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In recent years, 2,3-butanediol (BDO) has been recognized as a valuable platform molecule suitable for production of bulk and fine chemicals. BDO can be efficiently produced biotechnologically in high titer. Recovery of high-boiling and highly polar BDO from the fermentation broths presents the major challenge. In this study, we propose adsorption of BDO onto polymers bearing phenylboronate groups as a method for selective and efficient recovery. We optimized the adsorption parameters, such as NaOH concentration and the dilution factor, using solutions of commercially available BDO. Under the optimized conditions, nearly quantitative complexation of phenylboronates occurs, which corresponds to a loading of 250 mgBDO gPolymer−1. Desorption of BDO into acidic and neutral solutions was efficient. Most importantly, an 80%EtOH/20%H2O mixture can be used to recover BDO with up to 93% desorption efficiency. Two fermentation broths obtained with Bacillus licheniformis DSM 8785 were applied for adsorption studies. The material capacities upon adsorption from the fermentation broths were as high as from the model solutions. BDO uptake from the fermentation broth was very selective, with only negligible co-adsorption of other components, such as acetoin, lactate or succinate. Utilization of the polymer in three adsorption and two desorption cycles proved recyclability of the material. 1H, 11B, and 13C MAS NMR were used to monitor the adsorption and desorption processes. This work shows a high potential of the polymers bearing phenylboronate functionalities for adsorptive recovery of BDO from fermentation broths.

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

The growing need for industrial production of bio-based acetoin and 2,3-butanediol (2,3-BD) is due to both environmental concerns, and their widespread use in the food, pharmaceutical, and chemical industries. Acetoin is a common spice added to many foods, but also a valuable reagent in many chemical syntheses. Similarly, 2,3-BD is an indispensable chemical on the platform in the production of synthetic rubber, printing inks, perfumes, antifreeze, and fuel additives. This state-of-the-art review focuses on representatives of the genus Bacillus as prospective producers of acetoin and 2,3-BD. They have the following important advantages: non-pathogenic nature, unpretentiousness to growing conditions, and the ability to utilize a huge number of substrates (glucose, sucrose, starch, cellulose, and inulin hydrolysates), sugars from the composition of lignocellulose (cellobiose, mannose, galactose, xylose, and arabinose), as well as waste glycerol. In addition, these strains can be improved by genetic engineering, and are amenable to process optimization. Bacillus spp. are among the best acetoin producers. They also synthesize 2,3-BD in titer and yield comparable to those of the pathogenic producers. However, Bacillus spp. show relatively lower productivity, which can be increased in the course of challenging future research. [ABSTRACT FROM AUTHOR]

Gas fermentation by acetogens of the genus Clostridium is an attractive technology since it affords the production of biochemicals and biofuels from industrial waste gases while contributing to mitigate the carbon cycle alterations. The acetogenic model organisms C. ljungdahlii and C. autoethanogenum have already been used in large scale industrial fermentations. Among the natural products, ethanol production has already attained industrial scale. However, some acetogens are also natural producers of 2,3-butanediol (2,3-BDO), a platform chemical of relevant industrial interest. Here, we have developed a lab-scale screening campaign with the aim of enhancing 2,3-BDO production. Our study generated comparable data on growth and 2,3-BDO production of several batch gas fermentations using C. ljungdahlii and C. autoethanogenum grown on different gas substrates of primary applicative interest (CO2 · H2, CO · CO2, syngas) and on different media featuring different compositions as regards trace metals, mineral elements and vitamins. CO · CO2 fermentation was found to be preferable for the production of 2,3-BDO, and a fair comparison of the strains cultivated in comparable conditions revealed that C. ljungdahlii produced 3.43-fold higher titer of 2,3-BDO compared to C. autoethanogenum. Screening of different medium compositions revealed that mineral elements, Zinc and Iron exert a major positive influence on 2,3-BDO titer and productivity. Moreover, the CO2 influence on CO fermentation was explored by characterizing C. ljungdahlii response with respect to different gas ratios in the CO · CO2 gas mixtures. The screening strategies undertaken in this study led to the production of 2.03 ± 0.05 g/L of 2,3-BDO, which is unprecedented in serum bottle experiments. [ABSTRACT FROM AUTHOR]

BACKGROUND: For the efficient use of cassava as industrial material in the fermentation business, an integrated process was developed to produce 2,3‐butanediol (2,3‐BDO) and bioethanol (BE) by a highly efficient and economical method. After separating the liquefied cassava into a solid and a liquid part, the liquid part was used to produce 2,3‐BDO, and the solid part was then used to produce BE. RESULTS: The integrated process efficiently fermented 1000 g of dried cassava to 288 g of 2,3‐BDO (120.1 g L−1 titer and 2.39 L fermented broth) and 57 g of BE (38.4 g L−1 titer and 1.49 L fermented broth). Also, fermentable sugar of cassava was 100% available for this process, and the byproduct of BE production, condensed distillers solubles (CDS), was used as a potential nutrient source for the economical production of 2,3‐BDO. CONCLUSION: An integrated process with simultaneous saccharification and fermentation (SSF), pH shift, and rpm shift was developed to enhance 2,3‐BDO production and to reduce the production cost of 2,3‐BDO and BE. The strategy of employing cassava as a starchy biomass‐based biorefinery resource for 2,3‐BDO and BE production was successfully performed. © 2021 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

2,3-Butanediol (2,3-BD) is a promising platform chemical, produced from microbial cells. Oxygen availability is a crucial factor driving the formation and proportion of 2,3-BD and acetoin in 2,3-BD producing bacterial strains. In this study, the ability of B. subtills GD5 to produce 2,3-BD in optimized sucrose-based media was evaluated, by investigating the impact of carbon to nitrogen (C/N) ratio and the effectiveness of alternative low-cost nitrogen sources (corn steep liquor, soybean meal, and ammonium sulphate). Subsequently, different dissolved oxygen (DO) controlling regimes were assessed in batch bioreactor fermentations. The best fermentation outcomes were obtained with uncontrolled DO, achieving 5.88 g/L of optically pure (R,R)-2,3-BD (~100% purity), accompanied by a production yield of 0.43 g/g, and a productivity of 0.2 g/L/h. Additionally, the influence of the DO controlling regime on B. subtills key enzymes involved in the reverse activity of acetoin reductase was also monitored. A fed-batch process under the most suitable DO conditions was carried out to improve 2,3-BD production, achieving 42.31 g/L 2,3-BD with a production yield of 0.52 g/g. Thus, B. subtilis GD5 is a promising strain for the efficient production of pure chiral (R,R)-2,3-BD under uncontrolled DO conditions, using alternative low-cost nitrogen sources.

The goal of this study was to improve resource use efficiency in agricultural systems and agro-based industries, reduce wastes that go to landfills and incinerators, and consequently, improve the economics of 2,3-butanediol (2,3-BD) production. This study evaluated the feasibility of 2,3-BD production by replacing the mineral nutrients, and buffers with anaerobic digestate (ADE), poultry-litter (PLBC)- and forage-sorghum (FSBC)-derived biochars. Fermentation media formulations with ADE and 5–20 g/L PLBC or FSBC were evaluated for 2,3-BD production using Paenibacillus polymyxa as a biocatalyst. An optimized medium containing nutrients and buffers served as control. While 2,3-BD production in the ADE cultures was 0.5-fold of the maximum generated in the control cultures, 2,3-BD produced in the PLBC and FSBC cultures were ~1.3-fold more than the control (33.6 g/L). Cost analysis showed that ADE and biochar can replace mineral nutrients and buffers in the medium with the potential to make bio-based 2,3-BD production profitably feasible.

