.5 g/L). Furfural and HMF are predominantly converted to their less-toxic alcohols. E. hormaechei UW0SKVC1 produces 2,3-butanediol, acetoin, and acetol, among other compounds of industrial importance. E. hormaechei UW0SKVC1 produces as high as ~42 g/L 2,3-butanediol on 60 g/L glucose or lactose. The assembled genome consists of a 4,833,490-bp chromosome, with a GC content of 55.35%. Annotation of the assembled genome revealed 4586 coding sequences and 4516 protein-coding genes (average length 937-bp) involved in central metabolism, energy generation, biodegradation of xenobiotic compounds, production of assorted organic compounds, and drug resistance. E. hormaechei UW0SKVC1 shows considerable promise as a biocatalyst and a genetic repository of genes whose protein products may be harnessed for the efficient bioconversion of lignocellulosic biomass, abundant glycerol and lactose-replete whey permeate to value-added chemicals.

2,3-Butanediol (2,3-BD) is an alcohol highly demanded in the chemical, pharmaceutical, and food industries. Its microbial production, safe non-pathogenic producer strains, and suitable substrates have been avidly sought in recent years. The present study investigated 2,3-BD synthesis by the GRAS Bacillus licheniformis 24 using chicory inulin as a cheap and renewable substrate. The process appears to be pH-dependent. At pH 5.25, the synthesis of 2,3-BD was barely detectable due to the lack of inulin hydrolysis. At pH 6.25, 2,3-BD concentration reached 67.5 g/L with rapid hydrolysis of the substrate but was accompanied by exopolysaccharide (EPS) synthesis. Since inulin conversion by bacteria is a complex process and begins with its hydrolysis, the question of the acting enzymes arose. Genome mining revealed that several glycoside hydrolase (GH) enzymes from different CAZy families are involved. Five genes encoding such enzymes in B. licheniformis 24 were amplified and sequenced: sacA , sacB , sacC , levB, and fruA . Real-time RT-PCR experiments showed that the process of inulin hydrolysis is regulated at the level of gene expression, as four genes were significantly overexpressed at pH 6.25. In contrast, the expression of levB remained at the same level at the different pH values at all-time points. It was concluded that the sacC and sacA/fruA genes are crucial for inulin hydrolysis. They encode exoinulinase (EC 3.2.1.80) and sucrases (EC 3.2.1.26), respectively. The striking overexpression of sacB under these conditions led to increased synthesis of EPS; therefore, the simultaneous production of 2,3-BD and EPS cannot be avoided.

Rhizoctonia solani (RS) is a pathogenic fungus that affects maize ( Zea mays L.) plants and causes banded leaf and sheath blight (BLSB) with severe consequences leading to significant economic losses. Contrarily, rhizobacteria produce numerous volatile organic compounds (VOCs) that help in devising the environment-friendly mechanism for promoting plant growth and stress alleviation without having physical contact with plants. In the present study, 15 rhizobacterial strains were tested for their antagonism against RS. The antagonistic potential of VOCs of the tested plant growth-promoting rhizobacteria (PGPR) strains ranged from 50% to 80% as compared to the control (without PGPR). Among these 15 strains, the maximum (80%) antagonistic activity was exhibited by Pseudomonas pseudoalcaligenes SRM-16. Thus, the potential of VOCs produced by P. pseudoalcaligenes SRM-16 to alleviate the BLSB disease in maize was evaluated. A pot experiment was conducted under greenhouse conditions to observe the effect of VOCs on disease resistance of BLSB-infected seedlings. Overall, maize seedlings exposed to VOCs showed a significant increase in disease resistance as indicated by a reduced disease score than that of unexposed infected plants. The VOCs-exposed maize exhibited lower (11.6%) disease incidence compared to the non-inoculated maize (14.1%). Moreover, plants exposed to VOCs displayed visible improvements in biomass, photosynthetic pigments, osmoregulation, and plant antioxidant and defense enzyme activities compared to the healthy but unexposed seedlings. Simultaneous application of RS and VOCs enhanced superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), phenylalanine ammonia lyase (PAL), ascorbate peroxidase (APX), and polyphenol oxidase (PPO) activities by 96.7%, 266.6%, 313.7%, 246.6%, 307%, and 149.7%, respectively, in the roots and by 81.6%, 246.4%, 269.5%, 269.6%, 329%, and 137.6%, respectively, in the shoots, relative to those of the control plants. The binding affinity of the VOCs (2-pentylfuran, 2,3-butanediol, and dimethyl disulfide) with CRZ1 and S9 protein receptors of RS was assessed by deploying in silico methods. Overall, 2-pentylfuran exhibited a binding affinity with both the selected receptors of RS, while 2,3-butanediol and dimethyl disulfide were able to bind S9 protein only. Hence, it can be deduced that S9 protein receptors are more likely the target RS receptors of bacterial VOCs to inhibit the proliferation of RS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yasmin, Shah, Mumtaz, Ilyas, Rashid, Alsahli and Chung.)

