Background Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains limited despite growing global demands. Low yields of the critical taxadiene precursor remains a key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to relieve this bottleneck. Results Multi-copy chromosomal integration of TASY harbouring a selection of fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at microscale, compared to expressing a single episomal copy of TASY. The scalability of the process was highlighted through achieving similar titres during scale up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively at 20 and 30 °C. Maximum taxadiene titres of 129 ± 15 mg/L and 127 mg/L were achieved through shake flask and bioreactor cultivations, respectively, of the optimal strain at a reduced temperature of 20 °C. Conclusions The results of this study highlight the benefit of employing a combination of molecular biology and bioprocess tools during synthetic pathway development, with which TASY activity was successfully improved by 6.5-fold compared to the highest literature titre in S. cerevisiae cell factories.

Thanks to its sophistication, the CRISPR/Cas system has been a widely used yeast genome editing method. However, CRISPR methods generally rely on preassembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose an alternative method, Assembly and CRISPR-targeted in vivo Editing (ACtivE), that only relies on in vivo assembly of linear DNA fragments for plasmid and donor DNA construction. Thus, depending on the user’s need, these parts can be easily selected and combined from a repository, serving as a toolkit for rapid genome editing without any expensive reagent. The toolkit contains verified linear DNA fragments, which are easy to store, share, and transport at room temperature, drastically reducing expensive shipping costs and assembly time. After optimizing this technique, eight loci proximal to autonomously replicating sequences (ARS) in the yeast genome were also characterized in terms of integration and gene expression efficiencies and the impacts of the disruptions of these regions on cell fitness. The flexibility and multiplexing capacity of the ACtivE were shown by constructing a β-carotene pathway. In only a few days, >80% integration efficiency for single gene integration and >50% integration efficiency for triplex integration were achieved on Saccharomyces cerevisiae BY4741 from scratch without using in vitro DNA assembly methods, restriction enzymes, or extra cloning steps. This study presents a standardizable method to be readily employed to accelerate yeast genome engineering and provides well-defined genomic location alternatives for yeast synthetic biology and metabolic engineering purposes.

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP‐20, XAD7HP, and HP‐2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi‐continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α‐yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell‐waste reactive oxygen species concentrations from the yeast were 1.5‐fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi‐continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.

The COVID-19 pandemic has had an enormous impact on economies and health systems globally, therefore a top priority is the development of increasingly better diagnostic and surveillance alternatives to slow down the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In order to establish massive testing and contact tracing policies, it is crucial to have a clear view of the diagnostic options available and their principal advantages and drawbacks. Although classical molecular methods such as RT-qPCR are broadly used, diagnostic alternatives based on technologies such as LAMP, antigen, serological testing, or the application of novel technologies such as CRISPR-Cas for diagnostics, are also discussed. The present review also discusses the most important automation strategies employed to increase testing capability. Several serological-based diagnostic kits are presented, as well as novel nanotechnology-based diagnostic methods. In summary, this review provides a clear diagnostic landscape of the most relevant tools to track COVID-19.

The COVID-19 pandemic has become a global challenge for the healthcare systems of many countries with 6 million people having lost their lives and 530 million more having tested positive for the virus. Robust testing and a comprehensive track and trace process for positive patients are essential for effective pandemic control, leading to high demand for diagnostic testing. In order to comply with demand and increase testing capacity worldwide, automated workflows have come into prominence as they enable highthroughput screening, faster processing, exclusion of human error, repeatability, reproducibility and diagnostic precision. The gold standard for COVID-19 testing so far has been RT-qPCR, however, different SARS-CoV-2 testing methods have been developed to be combined with high throughput testing to improve diagnosis. Case studies in China, Spain and the United Kingdom have been reviewed and automation has been proven to be promising for mass testing. Free and Open Source scientific and medical Hardware (FOSH) plays a vital role in this matter but there are some challenges to be overcome before automation can be fully implemented. This review discusses the importance of automated high-throughput testing, the different equipment available, the bottlenecks of its implementation and key selected case studies that due to their high effectiveness are already in use in hospitals and research centres.