Biotechnological production of 2,3-butanediol (2,3-BD), a versatile platform bio-chemical and a potential biofuel, is limited due to by-product toxicity. In this study, we aimed to redirect the metabolic flux toward 2,3-BD in Enterobacter aerogenes (E. aerogenes) by increasing the intracellular NADH pool. Increasing the NADH/NAD+ ratio by knocking out the NADH dehydrogenase genes (nuoC/nuoD) enhanced 2,3-BD production by up to 67% compared with wild-type E. aerogenes. When lactate dehydrogenase (ldh) was knocked out, the yield of 2,3-BD was increased by 71.2% compared to the wild type. Metabolic flux analysis revealed that upregulated expression of the sRNA RyhB led to a noteworthy shift in metabolism. The 2,3-BD titer of the best mutant Ea-2 was almost seven times higher than that of the parent strain in a 5-L fermenter. In this study, an effective metabolic engineering strategy for improved 2,3-BD production was implemented by increasing the NADH/NAD+ ratio and blocking competing pathways.

Salt stress is one of the major abiotic stresses that affects plant growth and development. The use of plant growth-promoting rhizobacteria to mitigcate salt stress damage in plants is an important way to promote crop growth under salt stress conditions. Rahnella aquatilis JZ-GX1 is a plant growth-promoting rhizobacterial strain, but it is not clear whether it can improve the salt tolerance of plants, and in particular, the role of volatile substances in plant salt tolerance is unknown. We investigated the effects of volatile organic compounds (VOCs) from JZ-GX1 on the growth performance, osmotic substances, ionic balance and antioxidant enzyme activities of acacia seedlings treated with 0 and 100mm NaCl and explored the VOCs associated with the JZ-GX1 strain. The results showed that compared to untreated seedlings, seedlings exposed to plant growth-promoting rhizobacterium JZ-GX1 via direct contact with plant roots under salt stress conditions exhibited increases in fresh weight, lateral root number and primary root length equal to approximately 155.1, 95.4, and 71.3%, respectively. Robinia pseudoacacia seedlings exposed to VOCs of the JZ-GX1 strain showed increases in biomass, soil and plant analyser development values and lateral root numbers equal to 132.1, 101.6, and 166.7%, respectively. Additionally, decreases in malondialdehyde, superoxide anion (O2−) and hydrogen peroxide (H2O2) contents and increases in proline contents and superoxide dismutase, peroxidase and glutathione reductase activities were observed in acacia leaves. Importantly, the sodium-potassium ratios in the roots, stems, and leaves of acacia exposed to VOCs of the JZ-GX1 strain were significantly lower than those in the control samples, and this change in ion homeostasis was consistent with the upregulated expression of the (Na+, K+)/H+ reverse cotransporter RpNHX1 in plant roots. Through GC-MS and creatine chromatography, we also found that 2,3-butanediol in the volatile gases of the JZ-GX1 strain was one of the important signaling substances for improving the salt tolerance of plants. The results showed that R. aquatilis JZ-GX1 can promote the growth and yield of R. pseudoacacia under normal and salt stress conditions. JZ-GX1 VOCs have good potential as protectants for improving the salt tolerance of plants, opening a window of opportunity for their application in salinized soils.

Salt stress is one of the major abiotic stresses that affects plant growth and development. The use of plant growth-promoting rhizobacteria to mitigcate salt stress damage in plants is an important way to promote crop growth under salt stress conditions. Rahnella aquatilis JZ-GX1 is a plant growth-promoting rhizobacterial strain, but it is not clear whether it can improve the salt tolerance of plants, and in particular, the role of volatile substances in plant salt tolerance is unknown. We investigated the effects of volatile organic compounds (VOCs) from JZ-GX1 on the growth performance, osmotic substances, ionic balance and antioxidant enzyme activities of acacia seedlings treated with 0 and 100mm NaCl and explored the VOCs associated with the JZ-GX1 strain. The results showed that compared to untreated seedlings, seedlings exposed to plant growth-promoting rhizobacterium JZ-GX1 via direct contact with plant roots under salt stress conditions exhibited increases in fresh weight, lateral root number and primary root length equal to approximately 155.1, 95.4, and 71.3%, respectively. Robinia pseudoacacia seedlings exposed to VOCs of the JZ-GX1 strain showed increases in biomass, soil and plant analyser development values and lateral root numbers equal to 132.1, 101.6, and 166.7%, respectively. Additionally, decreases in malondialdehyde, superoxide anion (O2−) and hydrogen peroxide (H2O2) contents and increases in proline contents and superoxide dismutase, peroxidase and glutathione reductase activities were observed in acacia leaves. Importantly, the sodium-potassium ratios in the roots, stems, and leaves of acacia exposed to VOCs of the JZ-GX1 strain were significantly lower than those in the control samples, and this change in ion homeostasis was consistent with the upregulated expression of the (Na+, K+)/H+ reverse cotransporter RpNHX1 in plant roots. Through GC-MS and creatine chromatography, we also found that 2,3-butanediol in the volatile gases of the JZ-GX1 strain was one of the important signaling substances for improving the salt tolerance of plants. The results showed that R. aquatilis JZ-GX1 can promote the growth and yield of R. pseudoacacia under normal and salt stress conditions. JZ-GX1 VOCs have good potential as protectants for improving the salt tolerance of plants, opening a window of opportunity for their application in salinized soils. [ABSTRACT FROM AUTHOR]

Biotechnological production of 2,3-butanediol (2,3-BD), a versatile platform bio-chemical and a potential biofuel, is limited due to by-product toxicity. In this study, we aimed to redirect the metabolic flux toward 2,3-BD in Enterobacter aerogenes (E. aerogenes) by increasing the intracellular NADH pool. Increasing the NADH/NAD+ ratio by knocking out the NADH dehydrogenase genes (nuoC / nuoD) enhanced 2,3-BD production by up to 67% compared with wild-type E. aerogenes. When lactate dehydrogenase (ldh) was knocked out, the yield of 2,3-BD was increased by 71.2% compared to the wild type. Metabolic flux analysis revealed that upregulated expression of the sRNA RyhB led to a noteworthy shift in metabolism. The 2,3-BD titer of the best mutant Ea-2 was almost seven times higher than that of the parent strain in a 5-L fermenter. In this study, an effective metabolic engineering strategy for improved 2,3-BD production was implemented by increasing the NADH/NAD+ ratio and blocking competing pathways. [ABSTRACT FROM AUTHOR]

: 2,3-Butanediol (2,3-BD) is a reagent with remarkable commercial use as a platform chemical in numerous industries. The present study aims to determine the capabilities of non-pathogenic and cellulolytic Bacillus licheniformis 24 as a 2,3-BD producer. By applying the Plackett–Burman design and response surface methodology through central composite design (CCD), a complex optimization of medium and process parameters was conducted. Thus, among ten studied factors of medium content, four components were evaluated with a significant positive effect on 2,3-BD formation. Their optimal values for 2,3-BD production (yeast extract, 13.38 g/L; tryptone, 6.41 g/L; K2HPO4, 4.2 g/L; MgSO4, 0.32 g/L), as well as the optimal temperature (37.8 ◦C), pH (6.23) and aeration rate (3.68 vvm) were predicted by CCD experiments and validated in a series of batch processes. In optimized batch fermentation of 200 g/L of glucose 91.23 g/L of 2,3-BD was obtained, with the overall productivity of 1.94 g/L/h and yield of 0.488 g/g. To reveal the maximum 2,3-BD tolerance of B. licheniformis 24, fed-batch fermentation was carried out. The obtained 138.8 g/L of 2,3-BD with a yield of 0.479 g/g and productivity of 1.16 g/L/h ranks the strain among the best 2,3-BD producers. [ABSTRACT FROM AUTHOR]

