Dynamic upregulation of the rate-limiting enzyme for valerolactam biosynthesis in Corynebacterium glutamicum.

Valerolactam is a monomer used to manufacture high-value nylon-5 and nylon-6,5. However, the biological production of valerolactam has been limited by the inadequate efficiency of enzymes to cyclize 5-aminovaleric acid to produce valerolactam. In this study, we engineered Corynebacterium glutamicum with a valerolactam biosynthetic pathway consisting of DavAB from Pseudomonas putida to convert L-lysine to 5-aminovaleric acid and ß-alanine CoA transferase (Act) from Clostridium propionicum to produce valerolactam from 5-aminovaleric acid. Most of the L-lysine was converted into 5-aminovaleric acid, but promoter optimization and increasing the copy number of Act were insufficient to significantly improve the titer of valerolactam. To eliminate the bottleneck at Act, we designed a dynamic upregulation system (a positive feedback loop based on the valerolactam biosensor ChnR/Pb). We used laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range, and the engineered ChnR-B1/Pb-E1 system was used to overexpress the rate-limiting enzymes (Act/ORF26/CaiC) that cyclize 5-aminovaleric acid into valerolactam. In glucose fed-batch culture, we obtained 12.33 g/L valerolactam from the dynamic upregulation of Act, 11.88 g/L using ORF26, and 12.15 g/L using CaiC. Our engineered biosensor (ChnR-B1/Pb-E1 system) was also sensitive to 0.01-100 mM caprolactam, which suggests that this dynamic upregulation system can be used to enhance caprolactam biosynthesis in the future.

Dynamics of microbial communities and metabolites in ganjang, a traditional Korean fermented soy sauce, during fermentation.

Six different ganjang batches were prepared and the microbial communities in the ganjang samples and raw materials (meju and solar salts), as well as the metabolites generated during fermentation were analyzed. The varying amounts of raw materials used differentially affected the microbial communities. Halophilic or halotolerant microbes derived from solar salts were abundant during middle or late fermentation. By contrast, non-halophilic microbes derived from meju were abundant mainly during early fermentation. Debaryomyces, Tetragenococcus, and Staphylococcus were abundant in all ganjang batches, which suggested that these may be the most common microbes involved in ganjang fermentation. The salt concentrations did not affect the abundance of Debaryomyces, which was abundant in all ganjang batches. Tetragenococcus was abundant in low salt ganjang, whereas Staphylococcus was abundant in high salt ganjang. Metabolite analysis revealed that carbohydrate concentrations were high in ganjang prepared using high amounts of meju. The level of lactate, which may be largely produced by Tetragenococcus, in low salt ganjang was high because of high microbial activity. The amino acid concentrations of the ganjang batches were mainly associated with meju quantity, but not salt concentration. These results indicated that the production of amino acids may be associated with indigenous proteases in meju, but not microbial activities during ganjang fermentation.

Cupriavidus lacunae sp. nov., isolated from pond-side soil.

A Gram-stain negative, strictly aerobic, mesophilic bacterial strain, designated strain S23T, was isolated from pond-side soil of an artificial pond in South Korea. Cells were observed to be peritrichously flagellated short rods showing positive oxidase and catalase activities. Growth of strain S23T was observed at 15-37 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0-8.0) and 0-2% (w/v) NaCl (optimum, 0-0.5%). The major respiratory quinone was identified as ubiquinone-8 and the major fatty acids were identified as C16:0, cyclo-C17:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The G + C content of the genomic DNA was determined to be 65.1 mol%. Phosphatidylglycerol, phosphatidylethanolamine and an unidentified phospholipid were detected as the major polar lipids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain S23T formed a phyletic lineage with Cupriavidus necator N-1T within the genus Cupriavidus. Strain S23T is closely related to C. necator N-1T (99.2%), Cupriavidus basilensis DSM 11853T (98.8%), Cupriavidus alkaliphilus ASC-732T (98.8%) and Cupriavidus numazuensis TE26T (98.7%), based on 16S rRNA gene sequence similarities. However, the DNA-DNA relatedness values between strain S23T and the closely related type strains were less than 46%. On the basis of phenotypic, chemotaxonomic and molecular properties, strain S23T represents a novel species of the genus Cupriavidus, for which the name Cupriavidus lacunae sp. nov. is proposed. The type strain is S23T (KACC 19624T = JCM 32674T).

Amylibacter lutimaris sp. nov., isolated from sea-tidal flat sediment.

A Gram-stain-negative, aerobic bacterial strain, designated strain m18T, was isolated from a sea-tidal flat in South Korea. Cells were non-motile short rods showing oxidase and catalase activities. Growth of m18T was observed at 10-40 °C (optimum, 30 °C), pH 5.5-10.0 (optimum, pH 7.0) and 0.5-7.0 % (w/v) NaCl (optimum, 3.0 %). The major respiratory quinone was ubiquinone-10 and the major fatty acids of were summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c) and C16 : 0. The G+C content of the genomic DNA was 56.7 mol%. Phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid and four unidentified lipids were detected in m18T. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that m18T formed a tight phyletic lineage with the members of the genus Amylibacter. Strain m18T was most closely related to Amylibactercionae H-12T, Amylibacter ulvae 6Alg 255T and Amylibacter marinus 2-3T with 98.9, 96.1 and 95.5 % 16S rRNA gene sequence similarities, respectively. The DNA-DNA hybridization value between m18T and the type strain of A. cionae was 43.6±3.4 %. On the basis of phenotypic, chemotaxonomic and molecular properties, m18T represents a novel species of the genus Amylibacter, for which the name Amylibacterlutimaris sp. nov. is proposed. The type strain is m18T (KACC 19229T=JCM 32051T).