3-Hydroxybutanone (Acetoin, AC) and 2,3-butanediol (BD) are two essential four-carbon platform compounds with numerous pharmaceutical and chemical synthesis applications. AC and BD have two and three stereoisomers, respectively, while the application of the single isomer product in chemical synthesis is superior. AC and BD are glucose overflow metabolites produced by biological fermentation from a variety of microorganisms. However, the AC or BD produced by microorganisms using glucose is typically a mixture of various stereoisomers. This was discovered to be due to the simultaneous presence of multiple butanediol dehydrogenases (BDHs) in microorganisms, and AC and BD can be interconverted under BDH catalysis. In this paper, beginning with the synthesis pathways of microbial AC and BD, we review in detail the studies on the formation mechanisms of different stereoisomers of AC and BD, summarize the properties of different types of BDH that have been tabulated, and analyze the structural characteristics and affinities of different types of BDH by comparing them using literature and biological database data. Using microorganisms, recent research on the production of optically pure AC or BD was also reviewed. Limiting factors and possible solutions for chiral AC and BD production are discussed., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The valorization of fruit and vegetable residues (such as carrot discard) and their microbial conversion into 2,3-butanediol (BDO) can be considered as a very interesting way to reduce food waste and sustainably originate high value-added products. This work analyzes the valorization of carrot discard as feedstock for 2,3-butanediol (BDO) production by Paenibacillus polymyxa DSM 365. The influences of stirring and the presence of tryptone (nitrogen source) are studied. Furthermore, in order to evaluate the influence of the pre-culture medium (nitrogen source, nutrients, and pH) and the substrate, fermentation assays in simple and mixture semi-defined media (glucose, fructose, and/or galactose) were also carried out. As a result, 18.8 g/L BDO, with a BDO yield of 0.43 g/g (86% of its theoretical value), could be obtained from carrot discard enzymatic hydrolysate at 100 rpm, no tryptone, and pre-culture Häßler medium. No hydrothermal pre-treatment was necessary for BDO production from carrot discard, which increases the profitability of the process. Therefore, 18.8 g BDO, as well as 2.5 g ethanol and 2.1 g acetoin by-products, could be obtained from 100 g of carrot discard (dry matter).

Breast cancer patients are characterized by the oncobiotic transformation of multiple microbiome communities, including the gut microbiome. Oncobiotic transformation of the gut microbiome impairs the production of antineoplastic bacterial metabolites. The goal of this study was to identify bacterial metabolites with antineoplastic properties. We constructed a 30-member bacterial metabolite library and screened the library compounds for effects on cell proliferation and epithelial-mesenchymal transition. The metabolites were applied to 4T1 murine breast cancer cells in concentrations corresponding to the reference serum concentrations. However, yric acid, glycolic acid, d-mannitol, 2,3-butanediol, and trans-ferulic acid exerted cytostatic effects, and 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, and vanillic acid exerted hyperproliferative effects. Furthermore, 3-hydroxyphenylacetic acid, 4-hydroxybenzoic acid, 2,3-butanediol, and hydrocinnamic acid inhibited epithelial-to-mesenchymal (EMT) transition. We identified redox sets among the metabolites (d-mannitol-d-mannose, 1-butanol-butyric acid, ethylene glycol-glycolic acid-oxalic acid), wherein only one partner within the set (d-mannitol, butyric acid, glycolic acid) possessed bioactivity in our system, suggesting that changes to the local redox potential may affect the bacterial secretome. Of the nine bioactive metabolites, 2,3-butanediol was the only compound with both cytostatic and anti-EMT properties.

