Bacterial resistance to antimicrobials is a global health threat. Within the One Health context, water from regions with high antibiotic usage, such as clinical and urban areas, collects at wastewater treatment plants (WWTPs). In the WWTP, the activated sludge becomes a complex environment where various antimicrobials and microorganisms converge. While significant research has focused on the influent, activated sludge, and effluent, upstream and downstream sectors around the WWTP are often neglected. We conducted a systematic analysis using five publicly available metagenomic datasets (n=164) from different WWTP sectors and adjacent freshwater systems: upstream (n=14), influent (n=14), activated sludge (n=109), effluent (n=14), and downstream (n=13) to identify and characterise the microbiome, resistome, and mobilome. Opportunistic pathogenic bacteria, such as Pseudomonas, Aeromonas, and Acidovorax, were found in all WWTP sectors, with abundances exceeding 9% in the influent. ESKAPE pathogens, including Klebsiella pneumoniae and Enterobacter species, were identified in the effluent with abundances over 1%. We detected 230 antibiotic resistance genes (ARGs) throughout the WWTP. FTU and CKO β-lactamase gene families dominated the upstream, effluent, and downstream sectors, while the OXA β-lactamase gene family was highly abundant in the influent and activated sludge. ARGs, such as the OXA β-lactamase gene family, were linked to plasmids. Class-1 integrons, associated with the sul1 gene, a marker for anthropogenic pollution, were prevalent in the effluent and downstream sectors. Integrative elements (ICEclc, Tn4371, and PGI2), linked to ARGs, were identified in all sectors, increasing AMR dissemination. These integrative elements conferred resistance to antibiotics, including sulfonamides, tetracyclines and carbapenems. Our findings highlight the presence of ARGs and mobile genetic elements in WWTPs and nearby freshwater systems, raising concerns about AMR transmission to humans, animals, and the environment. This study emphasises the need for effective AMR monitoring and strategies in wastewater treatment to protect public and environmental health.@en

Yeast metabolism can be engineered to produce xenobiotic compounds, such as cannabinoids, the principal isoprenoids of the plant Cannabis sativa, through heterologous metabolic pathways. However, yeast cell factories continue to have low cannabinoid production. This study employed an integrated omics approach to investigate the physiological effects of cannabidiol on S. cerevisiae CENPK2-1C yeast cultures. We treated the experimental group with 0.5 mM CBD and monitored CENPK2-1C cultures. We observed a latent-stationary phase post-diauxic shift in the experimental group and harvested samples in the inflection point of this growth phase for transcriptomic and metabolomic analysis. We compared the transcriptomes of the CBD-treated yeast and the positive control, identifying eight significantly overexpressed genes with a log fold change of at least 1.5 and a significant adjusted p-value. Three notable genes were PDR5 (an ABC-steroid and cation transporter), CIS1, and YGR035C. These genes are all regulated by pleiotropic drug resistance linked promoters. Knockout and rescue of PDR5 showed that it is a causal factor in the post-diauxic shift phenotype. Metabolomic analysis revealed 48 significant spectra associated with CBD-fed cell pellets, 20 of which were identifiable as non-CBD compounds, including fatty acids, glycerophospholipids, and phosphate-salvage indicators. Our results suggest that mitochondrial regulation and lipidomic remodeling play a role in yeast’s response to CBD, which are employed in tandem with pleiotropic drug resistance (PDR). We conclude that bioengineers should account for off-target product C-flux, energy use from ABC-transport, and post-stationary phase cell growth when developing cannabinoid-biosynthetic yeast strains.@en

Background: Assembly algorithm choice should be a deliberate, well-justified decision when researchers create genome assemblies for eukaryotic organisms from third-generation sequencing technologies. While third-generation sequencing by Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio) has overcome the disadvantages of short read lengths specific to next-generation sequencing (NGS), third-generation sequencers are known to produce more error-prone reads, thereby generating a new set of challenges for assembly algorithms and pipelines. However, the introduction of HiFi reads, which offer substantially reduced error rates, has provided a promising solution for more accurate assembly outcomes. Since the introduction of third-generation sequencing technologies, many tools have been developed that aim to take advantage of the longer reads, and researchers need to choose the correct assembler for their projects. Results: We benchmarked state-of-the-art long-read de novo assemblers to help readers make a balanced choice for the assembly of eukaryotes. To this end, we used 12 real and 64 simulated datasets from different eukaryotic genomes, with different read length distributions, imitating PacBio continuous long-read (CLR), PacBio high-fidelity (HiFi), and ONT sequencing to evaluate the assemblers. We include 5 commonly used long-read assemblers in our benchmark: Canu, Flye, Miniasm, Raven, and wtdbg2 for ONT and PacBio CLR reads. For PacBio HiFi reads, we include 5 state-of-the-art HiFi assemblers: HiCanu, Flye, Hifiasm, LJA, and MBG. Evaluation categories address the following metrics: reference-based metrics, assembly statistics, misassembly count, BUSCO completeness, runtime, and RAM usage. Additionally, we investigated the effect of increased read length on the quality of the assemblies and report that read length can, but does not always, positively impact assembly quality. Conclusions: Our benchmark concludes that there is no assembler that performs the best in all the evaluation categories. However, our results show that overall Flye is the best-performing assembler for PacBio CLR and ONT reads, both on real and simulated data. Meanwhile, best-performing PacBio HiFi assemblers are Hifiasm and LJA. Next, the benchmarking using longer reads shows that the increased read length improves assembly quality, but the extent to which that can be achieved depends on the size and complexity of the reference genome.

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Combinatorial pathway optimization is an important tool in metabolic flux optimization. Simultaneous optimization of a large number of pathway genes often leads to combinatorial explosions. Strain optimization is therefore often performed using iterative design-build-test-learn (DBTL) cycles. The aim of these cycles is to develop a product strain iteratively, every time incorporating learning from the previous cycle. Machine learning methods provide a potentially powerful tool to learn from data and propose new designs for the next DBTL cycle. However, due to the lack of a framework for consistently testing the performance of machine learning methods over multiple DBTL cycles, evaluating the effectiveness of these methods remains a challenge. In this work, we propose a mechanistic kinetic model-based framework to test and optimize machine learning for iterative combinatorial pathway optimization. Using this framework, we show that gradient boosting and random forest models outperform the other tested methods in the low-data regime. We demonstrate that these methods are robust for training set biases and experimental noise. Finally, we introduce an algorithm for recommending new designs using machine learning model predictions. We show that when the number of strains to be built is limited, starting with a large initial DBTL cycle is favorable over building the same number of strains for every cycle.

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