The aim of this study was to validate an analytical method for the simultaneous quantification of 2,3-butanediol, glycerol, acetoin, ethanol and phosphate (PO43-) by high-performance liquid chromatography (HPLC) coupled with a refractive index detector. The validation was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) - Guideline Q2 (R1). The parameters of linearity, limits of detection and quantification, precision and accuracy were evaluated. The accuracy was evaluated using standards solutions and a microbial cultivation sample. The linearity (r ≥ 0.99, n = 3) of the method was defined in the range from 0.18 to 2.52 g L−1 for phosphate (PO43-); 0.5 to 10.0 g L−1 for glycerol, acetoin and ethanol; 0.375 to 7.5 g L−1 for meso-2,3-butanediol; and 0.125 to 2.5 g L−1 for (S,S)- or (R,R)-2,3-butanediol. The limits of detection and quantification were below the concentration range used in the method. The average recovery rate, intra-day and inter-day precisions for all compounds were 98.71, 0.09 and 0.50%, respectively. The results for the accuracy ranged between 97.97 and 101.18% for all compounds in the standard solution. The method was demonstrated to be precise, specific, and reproducible for the quantification of all compounds evaluated, ensuring the reliability. Further, this method was successfully applied to samples from bioprocesses with low matrix effects and an average recovery of 99.55%. [ABSTRACT FROM AUTHOR]

[Display omitted] • Pinch technology reduces 8% of BDO and 9–10% of MEK production costs. • Bio-based BDO is economically viable with a minimum selling price of US$ 1.54/kg. • Extractive MEK separation is the simplest process despite slightly higher production costs. • Renewable MEK is approximately 16–24% costlier than the petrochemical route. • Sugarcane (∼50 %), utilities (∼20 %), and direct cost (∼25 %) are major MEK operating costs. Butanediols are versatile platform chemicals that can be transformed into a spectrum of valuable products. This study examines the techno-commercial feasibility of an integrated biorefinery for fermentative production of 2,3-butanediol (BDO) from sucrose of sugarcane (SC), followed by chemo-catalytic upgrading of BDO to a carbon-conservative derivative, methyl ethyl ketone (MEK), with established commercial demand. The techno-economics of three process configurations are compared for downstream MEK separation from water and co-product, isobutyraldehyde (IBA): (I) heterogeneous azeotropic distillation of MEK-water and extractive separation of (II) MEK and (III) MEK-IBA from water using p- xylene as a solvent. The thermal efficiency of these manufacturing processes is further improved using pinch technology. The implementation of pinch technology reduces 8% of BDO and 9–10% of MEK production costs. Despite these improvements, raw material and utility costs remain substantial. The capital expenditure is notably higher for MEK production from SC than BDO alone due to additional processing steps. The extraction based MEK separation is the simplest process configuration despite marginally higher capital requirements and utility consumption with slightly higher production costs than MEK-water azeotropic distillation. Economic analysis suggests that bio-based BDO is cost-competitive with its petrochemical counterpart, with a minimum gross unitary selling price of US$ 1.54, assuming a 15% internal rate of return over five-year payback periods. However, renewable MEK is approximately 16–24% costlier than the petrochemical route. Future strategies must focus on reducing feedstock costs, improving BDO fermentation efficacy, and developing a low-cost downstream separation process to make renewable MEK commercially viable. [ABSTRACT FROM AUTHOR]

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 Δ hycEFG 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 Δ ldhA Δ hycEFG 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 Δ ldhA Δ adhE Δ hycEFG 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. • Hydrogenase-3 responsible for H 2 generation in Klebsiella pneumoniae. • Knockout of hycEFG promotes 1,3-propanediol and 2,3-butanediol production. • The conversion ratio of 1,3-propanediol was 0.628 mol/mol for Kp Δ ldhA Δ hycEFG. • The conversion ratio of 2,3-butanediol was 0.730 mol/mol for Kp Δ ldhA Δ adhE Δ hycEFG. [ABSTRACT FROM AUTHOR]

This study evaluates different fermentation strategies to produce 2,3-butanediol (2,3-BD) from banana industry waste, such as whole bananas (fruit + peels) and banana peels, selecting the most favorable from a technical and economic point of view. Both residues have enough free sugars (17.8 %–35.8 %), glucan (11.0 %–14.2 %) and hemicellulose (2.8 %–6.3 %), to be promising substrates for 2,3-BD fermentation. Saccharification was studied by comparing enzymatic hydrolysis, hydrothermal pretreatment, and hydrothermal pretreatment followed by enzymatic hydrolysis. Different fermentation scenarios were also compared regarding the 2,3-BD yield and productivity: Separate Hydrolysis and Fermentation (SHF), Simultaneous Saccharification and Fermentation (SSF), and direct fermentation without prior saccharification using Paenibacillus polymyxa DSM-365 as the fermenting microorganism. The results showed that the pretreatment step was not necessary to improve the release of fermentable sugars. Enzymatic hydrolysis was the most effective alternative for maximizing sugar recovery, reaching sugar concentrations of 18.1 g/L (recovery: 92.5 %) for banana peels and 33.3 g/L (recovery: ∼100 %) for whole bananas. The SSF strategy led to higher 2,3-BD concentrations of 15.0 g/L and 26.6 g/L for banana peels and whole bananas, respectively. The preliminary economic analysis indicated that SSF and direct fermentation could be the more cost-effective process alternatives for banana peels and whole bananas, respectively. Thus, it was demonstrated that banana waste is an interesting resource for the production of 2,3-BD. The bioprocess can be competitive when using a low-cost raw material and reducing the number of process steps compared to traditional technologies. [Display omitted] • Valorizing Banana Wastes for Sustainable 2,3-Butanediol Production. • Comparison of three fermentation scenarios: SHF, SSF and direct fermentation. • Techno-economic comparison of three 2,3-Butanediol fermentation scenarios. • Enzymatic saccharification eliminates extensive pretreatment. • Maximum 2,3-Butanediol obtained of 26.6 g/L using whole banana in SSF. [ABSTRACT FROM AUTHOR]

Abstract Background Biobutanol has great potential as biofuel of the future. However, only a few organisms have the natural ability to produce butanol. Amongst them, Clostridium spp. are the most efficient producers. The high toxicity of biobutanol constitutes one of the bottlenecks within the biobutanol production process which often suffers from low final butanol concentrations and yields. Butanol tolerance is a key driver for process optimisation and, therefore, in the search for alternative butanol production hosts. Many Lactobacillus species show a remarkable tolerance to solvents and some Lactobacillus spp. are known to naturally produce 2-butanol from meso-2,3-butanediol (meso-2,3-BTD) during anaerobic sugar fermentations. Lactobacillus diolivorans showed already to be highly efficient in the production of other bulk chemicals using a simple two-step metabolic pathway. Exactly, the same pathway enables this cell factory for 2-butanol production. Results Due to the inability of L. diolivorans to produce meso-2,3-BTD, a two-step cultivation processes with Serratia marcescens has been developed. S. marcescens is a very efficient producer of meso-2,3-BTD from glucose. The process yielded a butanol concentration of 10 g/L relying on wild-type bacterial strains. A further improvement of the maximum butanol titer was achieved using an engineered L. diolivorans strain overexpressing the endogenous alcohol dehydrogenase pduQ. The two-step cultivation process based on the engineered strain led to a maximum 2-butanol titer of 13.4 g/L, which is an increase of 34%. Conclusion In this study, L. diolivorans is for the first time described as a good natural producer for 2-butanol from meso-2,3-butanediol. Through the application of a two-step cultivation process with S. marcescens, 2-butanol can be produced from glucose in a one-vessel, two-step microbial process.