Bacterial cellulose (BC) is a bio-produced nanostructure material widely used in biomedical, food, and paper-manufacturing industries. However, low production efficiency and high-cost have limited its industrial applications. This study aimed to examine the level of improvement in BC production by co-culturing Bacillus cereus and Komagataeibacter xylinus. The BC yield in corn stover enzymatic hydrolysate was found to be obviously enhanced from 1.2 to 4.4 g/L after the aforementioned co-culturing. The evidence indicated that acetoin (AC) and 2,3-butanediol (2,3-BD) produced by B. cereus were the key factors dominating BC increment. The mechanism underlying BC increment was that AC and 2,3-BD increased the specific activity of AC dehydrogenase and the contents of adenosine triphosphate (ATP) and acetyl coenzyme A (acetyl-CoA), thus promoting the growth and energy level of K. xylinus. Meanwhile, the immobilization of BC could also facilitate oxygen acquisition in B. cereus under static conditions. This study was novel in reporting that the co-culture could effectively enhance BC production from the lignocellulosic enzymatic hydrolysate., 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 © 2023 Elsevier Ltd. All rights reserved.)

The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol (2,3-BDO), organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene ( acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a Δ acoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sheng, Madika, Lau, Zhang and Minton.)

Sugarcane industry is a major agricultural sector capable of producing sugars with byproducts including straw, bagasse, and molasses. Sugarcane byproducts are no longer wastes since they can be converted into carbon-rich resources for biorefinery if pretreatment of these is well established. Considerable efforts have been devoted to effective pretreatment techniques for each sugarcane byproduct to supply feedstocks in microbial fermentation to produce value-added fuels, chemicals, and polymers. These value-added chains, which start with low-value industrial wastes and end with high-value products, can make sugarcane-based biorefinery a more viable option for the modern chemical industry. In this review, recent advances in sugarcane valorization techniques are presented, ranging from sugarcane processing, pretreatment, and microbial production of value-added products. Three lucrative products, ethanol, 2,3-butanediol, and polyhydroxyalkanoates, whose production from sugarcane wastes has been widely researched, are being explored. Future studies and development in sugarcane waste biorefinery are discussed to overcome the challenges remaining., 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 © 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.)

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.

Background: Flux Balance Analysis (FBA) is a well-known bioinformatics tool for metabolic engineering design. Previously, we have successfully used single-level FBA to design metabolic fluxes in Bacillus subtilis to enhance (R,R)-2,3-butanediol (2,3-BD) production from glycerol. OptKnock is another powerful technique for devising gene deletion strategies to maximize microbial growth coupling with improved biochemical production. It has never been used in B. subtilis. In this study, we aimed to compare the use of single-level FBA and OptKnock for designing enhanced 2,3-BD production from glycerol in B. subtilis., Results: Single-level FBA and OptKnock were used to design metabolic engineering approaches for B. subtilis to enhance 2,3-BD production from glycerol. Single-level FBA indicated that deletion of ackA, pta, lctE, and mmgA would improve the production of 2,3-BD from glycerol, while OptKnock simulation suggested the deletion of ackA, pta, mmgA, and zwf. Consequently, strains LM01 (single-level FBA-based) and MZ02 (OptKnock-based) were constructed, and their capacity to produce 2,3-BD from glycerol was investigated. The deletion of multiple genes did not negatively affect strain growth and glycerol utilization. The highest 2,3-BD production was detected in strain LM01. Strain MZ02 produced 2,3-BD at a similar level as the wild type, indicating that the OptKnock prediction was erroneous. Two-step FBA was performed to examine the reason for the erroneous OptKnock prediction. Interestingly, we newly found that zwf gene deletion in strain MZ02 improved lactate production, which has never been reported to date. The predictions of single-level FBA for strain MZ02 were in line with experimental findings., Conclusions: We showed that single-level FBA is an effective approach for metabolic design and manipulation to enhance 2,3-BD production from glycerol in B. subtilis. Further, while this approach predicted the phenotypes of generated strains with high precision, OptKnock prediction was not accurate. We suggest that OptKnock modelling predictions be evaluated by using single-level FBA to ensure the accuracy of metabolic pathway design. Furthermore, the zwf gene knockout resulted in the change of metabolic fluxes to enhance the lactate productivity., (© 2023. The Author(s).)

