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Klebsiella pneumoniae synthesize large amounts of l-2,3-butanediol (l-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an l-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward l-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of l-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.

The acetolactate synthase (als)-deficient mutant of Klebsiella pneumoniae fails to produce 1,3-propanediol (1,3-PD) or 2,3-butanediol (2,3-BD), and is defective in glycerol metabolism. In an effort to recover production of the industrially valuable 1,3-PD, we introduced the Zymomonas mobilis pyruvate decarboxylase (pdc) and aldehyde dehydrogenase (aldB) genes into the als-deficient mutant to activate the conversion of pyruvate to ethanol. Heterologous expression of pdc and aldB efficiently recovered glycerol metabolism in the 2,3-BD synthesis-defective mutant, enhancing the production of 1,3-PD by preventing the accumulation of pyruvate. Production of 1,3-PD in the pdc- and aldB-expressing als-deficient mutant was further enhanced by increasing the aeration rate. This system uses metabolic engineering to produce 1,3-PD while minimizing the generation of 2,3-BD, offering a breakthrough for the industrial production of 1,3-PD from crude glycerol.

Klebsiella pneumoniae was engineered to produce isobutanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ∆ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced isobutanol (160 mg l−1) from crude glycerol. To increase the yield of isobutanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of isobutanol from 160 to 320 mg l−1. This represents the first successful attempt to produce isobutanol from crude glycerol.

In this study, β-glucosidase from Aspergillus niger was pretreated with cellobiose and glucose to prevent loss of enzyme activity, and pretreated β-glucosidase was immobilized on silica gel as a carrier by covalent binding. To enhance the activity of immobilized β-glucosidase, the effects of substrate concentration and reaction conditions, including temperature, time, and agitation speed, were investigated. The optimal concentrations of cellobiose and glucose, temperature, time, and agitation speed were determined to be 0.02 M, 40 °C, 20 min, and 130 rpm, respectively. The activity of immobilized β-glucosidase after pretreatment was increased to about 176% of that of non-pretreated β-glucosidase. In addition, the optimal pH and temperature of the non-pretreated and pretreated immobilized β-glucosidases were both pH 5.5 and 65 °C, respectively. Moreover, the immobilized β-glucosidases were used repeatedly 20 times, and the enzyme activities were maintained at levels higher than 80% of their initial activities.

To obtain mutant strains producing high levels of cellulases (FPase and CMCase) and β-glucosidase, Trichoderma reesei KCTC 6950 was mutated by proton beam irradiation. Five mutants were selected out of 1,000 mutants of T.reesei treated with proton beam irradiation, based on their ability for enzyme production on a plate screening medium. In submerged cultures containing Mandel’s fermentation medium, the mutant strain T-2 (MT-2) demonstrated a 165% increase in the activity of FPase, a 146% increase in the activity of CMCase, and a 313% increase in the activity of β-glucosidase, compared with the wild type strain. Additionally, the properties of high level β-glucosidase produced by MT-2 were the same as those of the wild type strain, e.g., an optimum pH of 4.8, and an optimum temperature of 65 °C. Moreover, the protein concentrations of β-glucosidase produced by the wild type strain and MT-2 were measured by SDS-PAGE, and then β-glucosidase activities were detected by the MUG-zymogram assay.

In this study, the effects of glycerol on cephalosporin C production by Acremonium chrysogenum M35 were evaluated. The addition of glycerol increased cephalosporin production by up to 12-fold. Glycerol caused the upregulation of the transcription of the isopenicillin synthase (pcbC) and transporter (cefT) genes in early exponential phase, and affected the cell morphology since hyphal fragments differentiated into arthrospores. These results indicate that glycerol effectively enhances cephalosporin C production via stimulation of cell differentiation.

A Bacillus species that produces 2,3-butanediol (2,3-BD), termed BRC1, was newly isolated, and a 2,3-BD dehydrogenase (Bdh) from this species was identified and characterized at the molecular and biochemical level. Sequence analysis revealed that Bdh is homologous to D-2,3-BD dehydrogenases. An analysis of the enzymatic properties of Bdh overexpressed in Escherichia coli confirmed the molecular results, showing preferred activity toward D-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Overexpression of bdh under the control of a xylose-inducible promoter resulted in increased enzyme activity and enhanced 2,3-BD production in Bacillus sp. BRC1. Additionally, a hydrolysate of cellulosic material, (empty palm fruit bunches), was successfully used for the enhanced production of 2,3-BD in the recombinant Bacillus strain.

Klebsiella pneumoniae was engineered to produce 2-butanol from crude glycerol as a sole carbon source by expressing acetolactate synthase (ilvIH), keto-acid reducto-isomerase (ilvC) and dihydroxy-acid dehydratase (ilvD) from K. pneumoniae, and α-ketoisovalerate decarboxylase (kivd) and alcohol dehydrogenase (adhA) from Lactococcus lactis. Engineered K. pneumonia, ∆ldhA/pBR-iBO (ilvIH–ilvC–ilvD–kivd–adhA), produced 2-butanol (160 mg l−1) from crude glycerol. To increase the yield of 2-butanol, we eliminated the 2,3-butanediol pathway from the recombinant strain by inactivating α-acetolactate decarboxylase (adc). This further engineering step improved the yield of 2-butanol from 160 to 320 mg l−1. This represents the first successful attempt to produce 2-butanol from crude glycerol.

Background: Use of lignocellulosic biomass has received attention lately because it can be converted into various versatile chemical compounds by biological processes. In this study, a two-step pretreatment with dilute sulfuric acid and aqueous ammonia was performed efficiently on rice straw to obtain fermentable sugar. The soaking in aqueous ammonia process was also optimized by a statistical method. Results: Response surface methodology was employed. The determination coefficient (R-2) value was found to be 0.9607 and the coefficient of variance was 6.77. The optimal pretreatment conditions were a temperature of 42.75 degrees C, an aqueous ammonia concentration of 20.93%, and a reaction time of 48 h. The optimal enzyme concentration for saccharification was 30 filter paper units. The crystallinity index was approximately 60.23% and the Fourier transform infrared results showed the distinct peaks of glucan. Ethanol production using Saccharomyces cerevisiae K35 was performed to verify whether the glucose saccharified from rice straw was fermentable. Conclusions: The combined pretreatment using dilute sulfuric acid and aqueous ammonia on rice straw efficiently yielded fermentable sugar and achieved almost the same crystallinity index as that of alpha-cellulose.