Abstract Background Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae. Results The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. 13C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production. Conclusion These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.

Abstract Fermentation of chemicals from lignocellulose hydrolysate is an effective way to alleviate environmental and energy problems. However, fermentation inhibitors in hydrolysate and weak inhibitor tolerance of microorganisms limit its development. In this study, atmospheric and room temperature plasma mutation technology was utilized to generate mutant strains of Enterobacter cloacae and screen for mutants with high inhibitor tolerance to acid hydrolysate of corncobs. A highly inhibitor-tolerant strain, Enterobacter cloacae M22, was obtained after fermentation with non-detoxified hydrolysate, and this strain produced 24.32 g/L 2,3-butanediol and 14.93 g/L organic acids. Compared with that of the wild-type strain, inhibitor tolerance was enhanced twofold with M22, resulting in improvement of 2,3-butanediol and organic acid production by 114% and 90%, respectively. This work presents an efficient method to screen for highly inhibitor-tolerant strains and evidence of a novel strain that can produce 2,3-butanediol and organic acids using non-detoxified acid hydrolysate of corncobs.

Abstract Background Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively. Results In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L−1 of AC and 17.08 g L−1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L−1 AC in one-step biocatalysis with the yield of 0.67 mol mol−1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L−1 of 2,3-BD with the yield of 0.80 mol mol−1. Conclusions The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.

Abstract Background 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. Results Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. Conclusions The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Abstract Background Serratia marcescens is a chitinolytic bacterium that can potentially be used for consolidated bioprocessing to convert chitin to value-added chemicals. Currently, S. marcescens is poorly characterized and studies on intracellular metabolic and regulatory mechanisms would expedite development of bioprocessing applications. Results In this study, our goal was to characterize the metabolic profile of S. marcescens to provide insight for metabolic engineering applications and fundamental biological studies. Hereby, we constructed a constraint-based genome-scale metabolic model (iSR929) including 929 genes, 1185 reactions and 1164 metabolites based on genomic annotation of S. marcescens Db11. The model was tested by comparing model predictions with experimental data and analyzed to identify essential aspects of the metabolic network (e.g. 138 essential genes predicted). The model iSR929 was refined by integrating RNAseq data of S. marcescens growth on three different carbon sources (glucose, N-acetylglucosamine, and glycerol). Significant differences in TCA cycle utilization were found for growth on the different carbon substrates, For example, for growth on N-acetylglucosamine, S. marcescens exhibits high pentose phosphate pathway activity and nucleotide synthesis but low activity of the TCA cycle. Conclusions Our results show that S. marcescens model iSR929 can provide reasonable predictions and can be constrained to fit with experimental values. Thus, our model may be used to guide strain designs for metabolic engineering to produce chemicals such as 2,3-butanediol, N-acetylneuraminic acid, and n-butanol using S. marcescens.

Abstract Background Production of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined. Results A shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space–time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h. Conclusions The presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.

Abstract Background Bacterial volatiles promote plant growth and elicit immunity responses in plants grown in two-compartment Petri dishes. Due to the limitations of bacterial volatile compound (BVC) treatments such as their high evaporation rates, it is convenient to apply BVCs in closed systems such as greenhouses. However, the concentrations of BVCs must be optimised. We therefore attempted to optimise BVC emissions from bacteria grown on solid medium and synthetic BVC treatment in order to maximise plant growth and induced resistance in a miniature greenhouse system. Results We cultivated the model BVC emitter Bacillus subtilis GB03 on complex medium for continuous treatment, which we placed near 1-week-old cucumber seedlings in a miniature greenhouse. Aboveground and belowground plant growth parameters were significantly increased at 1 and 2 weeks after treatment with BVCs released by B. subtilis GB03. Moreover, this treatment protected cucumber seedlings against the angular leaf spot pathogen Pseudomonas syringae pv. lachrymans. In addition, cucumber shoot growth was promoted in response to the slow release of BVCs from filter paper that had absorbed 1000 and 10 µM synthetic 2,3-butanediol, a key BVC from B. subtilis strain GB03. However, induced resistance was only elicited when 10 plates containing 10 µM 2,3-butanediol were utilised in the miniature greenhouse. The mechanism of induced resistance appears to involve the activation of the jasmonic acid signalling pathway. Conclusions To overcome the difficulties associated with treatment using a single application of BVC in the greenhouse, we optimised conditions for BVC application via consistent exposure in a slow-release system.

Acetoin is widely used in food and cosmetics industries as a taste and fragrance enhancer. To produce (R)-acetoin in Saccharomyces cerevisiae , acetoin biosynthetic genes encoding α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD) from Bacillus subtilis and water-forming NADH oxidase (NoxE) from Lactococcus lactis were integrated into delta-sequences in JHY605 strain, where the production of ethanol, glycerol, and (R , R)-2,3-butanediol (BDO) was largely eliminated. We further improved acetoin production by increasing acetoin tolerance by adaptive laboratory evolution, and eliminating other byproducts including meso-2,3-BDO and 2,3-dimethylglycerate, a newly identified byproduct. Ara1, Ypr1, and Ymr226c (named Ora1) were identified as (S)-alcohol-forming reductases, which can reduce (R)-acetoin to meso-2,3-BDO in vitro. However, only Ara1 and Ypr1 contributed to meso-2,3-BDO production in vivo. We elucidate that Ora1, having a substrate preference for (S)-acetoin, reduces (S)-α-acetolactate to 2,3-dimethylglycerate, thus competing with AlsD-mediated (R)-acetoin production. By deleting ARA1 , YPR1 , and ORA1 , 101.3 g/L of (R)-acetoin was produced with a high yield (96% of the maximum theoretical yield) and high stereospecificity (98.2%). • Saccharomyces cerevisiae was engineered for high-yield (R)-acetoin production. • (R)-acetoin tolerance was improved by adaptive laboratory evolution. • (S)-Alcohol-forming reductases producing meso-2,3-butanediol were identified. • A novel pathway reducing α-acetolactate to 2,3-dimethylglyceric acid was discovered. • By eliminating byproducts, (R)-acetoin production yield was improved up to 99.8%. [ABSTRACT FROM AUTHOR]

Bioethanol from lignocellulosic biomass (namely second-generation bioethanol) has been considered to be a great opportunity to increase production of biofuels from renewable sources without expanding the world planted area. Spathaspora passalidarum has shown great potential to convert sugars derived from lignocellulosic materials, which comprises hexoses and pentoses, into bioethanol. Besides its unusual ability to produce ethanol naturally from pentoses, this microorganism can also be used to obtain other products of interest. This review summarizes and discusses fermentative and kinetic parameters from the state of the art of ethanol production by S. passalidarum in synthetic medium and hemicellulosic hydrolysates. Additionally, S. passalidarum performance for xylose consumption and ethanol production are discussed in comparison with other wild yeasts and genetically modified Saccharomyces cerevisiae. In addition to ethanol, the production of other chemical blocks such as xylitol, glycerol, acetoin, and 2,3-butanediol are also discussed, elucidating the potential of S. passalidarum for application in biorefineries. The potential of S. passalidarum for studies of mutagenesis, evolutionary, and genetic modifications is also reviewed in this work. [ABSTRACT FROM AUTHOR]