Aquicoccus porphyridii gen. nov., sp. nov., isolated from a small marine red alga, Porphyridium marinum.

A Gram-stain-negative, non-motile and aerobic bacterial strain, designated L1 8-17T, was isolated from a marine alga, Porphyridium marinum, in South Korea. Cells of strain L1 8-17T were found to be oxidase- and catalase-positive cocci without flagella. Growth of strain L1 8-17T was observed at 20-40 °C (optimum, 37 °C), pH 6.0-10.0 (optimum, pH 7.0-8.0) and in the presence of 0-7 % (w/v) NaCl (optimum, 2-3 %). The isoprenoid quinone detected was only ubiquinone-10. Summed feature 8 (comprising C18 : 1ω7c/C18 : 1ω6c) and C16 : 0 were detected as major cellular fatty acids. The major polar lipids of strain L1 8-17T consisted of phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, an unidentified phospholipid and an unidentified lipid. The G+C content of the genomic DNA was 59.3 mol%. Strain L1 8-17T was most closely related to Marimonas arenosa CAU 1311T, Tropicibacter naphthalenivorans C02T and Donghicola eburneus SW-277T with 96.68, 96.68 and 96.60 % 16S rRNA gene sequence similarities, respectively, but the strain formed a phylogenetic lineage clearly distinct from them within the family Rhodobacteraceae. On the basis of phenotypic, chemotaxonomic and molecular properties, strain L1 8-17T represents a novel genus of the family Rhodobacteraceae, for which the name Aquicoccus porphyridii gen. nov., sp. nov. is proposed. The type strain of the type species is L1 8-17T (KACC 18806T=JCM 31543T).

Aestuariicoccus marinus gen. nov., sp. nov., isolated from sea-tidal flat sediment.

A Gram-stain-negative, strictly aerobic and halotolerant bacterial strain, designated strain NAP41T, was isolated from a sea tidal flat in the Yellow Sea of South Korea. Cells were non-motile cocci showing oxidase- and catalase-positive activities. Growth of strain NAP41T was observed at 15-40 °C (optimum, 37 °C), at pH 6.5-9.0 (optimum, pH 7.0-7.5) and in the presence of 0.5-12 % (w/v) NaCl (optimum, 2 %). Strain NAP41T contained summed feature 8 (comprising C18 : ω7c/C18 : 1ω6c) and C18 : 0 as the major fatty acids and ubiquinone-10 as the sole isoprenoid quinone. Phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, an unidentified aminolipid and three unidentified lipids were detected as the polar lipids. The G+C content of the genomic DNA was 56.0 mol%. Strain NAP41T was most closely related to Primorskyibacter insulae SSK3-2T, Thalassococcus lentus YCS-24T and Roseivivax lentus DSM 29430T with 96.67, 96.39 and 96.39 % 16S rRNA gene sequence similarities, respectively, and formed a phylogenetic lineage distinct from closely related taxa within the family Rhodobacteraceae with low bootstrap values. On the basis of phenotypic, chemotaxonomic and molecular properties, strain NAP41T represents a novel species of a novel genus of the family Rhodobacteraceae, for whichthe name Aestuariicoccus marinus gen. nov., sp. nov. is proposed. The type strain of the type species is NAP41T (KACC 18431T=JCM 30739T).

Paraburkholderia lacunae sp. nov., isolated from soil near an artificial pond.

A Gram-stain-negative, strictly aerobic bacterial strain, designated strain S27T, was isolated from soil near an artificial pond in South Korea. Cells were non-motile short rods showing oxidase- and catalase-positive activities. Growth of strain S27T was observed at 20-40°C (optimum, 30°C), pH 5.0-7.0 (optimum, pH 6.0), and 0-0.5% (w/v) NaCl (optimum, 0%). Ubiquinone-8 was detected as the sole respiratory quinone and the major fatty acids were C16:0, cyclo-C17:0, and cyclo-C19:0ω8c. The G + C content of the genomic DNA was 62.4 mol%. Phosphatidylglycerol, phosphatidylethanolamine, and an unidentified aminophospholipid were detected as the major polar lipids. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S27T formed a clearly distinct phyletic lineage from closely related Paraburkholderia species within the genus Paraburkholderia. Strain S27T was most closely related to Paraburkholderia rhynchosiae WSM3937T, Paraburkholderia ginsengiterrae DCY85T, Paraburkholderia fungorum NBRC 102489T, and Paraburkholderia graminis C4D1MT with 98.8%, 98.4%, 98.4%, and 97.7% 16S rRNA gene sequence similarities, respectively. The DNA-DNA relatedness level between strain S27T and the type strain of P. rhynchosiae was 36.8 ± 2.6%. On the basis of phenotypic, chemotaxonomic and molecular properties, strain S27T represents a novel species of the genus Paraburkholderia, for which the name Paraburkholderia lacunae sp. nov. is proposed. The type strain is S27T (KACC 19714 T = JCM 32721T).