Plant roots are colonized by rhizobacteria, and these soil microorganisms can not only stimulate plant growth but also increase tolerance to stress through the production of volatile organic compounds. However, little is known about the effect that these plant beneficial volatiles may have on bacteria. In this study, the effects on growth and oxidative status of different concentrations of three volatiles already reported to have a positive influence on plant growth (2-butanone, 3-methyl-1-butanol, and 2,3-butanediol) were determined in A. thaliana and Rhizobium sp. strain E20-8 via airborne exposure in the presence and absence of Cd. It was expected to ascertain if the plant and the bacterium are influenced in the same way by the volatiles, and if exposure to stress (Cd) shifts the effects of volatiles on plants and bacteria. Results showed the antioxidant activity of the volatiles protecting the plant cell metabolism from Cd toxicity and increasing plant tolerance to Cd. Effects on bacteria were less positive. The two alcohols (3-methyl-1-butanol and 2,3-butanediol) increased Cd toxicity, and the ketone (2-butanone) was able to protect Rhizobium from Cd stress, constituting an alternative way to protect soil bacterial communities from stress. The application of 2-butanone thus emerges as an alternative way to increase crop production and crop resilience to stress in a more sustainable way, either directly or through the enhancement of PGPR activity.

As a valuable platform chemical, 2,3-Butanediol (2,3-BDO) has a variety of industrial applications, and its microbial production is particularly attractive as an alternative to petroleum-based production. In this study, the regulation of intracellular carbon flux and NADH/NAD + was used to increase the 2,3-BDO production of Enterobacter aerogenes. The genes encoding lactate dehydrogenase (ldh) and pyruvate formate lyase (pfl) were disrupted using the λ-Red recombination method and CRISPR-Cas9 to reduce the production of several byproducts and the consumption of NADH. Knockout of ldh or pfl increased intracellular NADH/NAD + by 111 % and 113 %, respectively. Moreover, two important genes in the 2,3-BDO biosynthesis pathway, acetolactate synthase (budB) and acetoin reductase (budC), were overexpressed in E. aerogenes to further amply the metabolic flux toward 2,3-BDO production. And the overexpression of budB or budC increased intracellular NADH/NAD + by 46 % and 57 %, respectively. In shake-flask cultivation with sucrose as carbon source, the 2,3-BDO titer of the IAM1183-LPBC was 3.55 times that of the wild type. In the 5-L fermenter, the maximal 2,3-BDO production produced by the IAM1183-LPBC was 2.88 times that of the original strain. This work offers new ideas for promoting the biosynthesis of 2,3-BDO for industrial 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 © 2022 Elsevier B.V. All rights reserved.)

As part of the transition from a fossil resources-based economy to a bio-based economy, the production of platform chemicals by microbial cell factories has gained strong interest. 2,3-butanediol (2,3-BDO) has various industrial applications, but its production by microbial fermentation poses multiple challenges. We have engineered the bacterial 2,3-BDO synthesis pathway, composed of AlsS, AlsD and BdhA, in a pdc-negative version of an industrial Saccharomyces cerevisiae yeast strain. The high concentration of glycerol caused by the excess NADH produced in the pathway from glucose to 2,3-BDO was eliminated by overexpression of NoxE and also in a novel way by combined overexpression of NDE1, encoding mitochondrial external NADH dehydrogenase, and AOX1, encoding a heterologous alternative oxidase expressed inside the mitochondria. This was combined with strong downregulation of GPD1 and deletion of GPD2, to minimize glycerol production while maintaining osmotolerance. The HGS50 strain produced a 2,3-BDO titer of 121.04 g/L from 250 g/L glucose, the highest ever reported in batch fermentation, with a productivity of 1.57 g/L.h (0.08 g/L.h per gCDW) and a yield of 0.48 g/g glucose or with 96% the closest to the maximum theoretical yield ever reported. Expression of Lactococcus lactis NoxE, encoding a water-forming NADH oxidase, combined with similar genetic modifications, as well as expression of Candida albicans STL1, also minimized glycerol production while maintaining high osmotolerance. The HGS37 strain produced 130.64 g/L 2,3-BDO from 280 g/L glucose, with productivity of 1.58 g/L.h (0.11 g/L.h per gCDW). Both strains reach combined performance criteria adequate for industrial implementation., (© 2022. The Author(s).)