BACKGROUND: Production of biobased chemicals from renewable resources is a green starring approach that serves as a substitute to petroleum derivatives. Bioglycerol, with its growing production as a co‐product of biodiesel, is an attractive low‐cost feedstock for the synthesis of platform chemicals by microbial fermentation. 2,3‐butanediol (2,3‐BDO) is amongst the top biorefinery platform chemicals that can be produced by glycerol fermentation. RESULTS: The 'Circular Economy' concept is demonstrated by converting the by‐product bioglycerol using a cell‐free extract of Enterobacter aerogenes NCIM 2695 (National Collection of Industrial Microorganisms, NCIM), yielding 22 g L−1 2,3‐BDO, in 96 h, 98% atom economy and 0.4 g/g E factor. The cell‐free bioglycerol conversion to 2,3‐BDO was proved using a modified procedure for determining glycerol dehydrogenase enzyme assay by protein analysis and it was also shown to be cell‐bounded. CONCLUSION: Our study offers an effective utilization of the leftover material (i.e. cell‐free extract) that biocatalysed C3 to C4 diol, which adds value to the overall economics of the process using only crude glycerol (C3) itself as a fermentative medium. © 2020 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

The demand for renewable energy is increasing. Klebsiella pneumoniae is one of the most promising strains to produce 2,3-butanediol (2,3-BD). Compared with chemical methods, the biological production of 2,3-BD has the characteristics of substrate safety, low cost, and low energy consumption. However, excessive glucose concentrations can cause damage to cells. Therefore, this study investigated the effect of sRNA-SgrS as a sugar transport regulator on the fermentative production of 2,3-BD by K. pneumoniae in response to sugar stress. We designed multiple mutants of K. pneumoniae HD79 to redistribute its carbon flux to produce 2,3-BD. It was found that the 2,3-BD yield of sgrS overexpressed strain decreased by 44% compared with the original strain. The results showed that a high concentration of sRNA-SgrS could accelerate the degradation of ptsG mRNA (encoding the glucose transporter EIICBGlc) and downregulate the expression levels of the budA gene (encoding the α-acetyllactate decarboxylase) and the budB gene (encoding the α-acetyllactate synthase) and budC gene (encoding the 2,3-BD dehydrogenase) but had no effect on the ack gene (encoding the acetate kinase) and the ldh gene (encoding the lactate dehydrogenase). It provides a theoretical basis and a technical reference for understanding the complex regulation mechanism of sRNA in microorganisms and the genetics and breeding in industrial fermentation engineering.

Although the yeast Saccharomyces cerevisiae has been used to produce various bio-based chemicals, including solvents and organic acids, most of these products inhibit yeast growth at high concentrations. In general, it is difficult to rationally improve stress tolerance in yeast by modifying specific genes, because many of the genes involved in stress response remain unidentified. Previous studies have reported that various forms of stress tolerance in yeast were improved by introducing random mutations, such as DNA point mutations and DNA structural mutations. In this study, we developed a novel mutagenesis strategy that allows for the simultaneous performance of these two types of mutagenesis to construct a yeast variant with high 2,3-butanediol (2,3-BDO) tolerance. The mutations were simultaneously introduced into S. cerevisiae YPH499, accompanied by a stepwise increase in the concentration of 2,3-BDO. The resulting mutant YPH499/pol3δ/BD_392 showed 4.9-fold higher cell concentrations than the parental strain after 96 h cultivation in medium containing 175 g/L 2,3-BDO. Afterwards, we carried out transcriptome analysis to characterize the 2,3-BDO-tolerant strain. Gene ontology enrichment analysis with RNA sequence data revealed an increase in expression levels of genes related to amino acid metabolic processes. Therefore, we hypothesize that the yeast acquired high 2,3-BDO tolerance by amino acid function. Our research provides a novel mutagenesis strategy that achieves efficient modification of the genome for improving tolerance to various types of stressors. Image 1 [ABSTRACT FROM AUTHOR]

• Nonsterile food waste was fermentated to produce 2,3-butanediol (2,3-BDO). • Fermentation was conducted under alkaline and thermophilic condition. • Novel Bacillus licheniformis YNP5-TSU from Yellowstone National Park was used. • YNP5-TSU produced high yields of 2,3-BDO (0.38–0.41 g/g) from food waste sugars. • YNP5-TSU could ferment food waste without addition of external nutrients. Conversion of food waste into 2,3-butanediol (2,3-BDO) via microbial fermentation provides a promising way to reduce waste disposal to landfills and produce sustainable chemicals. However, sterilization of food waste, an energy- and capital-costly process, is generally required before fermentation to avoid any contamination, which reduces the energy net output and economic feasibility of food waste fermentation. In this study, we investigated the non-sterile fermentation of food waste to produce 2,3-BDO using a newly isolated thermophilic and alkaliphilic B. licheniformis YNP5-TSU. Three unitary food waste samples (i.e., pepper, pineapple, cabbage wastes) and one miscellaneous food waste mixture were respectively inoculated with B. licheniformis YNP5-TSU under non-sterile conditions. At 50 °C and an initial pH of 9.0, B. licheniformis YNP5-TSU was able to consume all sugars in food waste and produce 5.2, 5.9, 5.9 and 4.3 g/L of 2,3-BDO within 24 h from pepper, pineapple, cabbage and miscellaneous wastes, respectively, corresponding to a yield of 0.40, 0.38, 0.41 and 0.41 g 2,3-BDO/g sugar. These 2,3-BDO concentrations and yields from the non-sterile fermentations were comparable to those from the traditional sterile fermentations, which produced 4.0–6.8 g/L of 2,3-BDO with yields of 0.31–0.48 g 2,3-BDO/g sugar. Moreover, B. licheniformis was able to ferment various food wastes (pepper, pineapple and miscellaneous wastes) without any external nutrient addition and produce similar 2,3-BDO quantities. The non-sterile fermentation of food waste using novel thermophilic and alkaliphilic B. licheniformis YNP5-TSU provides a robust and energy-efficient approach to convert food waste to high-value chemicals. [ABSTRACT FROM AUTHOR]

High alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the gut microbiota had been demonstrated to be the causative agent of fatty liver disease (FLD). However, the catabolic pathways for alcohol production in vivo remain unclear. Here, we characterized the genome of HiAlc and medium alcohol-producing (MedAlc) Kpn and constructed an adh (an essential gene encoding alcohol dehydrogenase) knock-out HiAlc Kpn W14 strain (W14Δadh) using CRISPR-Cas9 system. Subsequently, we established the mouse model via gavage administration of HiAlc Kpn W14 and W14 Δadh strains, respectively. Proteome and metabolome analysis showed that 10 proteins and six major metabolites involved in the 2,3-butanediol fermentation pathway exhibited at least a three-fold change or greater during intestinal growth. Compared with HiAlc Kpn W14-fed mice, W14Δadh-fed mice with weak alcohol-producing ability did not show apparent pathological changes at 4 weeks, although some steatotic hepatocytes were observed at 12 weeks. Our data demonstrated that carbohydrate substances are catabolized to produce alcohol and 2,3-butanediol via the 2,3-butanediol fermentation pathway in HiAlc Kpn, which could be a promising clinical diagnostic marker. The production of high amounts of endogenous alcohol is responsible for the observed steatosis effects in hepatocytes in vivo.

