Amplicon sequencing is a widely used method for surveying biological diversity. However, the technique is disturbed by PCR bias leading to errors in community composition analyses. In this study, microbial community composition was evaluated in twenty-eight locations of hot spring water with temperatures between 87-48°C at Nakabusa Hot Springs, Japan, using amplicon sequencing analysis with the V4 region of the 16S rRNA gene. In discrepancy with the greenish color and the absorption spectra of the microbial samples, the relative abundance of amplicon sequence variants (ASVs) in the major photosynthetic organisms, Chloroflexus spp., were scarce in any sample when using the annealing temperature of 50°C in amplicon PCR. Changing the annealing temperature to 68ºC significantly improved the detection efficiency of Chloroflexus ASVs, and the obtained numbers were consistent with the presence of the photosynthetic pigments. The abundance of many other microbial ASVs was also dependent on the annealing temperature. The log ratio in the abundance of major ASVs between two annealing temperatures was correlated with the GC content of the 16S rRNA gene, suggesting that even some other major ASVs in the community are seriously affected by PCR bias due to the GC content. Combined usage of results from two different annealing temperatures, rather than a result using a single annealing temperature, seems to be a better way to obtain community structure information with less PCR bias in thermophilic organisms of high 16S rRNA GC content.

Serratia nuclease Nuc A is a non-specific nucleotide hydrolase that has been widely used in large-scale protein purification or eliminating nucleic acid contamination from purified proteins. To enhance the enzyme production, the Serratia nuclease gene was synthesized and expressed in Bacillus licheniformis 2709, a robust strain capable of secreting native and heterologous proteins selectively or non-selectively. To further increase the secretory expression level of the enzyme, different strong promoters and signal peptides were fused with the mature Nuc A-encoding gene at various genetic loci. The highest expression level of Nuc A was observed under the control of regulatory elements P_aprE, which occur naturally in B. licheniformis 2709 for the alkaline protease (AprE) expression. Through maximizing the number of copies of P_aprE-nucA expression cassette at different integration sites, the yield of nuclease Nuc A reached approximately 31954 U/mL after 60 hours of cultivation in shake flasks. The specific activity of the recombinant nuclease reached 1.58×107 U/mg, which is about 9 times higher than that expressed in Escherichia coli strain. Additionally, the recombinant Nuc A exhibited high catalytic activities in the pH range of 7-10. Furthermore, it was resistant to 0.2% SDS, 1.0 mM PMSF, and 0.4% Triton X-100. After 8 M Urea treatment, residual activity is measured. The high expression levels and positive characteristics of recombinant Nuc A provide an effective solution for large-scale production and industrial application of the nuclease.

Mycosporine-like amino acids (MAAs) are low-molecular-weight UV-protective compounds, and porphyra-334 and shinorine are common MAAs. Porphyra-334 is synthesized via the conjugation of mycosporine-glycine with threonine, whereas substitution with serine yields shinorine. The terrestrial cyanobacterium Nostoc commune KU002 (NIES-2538) produces 7-O-(β-arabinopyranosyl)-porphyra-334, and the mysABCD gene cluster responsible for MAA biosynthesis has been isolated. The heterologous expression of the mysABC genes from N. commune KU002 in Escherichia coli cells led to mycosporine-glycine production regardless of the culture medium supplemented with serine, threonine, or xylose. When the mysABCD genes from N. commune KU002 were expressed in E. coli cells, porphyra-334 production occurred, and shinorine production was observed upon serine supplementation in the culture medium. Notably, threonine and xylose supplementation in the culture medium increased the amounts of porphyra-334 in both cellular extracts and culture medium extracts. When the mysD gene was replaced with that from the shinorine producer Actinosynnema mirum JCM 3225, shinorine was primarily synthesized instead of porphyra-334. Interestingly, the transformant expressing the chimeric KU002-mysABC-JCM3225-mysD produced a novel MAA derivative with an absorption maximum at 334 nm and a molecular mass of 346 when cultured in the medium supplemented with threonine and xylose. These results suggest that the substrate specificity of MysD, which catalyzes the conjugation of mycosporine-glycine and serine or threonine, alters the production of porphyra-334 or shinorine and that the supplements added to the culture medium affect the amount and composition of MAAs produced in the E. coli transformant.

Methyl methacrylate (MMA), the primary raw material of acrylic resin, is an important polymeric material due to its increasing demand and ease of recycling. The most promising biosynthetic route for MMA involves the condensation of methanol with methacrylyl-CoA (MAA-CoA), an intermediate in the valine degradation pathway. The toxicity of MAA-CoA, poor stability and low activity of the heterologous pathway enzymes make this biosynthetic pathway less feasible. For enabling the evolutionary engineering of this pathway and its components (enzymes), we constructed a biosensor system in which the cellular level of key intermediate MAA-CoA can be evaluated in a high-throughput manner. With the aid of this MAA-CoA sensory system, we could establish the functional pathway from isobutyric acid to MAA-CoA. The sensor described in this paper should be valuable tool in the design-build-test-learn cycle for optimizing and breeding this MMA pathway.