Low-temperature biorefineries inhibit the multiplication of undesired microorganisms, improve product purity and reduce economic costs. Herein, to improve the 2,3-butanediol (2,3-BD) bioconversion efficiency from hemicellulose, a psychrotrophic hemicellulose-degrading strain Raoultella terrigena HC6 with high β-xylosidase activity 1520 U/mL was isolated and genetically modified. Xylan (hemicellulose replacement) was depolymerized into xylooligosaccharides (XOS) and xylose by HC6, which were further converted into 2,3-BD. Transcriptomic analysis revealed that β-xylosidase gene (xynB) and xylose isomerase gene (xylA), which are beneficial for increasing the carbon flux from xylan to 2,3-BD, were significantly upregulated 56.9-fold and 234-fold, respectively. A recombinant strain was constructed by overexpressing xynB in HC6, which obtained 0.389 g/g yield of 2,3-BD from hemicellulose extracted from corn straw at 15 °C. This study proposed a promised strategy for the bioconversion of agricultural waste into 2,3-BD at low temperatures and provides a basis for future efforts in the achievement of carbon neutrality., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Bread is Europe's most wasted food, and the second most wasted food after potatoes in UK. Bread waste (BW) is a clean source of high-quality fermentable sugars. In this study, the potential of Enterobacter ludwigii to accumulate 2,3-butanediol (BDO) from BW was evaluated. Initially, the optimal inoculum size and yeast extract concentration were determined, followed by extraction of sugars from BW using acid and enzymatic hydrolysis. A glucose yield of 330-530 g/kg BW was obtained, and the sugars released were utilised for BDO production by E. ludwigii. The fed-batch cultivation using pure glucose and glucose rich hydrolysates from acid and enzymatic hydrolysis resulted in BDO titres of 144.5, 135.4, and 138.8 g/L, after 96 h, with yield of 0.47, 0.42 and 0.48 g/g yield, respectively. The innovation of the work is valorisation of BW to BDO with a circular biorefining approach and thus, reducing BW disposal and associated environmental burden., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacterial wilt caused by Ralstonia solanacearum is a fatal disease that affects the production of tomatoes and many other crops worldwide. As an effective strategy to manage bacterial wilt, biological control agents using plant growth-promoting rhizobacteria (PGPR) are being developed. In this study, we screened 2,3-butanediol (BDO)-producing PGPR to control tomato bacterial wilt and investigated the action mechanism of the disease control agent. Of the 943 strains isolated from soil, Klebsiella pneumoniae strain JCK-2201 produced the highest concentration of 2,3-BDO. The culture broth of K. pneumoniae JCK-2201 did not show any direct activity on R. solanacearum in vitro , but a 100-fold dilution effectively controlled tomato bacterial wilt with a control value of 77% in vivo . Fermentation utilizing K. pneumoniae JCK-2201 was optimized to produce 48 g/L of meso-2,3-BDO, which is 50% of the sucrose conversion efficiency. In addition, the control efficacy and mechanism of meso-2,3-BDO produced by JCK-2201 in tomato bacterial wilt were determined by comparative analysis with Bacillus licheniformis DSM13 producing meso-2,3-BDO and B. licheniformis DSM13 ΔalsS that did not produce 2,3-BDO, as the step of converting pyruvate to α-acetolactate was omitted. Tomato seedlings treated with the K. pneumoniae JCK-2201 (500-fold dilution) and B. licheniformis DSM13 (100-fold dilution) culture broth produced meso-2,3-BDO that significantly reduced R. solanacearum -induced disease severity with control values of 55% and 63%, respectively. The formulated meso-2,3-BDO 9% soluble concentrate (SL; 1,000-fold dilution) showed 87% control against tomato bacterial wilt in the field condition. Klebsiella pneumoniae JCK-2201 and B. licheniformis DSM13 treatment induced the expression of plant defense marker genes, such as LePR1 , LePR2 , LePR5 , LePR3 , and PI-II , in the salicylic acid and jasmonic acid signaling pathways at 4 days after inoculation. These results show that 2,3-BDO-producing bacteria and 2,3-BDO are potential biological control agents that act through induction of resistance for controlling tomato bacterial wilt., Competing Interests: CS and HS were employed by the GS Caltex Corporation of South Korea. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kim, Park, Song, Song and Kim.)

