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High-copy-number plasmids are indispensable tools for gene overexpression studies in prokaryotes to engineer pathways or probe phenotypes of interest. The development of genetic tools for the industrially relevant Actinobacteria is of special interest, given their utility in producing keratolytic enzymes and biologically active natural products. Within the Actinobacteria, Streptomyces–Escherichia coli shuttle vectors based on the SCP2* and pIJ101 incompatibility groups are widely employed for molecular cloning and gene expression studies. Here, the sequences of two commonly used pIJ101-based Streptomyces–E. coli shuttle vectors, pEM4 and pUWL201, were determined using next-generation sequencing. These plasmids drive the expression of heterologous genes using the constitutive ermE*p promoter. pEM4 was found to be 8.3 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. pUWL201 was found to be 6.78 kbp long, containing a β-lactamase gene, thiostrepton resistance marker, the lacZɑ fragment, a ColE1 origin of replication and the Streptomyces pIJ101 origin of replication. Interestingly, the sequences for both pEM4 and pUWL201 exceed their previously reported size by 1.1 and 0.4 kbp, respectively. This report updates the literature with the corrected sequences for these shuttle vectors, ensuring their compatibility with modern synthetic biology cloning methodologies. 

Metagenome-assembled genomes were generated for two xenic cyanobacterial strains collected from aquatic sources in Kenya and sequenced by NovaSeq S4. Here, we report the classification and genome statistics of Microcystis panniformis WG22 and Limnospira fusiformis. 

The Nile perch (Lates niloticus L.) commercial fishery for Lake Victoria in East Africa is an important source of revenue and employment. We focused on shifts in food web structure and total mercury (THg) bioaccumulation and biomagnification in Nile perch, and lower food web items collected from Winam Gulf (Kenya) sampled 24 years apart (1998 and 2022). Stable isotope carbon (δ13C) values were higher in all species from 2022 compared to 1998. Stable nitrogen isotope (δ15N) values in baseline organisms were lower in 2022 compared to 1998. In Nile perch, δ15N values were correlated with total length, but the δ15N-length regressions were steeper in 1998 compared to 2022 except for one large (158 cm) Nile perch from 1998 with an uncharacteristically low δ15N value. Total Hg concentrations were lower in lower trophic species from 2022 compared to 1998. However, the THg bioaccumulation rate (as a function of fish length) in Nile perch was greater in 2022 compared to 1998 resulting in 24.2 % to 42.4 % higher wet weight dorsal THg concentrations in 2022 Nile perch for market slot size (50 to 85 cm) fish. The contrasting observations of increased THg bioaccumulation with size in 2022 against decreases in the rate of trophic increase with size and lower THg concentrations of lower food web items imply reduced fish growth and potential bioenergetic stressors on Winam Gulf Nile perch. All samples except 1 large Nile perch (139 cm total length collected in 2022) had THg concentrations below the European Union trade limit (500 ng/g wet weight). However, for more vulnerable individuals (women, children and frequent fish eaters), we recommend a decrease in maximum monthly meal consumption for 55–75 cm Nile perch from 16 meals per month calculated for 1998 to a limit of 8 meals per month calculated for 2022. 

ABSTRACT The Winam Gulf in the Kenyan region of Lake Victoria experiences prolific, year-round cyanobacterial harmful algal blooms (cyanoHABs) which pose threats to human, livestock, and ecosystem health. To our knowledge, there is limited molecular research on the gulf’s cyanoHABs, and thus, the strategies employed for survival and proliferation by toxigenic cyanobacteria in this region remain largely unexplored. Here, we used metagenomics to analyze the Winam Gulf’s cyanobacterial composition, function, and biosynthetic potential.Dolichospermumwas the dominant bloom-forming cyanobacterium, co-occurring withMicrocystisat most sites.MicrocystisandPlanktothrixwere more abundant in shallow and turbid sites. Metagenome-assembled genomes (MAGs) ofDolichospermumharbored nitrogen fixation genes, suggesting diazotrophy as a potential mechanism supporting the proliferation ofDolichospermumin the nitrogen-limited gulf. Over 300 biosynthetic gene clusters (BGCs) putatively encoding the synthesis of toxins and other secondary metabolites were identified across the gulf, even at sites where there were no visible cyanoHAB events. Almost all BGCs identified had no known synthesis product, indicating a diverse and novel biosynthetic repertoire capable of synthesizing harmful or potentially therapeutic metabolites.MicrocystisMAGs containedmcygenes encoding the synthesis of hepatotoxic microcystins which are a concern for drinking water safety. These findings illustrate the spatial variation of bloom-forming cyanobacteria in the Winam Gulf and their available strategies to dominate different ecological niches. This study underscores the need for further use of genomic techniques to elucidate the dynamics and mitigate the potentially harmful effects of cyanoHABs and their associated toxins on human, environmental, and economic health. 

ABSTRACT We report 40 metagenomic libraries collected from the Winam Gulf of Lake Victoria during May–July of 2022–2023 and an additional eight opportunistic libraries from adjacent Lakes Simbi, Naivasha, and regional river systems. The sampling period captured cyanobacterial bloom events – shedding insight onto community composition and genomic potential. 

Background/Goal/Aim The tetracenomycins are aromatic anticancer polyketides that inhibit peptide translation via binding to the large ribosomal subunit. Here, we expressed the elloramycin biosynthetic gene cluster in the heterologous host Streptomyces coelicolor M1146 to facilitate the downstream production of tetracenomycin analogs. Main Methods and Major Results We developed a BioBricks® genetic toolbox of genetic parts for substrate precursor engineering in S. coelicolor M1146::cos16F4iE. We cloned a series of integrating vectors based on the VWB, TG1, and SV1 integrase systems to interrogate gene expression in the chromosome. We genetically engineered three separate genetic constructs to modulate tetracenomycin biosynthesis: 1) the vhb hemoglobin from obligate aerobe Vitreoscilla stercoraria to improve oxygen utilization; (2) the accA2BE acetyl-CoA carboxylase to enhance condensation of malonyl-CoA; (3) lastly, the sco6196 acyltransferase, which is a “metabolic regulatory switch” responsible for mobilizing triacylglycerols to β-oxidation machinery for acetyl-CoA. In addition, we engineered the tcmO 8-O-methyltransferase and newly identified tcmD 12-O-methyltransferase from Amycolatopsis sp. A23 to generate tetracenomycins C and X. We also co-expressed the tcmO methyltransferase with oxygenase urdE to generate the analog 6-hydroxy-tetracenomycin C. Conclusions and Implications Altogether, this system is compatible with the BioBricks® [RFC 10] cloning standard for the co-expression of multiple gene sets for metabolic engineering of Streptomyces coelicolor M1146::cos16F4iE. This production platform improves access to potent analogs, such as tetracenomycin X, and sets the stage for the production of new tetracenomycins via combinatorial biosynthesis. This article is protected by copyright. All rights reserved