Heterologous production of a new lanthipeptide boletupeptin using a cryptic biosynthetic gene cluster of the myxobacterium Melittangium boletus.

Myxobacteria have comparatively large genomes that contain many biosynthetic genes with the potential to produce secondary metabolites. Based on genome mining, we discovered a new biosynthetic gene cluster of class III lanthipeptide in the genome of the myxobacterium Melittangium boletus. The biosynthetic gene cluster contained a precursor peptide-coding gene bolA, and a class III lanthipeptide synthetase-coding gene bolKC. The expression vector containing bolA and bolKC was constructed using synthetic DNA with codon-optimized sequences based on the commercially available vector pET29b. Co-expression of the two genes in the host Escherichia coli BL21(DE3) yielded a new class III lanthipeptide named boletupeptin. The structure of boletupeptin was proposed to have one unit of labionin, as determined by mass spectrometry experiments after reductive cleavage. This is the first report of a class III lanthipeptide from a myxobacterial origin.

Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT). The amino acid sequence of melA indicated similarity with that of known lanthipeptides mersacidin and lichenicidin A1 by the alignment. To perform heterologous production of new lanthipeptides, the expression vector containing the essential genes (melA and melM) was constructed by utilizing codon-optimized synthetic genes. The co-expression of two genes in the host bacterial cells of Escherichia coli BL21 (DE3) afforded new lanthipeptides named melittapeptins A-C. The structures of melittapeptins A-C including lanthionine/methyllanthionine bridge pattern were proposed based on protease digestion and MS/MS experiments. The native strain of M. boletus did not produce melittapeptins A-C, so heterologous production using the biosynthetic gene cluster was effective in obtaining the lanthipeptides. Melittapeptins A-C showed specific and potent antibacterial activity to the Gram-positive bacterium Micrococcus luteus. To the best of our knowledge, this is the first report of antibacterial lanthipeptides derived from myxobacterial origin. KEY POINTS: • New lanthipeptides melittapeptins were heterologously produced in Escherichia coli. • Melittapeptins showed specific antibacterial activity against Micrococcus luteus. • Melittapeptins were the first antibacterial lanthipeptides of myxobacterial origin.

Wastewater monitoring in tourist cities as potential sentinel sites for near real-time dynamics of imported SARS-CoV-2 variants.

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.