Abstract Background The biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear. Results In this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae. Conclusions These results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.

Abstract Background 2,3-Butanediol (BD) is a largely used fossil-based platform chemical. The yield and productivity of bio-based BD fermentative production must be increased and cheaper substrates need to be identified, to make bio-based BD production more competitive. As BD bioproduction occurs under microaerobic conditions, a fine tuning and control of the oxygen transfer rate (OTR) is crucial to maximize BD yield and productivity. Very few studies on BD bioproduction focused on the use of non-pathogenic microorganisms and of byproducts as substrate. The goal of this work was to optimize BD bioproduction by the non-pathogenic strain Bacillus licheniformis ATCC9789 by (i) identifying the ranges of volumetric and biomass-specific OTR that maximize BD yield and productivity using standard sugar and protein sources, and (ii) performing a preliminary evaluation of the variation in process performances and cost resulting from the replacement of glucose with molasses, and beef extract/peptone with chicken meat and bone meal, a byproduct of the meat production industry. Results OTR optimization with an expensive, standard medium containing glucose, beef extract and peptone revealed that OTRs in the 7–15 mmol/L/h range lead to an optimal BD yield (0.43 ± 0.03 g/g) and productivity (0.91 ± 0.05 g/L/h). The corresponding optimal range of biomass-specific OTR was equal to 1.4–7.9 $${\text{mmol}}_{{{\text{O}}_{2} }} /{\text{g}}_{\text{CDW}} /{\text{h}}$$ mmolO2/gCDW/h , whereas the respiratory quotient ranged from 1.8 to 2.5. The switch to an agro-industrial byproduct-based medium containing chicken meat and bone meal and molasses led to a 50% decrease in both BD yield and productivity. A preliminary economic analysis indicated that the use of the byproduct-based medium can reduce by about 45% the BD production cost. Conclusions A procedure for OTR optimization was developed and implemented, leading to the identification of a range of biomass-specific OTR and respiratory quotient to be used for the scale-up and control of BD bioproduction by Bacillus licheniformis. The switch to a byproduct-based medium led to a relevant decrease in BD production cost. Further research is needed to optimize the process of BD bioproduction from the tested byproduct-based medium.

Abstract Background Promoters regulate the expression of metabolic pathway genes to control the flux of metabolism. Therefore, fine-tuning of metabolic pathway gene expression requires an applicable promoter system. In this study, a dissolved oxygen-dependent nar promoter was engineered for fine-tuning the expression levels of biosynthetic pathway enzymes in Escherichia coli. To demonstrate the feasibility of using the synthetic nar promoters in production of biochemicals in E. coli, the d-lactate pathway consisting of one enzyme and the 2,3-butanediol (BDO) pathway consisting of three enzymes were investigated. Results The spacer sequence of 15 bp between the − 35 and − 10 elements of the upstream region of the wild-type nar promoter was randomized, fused to the GFP gene, transduced into E. coli, and screened by flow cytometry. The sorted synthetic nar promoters were divided into three groups according to fluorescence intensity levels: strong, intermediate, and weak. The selected three representative nar promoters of strong, intermediate, and weak intensities were used to control the expression level of the d-lactate and 2,3-BDO biosynthetic pathway enzymes in E. coli. When the ldhD gene encoding d-lactate dehydrogenase was expressed under the control of the strong synthetic nar promoter in fed-batch cultures of E. coli, the d-lactate titers were 105.6 g/L, 34% higher than those using the wild-type promoter (79.0 g/L). When the three 2,3-BDO pathway genes (ilvBN, aldB, and bdh1) were expressed under the control of combinational synthetic nar promoters (strong–weak–strong) in fed-batch cultures of E. coli, the titers of 2,3-BDO were 88.0 g/L, 72% higher than those using the wild-type promoter (51.1 g/L). Conclusions The synthetic nar promoters, which were engineered to have strong, intermediate, and weak intensities, were successfully applied to metabolic engineering of d-lactate and 2,3-BDO pathways in E. coli. By controlling expression levels of d-lactate and 2,3-BDO pathway enzymes using the synthetic nar promoters, the production of d-lactate and 2,3-BDO was increased over that using the wild-type promoter by 34 and 72%, respectively. Thus, this synthetic promoter module system will support the improved production of biochemicals and biofuels through fine-tuning of gene expression levels.

Abstract Background 2,3-butanediol (2,3-BD) is a bulk platform chemical with various potential applications such as aviation fuel. 2,3-BD has three optical isomers: (2R, 3R)-, (2S, 3S)- and meso-2,3-BD. Optically pure 2,3-BD is a crucial precursor for the chiral synthesis and it can also be used as anti-freeze agent due to its low freezing point. 2,3-BD has been produced in both native and non-native hosts. Several pathogenic bacteria were reported to produce 2,3-BD in mixture of its optical isomers including Klebsiella pneumoniae and Klebsiella oxytoca. Engineered hosts based on episomal plasmid expression such as Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis are not ideal for industrial fermentation due to plasmid instability. Results Pichia pastoris is generally regarded as safe and a well-established host for high-level heterologous protein production. To produce pure (2R, 3R)-2,3-BD enantiomer, we developed a P. pastoris strain by introducing a synthetic pathway. The alsS and alsD genes from B. subtilis were codon-optimized and synthesized. The BDH1 gene from S. cerevisiae was cloned. These three pathway genes were integrated into the genome of P. pastoris and expressed under the control of GAP promoter. Production of (2R, 3R)-2,3-BD was achieved using glucose as feedstock. The optical purity of (2R, 3R)-2,3-BD was more than 99%. The titer of (2R, 3R)-2,3-BD reached 12 g/L with 40 g/L glucose as carbon source in shake flask fermentation. The fermentation conditions including pH, agitation speeds and aeration rates were optimized in batch cultivations. The highest titer of (2R, 3R)-2,3-BD achieved in fed-batch fermentation using YPD media was 45 g/L. The titer of 2,3-BD was enhanced to 74.5 g/L through statistical medium optimization. Conclusions The potential of engineering P. pastoris into a microbial cell factory for biofuel production was evaluated in this work using (2R, 3R)-2,3-BD as an example. Engineered P. pastoris could be a promising workhorse for the production of optically pure (2R, 3R)-2,3-BD.

Background: Bacillus subtilis CS13 was previously isolated for 2,3-butanediol (2,3-BD) and poly-γ-glutamic acid (γ-PGA) co-production. When culturing this strain without L-glutamic acid in the medium, 2,3-BD is the main metabolic product. 2,3-BD is an important substance and fuel with applications in the chemical, food, and pharmaceutical industries. However, the yield and productivity for the B. subtilis strain should be improved for more efficient production of 2,3-BD. Results: The medium composition, which contained 281.1 g/L sucrose, 21.9 g/L ammonium citrate, and 3.6 g/L MgSO4·7H2O, was optimized by response surface methodology for 2,3-BD production using B. subtilis CS13. The maximum amount of 2,3-BD (125.5 ± 3.1 g/L) was obtained from the optimized medium after 96 h. The highest concentration and productivity of 2,3-BD were achieved simultaneously at an agitation speed of 500 rpm and aeration rate of 2 L/min in the batch cultures. A total of 132.4 ± 4.4 g/L 2,3-BD was obtained with a productivity of 2.45 ± 0.08 g/L/h and yield of 0.45 g2,3-BD/gsucrose by fed-batch fermentation. The meso-2,3-BD/2,3-BD ratio of the 2,3-BD produced by B. subtilis CS13 was 92.1%. Furthermore, 89.6 ± 2.8 g/L 2,3-BD with a productivity of 2.13 ± 0.07 g/L/h and yield of 0.42 g2,3-BD/gsugar was achieved using molasses as a carbon source. Conclusions: The production of 2,3-BD by B. subtilis CS13 showed a higher concentration, productivity, and yield compared to the reported generally recognized as safe 2,3-BD producers. These results suggest that B. subtilis CS13 is a promising strain for industrial-scale production of 2,3-BD. [ABSTRACT FROM AUTHOR]