In recent years, residual glycerol from biodiesel synthesis made this chemical a cheap, readily available carbon source to bioprocess, which is also a form to reduce costs in the fuel industry. We propose and describe a bioprocess using fluidized and packed-bed continuous bioreactors to convert this residual glycerol into value-added products such as 1,3-propanediol (1,3-PD) and 2,3-butanediol (2,3-BD), largely used in the chemical industry. The bacterium Klebsiella pneumoniae BLh-1, strain isolated by us, was immobilized in the permeable support of polyvinyl alcohol (LentiKats®). After testing different dilution rates (D) for all bioreactor configurations, the best obtained productivities of 1,3-PD was 8.69 g L -1  h -1 at a D = 0.45 h -1 , and 2.99 g L -1  h -1 at a D = 0.30 h -1 for 2,3-BD, both in the packed-bed configuration. In the fluidized-bed reactor, the highest productivity values achieved were 4.48 and 1.16 g L -1  h -1 for 1,3-PD and 2,3-BD, respectively, both at D = 0.33 h -1 . These results show the potential of setting up a bioprocess based on continuous cultures using immobilized K. pneumoniae BLh-1 in PVA matrices in order to efficiently convert the abundant surplus of glycerol into commercially important chemicals such as 1,3-PD and 2,3-BD., (© 2022 American Institute of Chemical Engineers.)

Background: 2,3-Butanediol (2,3-BD), a valuable compound used for chemicals, cosmetics, pesticides and pharmaceuticals, has been produced by various microbes. However, no high-temperature fermentation of the compound at high productivity has been reported., Methods: Thermotolerant xylose-utilizing microbes were isolated from 6 different districts in Laos and screened for a low accumulation of xylitol in a xylose medium at 37 ˚C. One isolate was found to produce 2,3-BD and identified by 16S rDNA sequencing. The 2,3-BD fermentation capacity was investigated at different temperatures using xylose and glucose as carbon sources, and the fermentation parameters were determined by a high-performance liquid chromatography system., Results: By screening for a low accumulation of xylitol in a xylose medium, one isolate that accumulated almost no xylitol was obtained. Further analyses revealed that the isolate is Cronobacter sakazakii and that it has the ability to produce 2,3-BD at high temperatures. When xylose and glucose were used, this strain, named C. sakazakii OX-25, accumulated 2,3-BD in a short period before the complete consumption of these sugars and then appeared to convert 2,3-BD to acetoin. The optimum temperature of the 2,3-BD fermentation was 42 ˚C to 45 ˚C, and the maximum yield of 2,3-BD was 0.3 g/g at 12 h in 20 g/l xylose medium and 0.4 g/g at 6 h in 20 g/l glucose medium at 42 ˚C. The 2,3-BD productivity of the strain was higher than the 2,3-BD productivities of other non-genetically engineered microorganisms reported previously, and the highest productivity was 0.6 g/l·h and 1.2 g/l·h for xylose and glucose, respectively., Conclusions: Among thermotolerant microbes isolated in Laos, we discovered a strain, C. sakazakii OX-25, that can convert xylose and glucose to 2,3-BD with high efficiency and high productivity at high temperatures, suggesting that C. sakazakii OX-25 has the potential for industrial application to produce 2,3-BD as an important platform chemical., (© 2022. The Author(s).)

