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The cellobiose-responsive regulator ClbR, a Zn(II)2Cys6 binuclear-cluster transcription factor, is a positive regulator of carbohydrate-active enzyme (CAZyme) genes responsive to cellulose in Aspergillus aculeatus. Because Zn(II)2Cys6 transcription factors tend to dimerize with proteins of the same family, we searched for a counterpart of ClbR and identified ClbR2, which is 42% identical to ClbR, as an interacting partner of ClbR by yeast two-hybrid screening. Genetic analyses suggested that ClbR and ClbR2 cooperatively regulate the expression of CAZyme genes in response to cellulose and 1,4-β-mannobiose in A. aculeatus. CAZyme genes under the control of the transcription factor ManR were regulated by ClbR and ClbR2, whereas those controlled by the transcription factor XlnR were regulated by ClbR, but not ClbR2. These findings suggest that ClbR participates in multiple regulatory pathways in A. aculeatus by altering an interacting factor. [ABSTRACT FROM AUTHOR]

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10–30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements. [ABSTRACT FROM AUTHOR]

Laminin_G_3 modules can exist together with family‐43 catalytic modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 module and a GH43 module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A‐Abf43B‐Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43‐1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 32‐α‐l‐arabinofuranosyl‐(1,5)‐α‐l‐arabinotriose but not for α‐1,5‐linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α‐1,5‐linked arabinooligosaccharides. RjGH43‐1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 module in RjGH43‐1 plays an important role in substrate hydrolysis as a carbohydrate‐binding module. [ABSTRACT FROM AUTHOR]

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host‐vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5′‐GmetATC‐3′, where the A nucleotide is Dam‐methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam−/dcm−) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 103 transformants/μg of DNA was obtained using a square‐wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin‐encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui. [ABSTRACT FROM AUTHOR]

Fungal cellulolytic and hemicellulolytic enzymes are promising tools for industrial hydrolysis of cellulosic biomass; however, the regulatory network underlying their production is not well understood. The recent discovery of the transcriptional activators ClrB and McmA in Aspergillus nidulans implied a novel regulatory mechanism driven by their interaction, experimental evidence for which was obtained from transcriptional and DNA-binding analyses in this study. It was found that ClrB was essential for induced expression of all the genes examined in this study, while McmA dependency of their expression was gene-dependent. DNA-binding studies revealed McmA assisted in the recruitment of ClrB to the cellulose-responsive element (CeRE) in the promoters of eglA and eglB, expression of which was significantly reduced in the mcmA mutant. The CCG triplet within the CeRE served as the recognition sequence for the ClrB monomer. In contrast, ClrB did not require McmA for binding as a homodimer to the CGGN8CCG sequences in the promoter of mndB, expression of which was affected less in the mcmA mutant than in all other examined genes. Thus, there are two types of ClrB-mediated regulation: McmA-assisted and McmA-independent. This novel McmA-ClrB synergistic system provides new insights into the complex regulatory network involved in cellulase and hemicellulase production. [ABSTRACT FROM AUTHOR]

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host-vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5'-G met ATC-3', where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam - /dcm - ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 10 3 transformants/μg of DNA was obtained using a square-wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin-encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)