Bacillus lichenformis is an industrially promising generally recognized as safe (GRAS) strain that can be used for the production of a valuable chemical, 2,3‐butanediol (BDO). Conventional gene deletion vectors and/or methods are time‐consuming and have poor efficiency. Therefore, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas9 mediated homologous recombination was used to engineer a newly isolated and UV‐mutagenized B. licheniformis 4071–15 strain. With the help of a CRISPR‐Cas9 system, this one‐step process could be used for the deletion of ldh gene within 4 days with high‐efficiency exceeding 60%. In addition, the sequential deletion of target genes for engineering studies was evaluated, and it was confirmed that a triple mutant strain (ldh, dgp, and acoR) could be obtained by repeated one‐step cycles. Furthermore, a practical metabolic engineering study was carried out using a CRISPR‐Cas9 system for the stereospecific production of (2R,3S)‐BDO. The predicted (2R,3R)‐butanediol dehydrogenase encoded by the gdh gene was selected as a target for the production of (2R,3S)‐BDO, and the mutant was successfully obtained. The results show that the stereospecific production of (2R,3S)‐BDO was possible with the gdh deletion mutant, while the 4071–15 host strain still generated 26% of (2R,3R)‐BDO. It was also shown that the 4071–15 Δgdh mutant could produce 115 g/L of (2R,3S)‐BDO in 64 hr by two‐stage fed‐batch fermentation. This study has shown the efficient development of a (2R,3S)‐BDO producing B. licheniformis strain based on CRISPR‐Cas9 and fermentation technologies. [ABSTRACT FROM AUTHOR]

• Novel inulinase producer of K. pneumoniae for 2,3-butanediol production was isolated. • The enzyme was an intracellular inulinase with an optimal temperature of 30 °C. • Concentration of 2,3-butanediol and acetoin reached 80.4 g/L in batch fermentation. • A productivity of 2.23 g/(L⋅h) and yield of 0.426 g/g sugar were obtained. Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin. [ABSTRACT FROM AUTHOR]

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• Ethanol pretreatment improved enzymatic hydrolysis and fermentation for OF-MSW. • The pretreatment agents, i.e., ethanol and acetic acid, were produced from OF-MSW. • The maximum glucose yield of 498.5 g/ kg OF-MSW was obtained. • From each kg of OF-MSW, biofuels equivalent to 257.4 mL gasoline was produced. • Biodegradable PHAs yield was 40 g/ kg OF-MSW. Municipal solid waste is an environmental threat worldwide; however, the organic fraction of municipal solid waste (OF-MSW) has a great potential for the generation of fuels and high-value products. In the current study, OF-MSW was utilized for the production of ethanol, hydrogen, as well as 2,3-butanediol, an octane booster, by using Enterobacter aerogenes. Furthermore, a promising alternative to non-biodegradable petrochemical-based polymers, polyhydroxyalkanoates (PHAs), was produced. The OF-MSW was first pretreated by an acetic acid catalyzed ethanol organosolv pretreatment at 120 and 160 °C followed by enzymatic hydrolysis of the residual solids. The residual unhydrolyzed solids resulting from enzymatic hydrolysis were further anaerobically digested for methane production. The enzymatic hydrolysis of the solids prepared at 120 °C for 60 min led to the production of hydrolysate with the highest glucose production yield of 498.5 g/kg dry untreated OF-MSW, which was fermented to 139.1 g 2,3-butanediol, 98.3 g ethanol, 28.6 g acetic acid, 71.4 L biohydrogen, and 40 g PHAs. Moreover, 23.1 L biomethane was produced through the anaerobic digestion of the enzymatic hydrolysis residue solids. Thus, appreciable amounts of energy (8236.9 kJ) and an eco-friendly bioplastic were produced by the valorization of carbon sources available in OF-MSW. [ABSTRACT FROM AUTHOR]

Vicinal diols and its derivatives can be exploited as model compounds for the investigation of radiation-induced free-radical transformations of hydroxyl-containing biomolecules such as carbohydrates, phospholipids, ribonucleotides, amino acids, and peptides. In this paper, for the first time, the prospects of isotope reinforcement approach in inhibiting free-radical transformations of hydroxyl-containing compounds in aqueous solutions are investigated on the example of radiolysis of 1,2-propanediol and 1,2-propanediol-2-d1 aqueous solutions. At an absorbed dose rate of 0.110 ± 0.003 Gy·s−1 a profound kinetic isotope effect (KIE) is observed for the non-branched chain formation of acetone, which is a final dehydration product of predominant carbon-centred radicals CH3·C(OH)CH2OH. In 0.1 and 1 M deaerated solutions at pH 7.00 ± 0.01, the values of KIE are 8.9 ± 1.7 and 15.3 ± 3.1, respectively. A rationale for the fact that a strong KIE takes place only in the case of chain processes, which may occur during free-radical transformations of vicinal diols, is also provided herein based on the results of 2-propanol and 2-propanol-2-d1 indirect radiolysis. Lastly, the lack of KIE is shown in the case of 2-butanone formation from 2,3-butanediol or 2,3-butanediol-2,3-d2. This indicates that the type (primary, secondary) of the β-carbonyl radicals formed as a result of CH3·C(OH)CH(OH)R (R = H, CH3) dehydration determines the manifestation of the effect. [ABSTRACT FROM AUTHOR]

In this study, the simultaneous production of hydrogen, ethanol, and 2,3-butanediol was assessed using three agro-industrial residues: cheese whey powder (CWP), wheat straw hydrolysate (WSH) and sugarcane molasses (SCM), by the Antarctic psychrophilic GA0F bacterium [EU636050], which is closely related to Pseudomonas antarctica [KX186936.1]. The main soluble metabolites produced in all the fermentations were ethanol and 2,3-butanediol. CWP demonstrated to be the most effective carbon source, since fermentation of this substrate resulted in the highest yields of H 2 (73.5 ± 10 cm3 g−1), ethanol (0.24 ± 0.03 g g−1) and 2,3-butanediol (0.42 ± 0.04 g g−1), followed by the use of SCM, whereas WSH showed to have an inhibitory effect during the fermentation process, showing the lowest production values. Our results demonstrated the ability of the Antarctic psychrophilic GA0F bacterium to produce valuable products using low-cost substrates at room temperature conditions. • Psychrophilic GA0F bacterium produces simultaneously H 2 , ethanol and 2,3-butanediol. • Three agro-industrial residues are used as carbon sources for biofuel production by Antarctic bacterium. • The highest biofuels yields were attained using cheese whey powder. • 2,3-butanediol and Ethanol yields ranged from 0.23 to 0.42 and 0.19–0.24 g g−1. [ABSTRACT FROM AUTHOR]

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms. • A pdc / mpc double deletion had a synergistic effect on global glucose metabolism. • Deletion of MPC is not lethal even in an aerobe such as A. oryzae. • Redirection of pyruvate metabolism shifted the carbon flow from anabolism to catabolism. • The pyruvate redirection strategy is useful for aerobic eukaryotes. [ABSTRACT FROM AUTHOR]