Pretreatment of lignocellulose is a vital step for biological production of bio-chemicals and bio-fuels. In this work, the pretreatment of Jerusalem artichoke stalk (JAS) by hydroxylammonium ionic liquids was evaluated based on pretreatment efficiency including polysaccharide recovery and enzymatic digestibility, and influence of ionic liquids on 2,3-butanediol fermentation using Bacillus subtilis. The results showed ethanolammonium acetate (EOAA) was efficient in JAS pretreatment, and maximum cell density was increased 25% when EOAA concentration was not greater than 0.3 mol/L in medium, while the total concentration of acetoin and 2,3-butanediol was 15% greater than the control at 0.1 mol/L EOAA. After the pretreatment under optimized conditions of 170 °C for 5-h and liquid-solid ratio of 18, about 87% cellulose and 75% hemicellulose were recovered, and glucose yield of 64% and xylose of 66% were obtained after 24-h hydrolysis of JAS residue by cellulase (15 FPU/g) with solid loading of 10 wt%., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Among abiotic stresses in plants, drought and chilling stresses reduce the supply of moisture to plant tissues, inhibit photosynthesis, and severely reduce plant growth and yield. Thus, the application of water stress-tolerant agents can be a useful strategy to maintain plant growth under abiotic stresses. This study assessed the effect of exogenous bio-based 2,3-butanediol (BDO) application on drought and chilling response in tomato and turfgrass, and expression levels of several plant signaling pathway-related gene transcripts. Bio-based 2,3-BDOs were formulated to levo-2,3-BDO 0.9% soluble concentrate (levo 0.9% SL) and meso-2,3-BDO 9% SL (meso 9% SL). Under drought and chilling stress conditions, the application of levo 0.9% SL in creeping bentgrass and meso 9% SL in tomato plants significantly reduced the deleterious effects of abiotic stresses. Interestingly, pretreatment with levo-2,3-BDO in creeping bentgrass and meso-2,3-BDO in tomato plants enhanced JA and SA signaling pathway-related gene transcript expression levels in different ways. In addition, all tomato plants treated with acibenzolar- S -methyl (as a positive control) withered completely under chilling stress, whereas 2,3-BDO-treated tomato plants exhibited excellent cold tolerance. According to our findings, bio-based 2,3-BDO isomers as sustainable water stress-tolerant agents, levo- and meso-2,3-BDOs, could enhance tolerance to drought and/or chilling stresses in various plants through somewhat different molecular activities without any side effects.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4 g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55 h of cultivation, with a substrate conversion ratio of 0.27 mol/mol glucose., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Brewers' spent grains (BSG) are a by-product of the brewing industry that is mainly used as feedstock; otherwise, it has to be disposed according to regulations. Due to the high content of glucose and xylose, after pretreatment and hydrolysis, it can be used as a main carbohydrate source for cultivation of microorganisms for production of biofuels or biochemicals like 2,3-butanediol or lactate. 2,3-Butanediol has applications in the pharmaceutical or chemical industry as a precursor for varnishes and paints or in the food industry as an aroma compound. So far, Klebsiella pneumoniae , Serratia marcescens , Clostridium sp. , and Enterobacter aerogenes are being used and investigated in different bioprocesses aimed at the production of 2,3-butanediol. The main drawback is bacterial pathogenicity which complicates all production steps in laboratory, pilot, and industrial scales. In our study, a gram-positive GRAS bacterium Paenibacillus polymyxa DSM 742 was used for the production of 2,3-butanediol. Since this strain is very poorly described in literature, bacterium cultivation was performed in media with different glucose and/or xylose concentration ranges. The highest 2,3-butanediol concentration of 18.61 g l -1 was achieved in medium with 70 g l -1 of glucose during 40 h of fermentation. In contrast, during bacterium cultivation in xylose containing medium there was no significant 2,3-butanediol production. In the next stage, BSG hydrolysates were used for bacterial cultivation. P. polymyxa DSM 742 cultivated in the liquid phase of pretreated BSG produced very low 2,3-butanediol and ethanol concentrations. Therefore, this BSG hydrolysate has to be detoxified in order to remove bacterial growth inhibitors. After detoxification, bacterium cultivation resulted in 30 g l -1 of lactate, while production of 2,3-butanediol was negligible. The solid phase of pretreated BSG was also used for bacterium cultivation after its hydrolysis by commercial enzymes. In these cultivations, P. polymyxa DSM 742 produced 9.8 g l -1 of 2,3-butanediol and 3.93 g l -1 of ethanol. On the basis of the obtained results, it can be concluded that different experimental setups give the possibility of directing the metabolism of P. polymyxa DSM 742 toward the production of either 2,3-butanediol and ethanol or lactate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Didak Ljubas, Novak, Trontel, Rajković, Kelemen, Marđetko, Grubišić, Pavlečić, Tominac and Šantek.)

Pichia pastoris , an important methylotrophic yeast, is currently mainly used for the expression of recombinant proteins and has great potential applications in the production of value-added compounds (e.g., chemical and natural products). However, the construction of P. pastoris cell factories is largely hindered by the lack of genetic tools for the manipulation of multigene biosynthetic pathways. Therefore, the present study aimed to establish a CRISPR-based synthetic biology toolkit for the integration and assembly of multigene biosynthetic pathways into the chromosome of P. pastoris . First, 23 intergenic regions were selected and characterized as potential integration sites, with a focus on the integration efficiency and heterologous gene expression levels. In addition, a panel of constitutive and methanol-inducible promoters with different strengths (weak, medium, and strong promoters) were characterized to control the expression of biosynthetic pathway genes to the desirable levels. With a series of gRNA plasmids (for single-locus, two-loci, and three-loci integration) and donor plasmids (containing homology arms for integration and promoters and terminators for driving heterologous gene expression) as major components, a CRISPR-based synthetic biology toolkit was established, which enabled the integration of one locus, two loci, and three loci with efficiencies as high as ∼100, ∼93, and ∼75%, respectively, in P. pastoris GS115 strain. Finally, the application of the toolkit was demonstrated by the construction of a series of P. pastoris cell factories, which could produce 2,3-butanediol, β-carotene, zeaxanthin, and astaxanthin with methanol as the sole carbon and energy source. The P. pastoris synthetic biology toolkit is highly standardized and can be employed to construct P. pastoris cell factories with high efficiency.

Firstly, 2,3-butanediol (2,3-BDO) is a chemical platform used in several applications. However, the pathogenic nature of its producers and the expensive feedstocks used limit its scale production. In this study, cane molasses was used for 2,3-BDO production by a nonpathogenic Clostridium ljungdahlii . It was found that cane molasses alone, without the addition of other ingredients, was favorable for use as the culture medium for 2,3-BDO production. Compared with the control (i.e., the modified DSMZ 879 medium), the differential genes are mainly involved in the pathways of carbohydrate metabolism, membrane transport, and amino acid metabolism in the case of the cane molasses alone. However, when cane molasses alone was used, cell growth was significantly inhibited by KCl in cane molasses. Similarly, a high concentration of sugars (i.e., above 35 g/L) can inhibit cell growth and 2,3-BDO production. More seriously, 2,3-BDO production was inhibited by itself. As a result, cane molasses alone with an initial 35 g/L total sugars was suitable for 2,3-BDO production in batch culture. Finally, an integrated fermentation and membrane separation process was developed to maintain high 2,3-BDO productivity of 0.46 g·L -1 ·h -1 . Meanwhile, the varied fouling mechanism indicated that the fermentation properties changed significantly, especially for the cell properties. Therefore, the integrated fermentation and membrane separation process was favorable for 2,3-BDO production by C. ljungdahlii using cane molasses.

This study aimed to investigate the elicitation of volatile organic compounds ( E )-2-hexenal and 2,3-butanediol on bioactive metabolites in Astragalus membranaceus var. mongholicus adventitious root cultures by adding them into the medium. The experiment was performed for 72 h and the roots were dynamically sampled for quantification of representative astragaloside IV, calycosin-7- O -β-d-glucoside (CG), ononin, and the gene expression. Compared with the controls, the combination of 2,3-butanediol and ( E )-2-hexenal advanced the peak accumulation of astragaloside IV and was the most effective, but their individual application delayed it. Meanwhile, 2,3-butanediol and ( E )-2-hexenal had no obviously promoting effect on the production of CG and ononin but chronologically changed their accumulation patterns. The underlying mechanism was uncovered by the correlation analysis between the metabolites and the gene expression, as did the identification of the target genes. Collectively, 2,3-butanediol and ( E )-2-hexenal were important cues shaping the production of bioactive products in the herbal plant.

Fermentation of food waste into 2,3-butanediol (2,3-BDO), a high-value chemical, is environmentally sustainable and an inexpensive method to recycle waste. Compared to traditional mesophilic fermentation, thermophilic fermentation can inhibit the growth of contaminant bacteria, thereby improving the success of food waste fermentation. However, the effects of sugar and nutrient concentrations in thermophilic food waste fermentations are currently unclear. Here, we investigated the effects of sugar and nutrients (yeast extract (YE) and peptone) concentrations on 2,3-BDO production from fermenting glucose and food waste media using the newly isolated thermophilic Bacillus licheniformis YNP5-TSU. When glucose media was used, fermentation was greatly affected by sugar and nutrient concentrations: excessive glucose (>70 g/L) slowed down the fermentation and low nutrients (2 g/L YE and 1 g/L peptone) caused fermentation failure. However, when food waste media were used with low nutrient addition, the bacteria consumed all 57.8 g/L sugars within 24 h and produced 24.2 g/L 2,3-BDO, equivalent to a fermentation yield of 0.42 g/g. An increase in initial sugar content (72.9 g/L) led to a higher 2,3-BDO titer of 36.7 g/L with a nearly theoretical yield of 0.47 g/g. These findings may provide fundamental knowledge for designing cost-effective food waste fermentation to produce 2,3-BDO.

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.