The dehydrogenation of 2,3-butanediol (BDO) to 3-hydroxybutanone (HBO) was studies over Li cation-doped CuZnAl catalysts. The catalyst samples were characterized by XRD, XPS, H2-TPR and SEM techniques. The characterization results showing that doping with Li cation obviously modified the surface morphology of CuZnAl catalysts; decreased the reduction temperature of CuZnAl catalysts; and finally increased the amount of Cu active sites. The reaction results showing that, Li modified CuZnAl catalysts obviously enhanced the selectivity of the dehydrogenation of BDO to HBO by inhibiting the dehydration of BDO. The Li(2%)–Cu(44%)–Zn(38%)–Al(16%) exhibited the highest activity for dehydrogenation of BDO, with the conversion rate of BDO is 72.4% and the selectivity of HBO is 95.9% at 260 °C. This catalyst shows excellent stability for more than 100 h without significant deactivation. [ABSTRACT FROM AUTHOR]

Production of 2,3-butanediol (2,3-BD) by Pantoea agglomerans strain BL1 was investigated using soybean hull hydrolysate as substrate in batch reactors. The cultivation media consisted of a mixture of xylose, arabinose, and glucose, obtained from the hemicellulosic fraction of the soybean hull biomass. We evaluated the influence of oxygen supply, pH control, and media supplementation on the growth kinetics of the microorganism and on 2,3-BD production. P. agglomerans BL1 was able to simultaneously metabolize all three monosaccharides present in the broth, with average conversions of 75% after 48 h of cultivation. The influence of aeration conditions employed demonstrated the mixed acid pathway of 2,3-BD formation by enterobacteria. Under fully aerated conditions (2 vvm of air), up to 14.02 g L−1 of 2.3-BD in 12 h of cultivation were produced, corresponding to yields of 0.53 g g−1 and a productivity of 1.17 g L−1 h−1, the best results achieved. These results suggest the production potential of 2,3-BD by P. agglomerans BL1, which has been recently isolated from an environmental consortium. The present work proposes a solution for the usage of the hemicellulosic fraction of agroindustry biomasses, carbohydrates whose utilization are not commonly addressed in bioprocess. [ABSTRACT FROM AUTHOR]

Bacillus subtilis naturally produces large amounts of 2,3-butanediol (2,3-BD) as a main by-product during poly-γ-glutamic acid (γ-PGA) production. 2,3-BD is a promising platform chemical in various industries, and co-production of the two chemicals has great economic benefits. Co-production of γ-PGA and 2,3-BD by a newly isolated B. subtilis CS13 was investigated here. The fermentation medium and culture parameters of the process were optimized using statistical methods. It was observed that sucrose, l-glutamic acid, ammonium citrate, and MgSO4·7H2O were favorable for γ-PGA and 2,3-BD co-production at culture pH of 6.5 and 37 °C. An optimal medium composed of 119.8 g/L sucrose, 48.8 g/L l-glutamic acid, 21.1 g/L ammonium citrate, and 3.2 g/L MgSO4·7H2O was obtained by response surface methodology (RSM). The results show that the titers of γ-PGA and 2,3-BD reached 27.8 ± 0.9 g/L at 24 h and 57.1 ± 1.3 g/L at 84 h with the optimized medium, respectively. γ-PGA and 2,3-BD production by B. subtilis CS13 was significantly enhanced in fed-batch fermentations. γ-PGA (36.5 ± 1.1 g/L, productivity of 1.22 ± 0.04 g/L/h) and 2,3-BD concentrations (119.6 ± 2.8 g/L, productivity of 2.49 ± 0.66 g/L/h) were obtained in the optimized medium with feeding sucrose. The co-production of 2,3-BD and γ-PGA provides a new perspective for industrial production of γ-PGA and 2,3-BD. Key points • A strategy for co-production of γ-PGA and 2,3-BD was developed. • The culture parameters for the co-production of γ-PGA and 2,3-BD were studied. • RSM was used to optimize the medium for γ-PGA and 2,3-BD co-production. • 36.5 g/L γ-PGA and 119.6 g/L 2,3-BD were obtained from the optimum medium in fed-batch fermentation. [ABSTRACT FROM AUTHOR]

Background: Whey is a major pollutant generated by the dairy industry. To decrease environmental pollution caused by the industrial release of whey, new prospects for its utilization need to be urgently explored. Here, we investigated the possibility of using whey powder to produce 2,3-butanediol (BDO), an important platform chemical. Results: Klebsiella oxytoca strain PDL-0 was selected because of its ability to efficiently produce BDO from lactose, the major fermentable sugar in whey. After deleting genes pox, pta, frdA, ldhD, and pflB responding for the production of by-products acetate, succinate, lactate, and formate, a recombinant strain K. oxytoca PDL-K5 was constructed. Fed-batch fermentation using K. oxytoca PDL-K5 produced 74.9 g/L BDO with a productivity of 2.27 g/L/h and a yield of 0.43 g/g from lactose. In addition, when whey powder was used as the substrate, 65.5 g/L BDO was produced within 24 h with a productivity of 2.73 g/L/h and a yield of 0.44 g/g. Conclusion: This study demonstrated the efficiency of K. oxytoca PDL-0 for BDO production from whey. Due to its non-pathogenicity and efficient lactose utilization, K. oxytoca PDL-0 might also be used in the production of other important chemicals using whey as the substrate. [ABSTRACT FROM AUTHOR]

2,3-Dihydroxyisovalerate is an intermediate of valine and leucine biosynthesis pathway; however, no natural microorganism has been found yet that can accumulate this compound. Klebsiella pneumoniae is a useful bacterium that can be used as a workhorse for the production of a range of industrially desirable chemicals. Dihydroxy acid dehydratase, encoded by the ilvD gene, catalyzes the reaction of 2-ketoisovalerate formation from 2,3-dihydroxyisovalerate. In this study, an ilvD disrupted strain was constructed which resulted in the inability to synthesize 2-ketoisovalerate, yet accumulate 2,3-dihydroxyisovalerate in its culture broth. 2,3-Butanediol is the main metabolite of K. pneumoniae and its synthesis pathway and the branched-chain amino acid synthesis pathway share the same step of the α-acetolactate synthesis. By knocking out the budA gene, carbon flow into the branched-chain amino acid synthesis pathway was upregulated, which resulted in a distinct increase in 2,3-dihydroxyisovalerate levels. Lactic acid was identified as a by-product of the process and by blocking the lactic acid synthesis pathway, a further increase in 2,3-dihydroxyisovalerate levels was obtained. The culture parameters of 2,3-dihydroxyisovalerate fermentation were optimized, which include acidic pH and medium level oxygen supplementation to favor 2,3-dihydroxyisovalerate synthesis. At optimal conditions (pH 6.5, 400 rpm), 36.5 g/L of 2,3-dihydroxyisovalerate was produced in fed-batch fermentation over 45 h, with a conversion ratio of 0.49 mol/mol glucose. Thus, a biological route of 2,3-dihydroxyisovalerate production with high conversion ratio and final titer was developed, providing a basis for an industrial process. Key Points • A biological route of 2,3-dihydroxyisovalerate production was setup. • Disruption of budA causes 2,3-dihydroxuisovalerate accumulation in K. pneumoniae. • Disruption of ilvD prevents 2,3-dihydroxyisovalerate reuse by the cell. • 36.5 g/L of 2,3-dihydroxyisovalerate was obtained in fed-batch fermentation. [ABSTRACT FROM AUTHOR]