Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-ylacetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol ®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L.

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.

As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker's yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of preplaced gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.

Simple and effective molecular diagnostic methods have gained importance due to the devastating effects of the COVID-19 pandemic. Various isothermal one-pot COVID-19 detection methods have been proposed as favorable alternatives to standard RT-qPCR methods as they do not require sophisticated and/or expensive devices. However, as one-pot reactions are highly complex with a large number of variables, determining the optimum conditions to maximize sensitivity while minimizing diagnostic cost can be cumbersome. Here, statistical design of experiments (DoE) was employed to accelerate the development and optimization of a CRISPR/Cas12a-RPA-based one-pot detection method for the first time. Using a definitive screening design, factors with a significant effect on performance were elucidated and optimized, facilitating the detection of two copies/μL of full-length SARS-CoV-2 (COVID-19) genome using simple instrumentation. The screening revealed that the addition of a reverse transcription buffer and an RNase inhibitor, components generally omitted in one-pot reactions, improved performance significantly, and optimization of reverse transcription had a critical impact on the method’s sensitivity. This strategic method was also applied in a second approach involving a DNA sequence of the N gene from the COVID-19 genome. The slight differences in optimal conditions for the methods using RNA and DNA templates highlight the importance of reaction-specific optimization in ensuring robust and efficient diagnostic performance. The proposed detection method is automation-compatible, rendering it suitable for high-throughput testing. This study demonstrated the benefits of DoE for the optimization of complex one-pot molecular diagnostics methods to increase detection sensitivity.

Metabolic Engineering 23Highlights 27• Maximum taxadiene titre of 129 ± 15 mg/L in Saccharomyces cerevisiae at 20 °C 28• Integrating fusion protein tagged-taxadiene synthase improved taxadiene titre. 29• Consistent taxadiene titres were achieved at the micro-and mini-bioreactor scales. 30 Abstract 31Cost-effective production of the highly effective anti-cancer drug, paclitaxel (Taxol®), remains 32 limited despite growing global demands. Low yields of the critical taxadiene precursor remains a 33 key bottleneck in microbial production. In this study, the key challenge of poor taxadiene synthase 34 (TASY) solubility in S. cerevisiae was revealed, and the strains were strategically engineered to 35 relieve this bottleneck. Multi-copy chromosomal integration of TASY harbouring a selection of 36 fusion solubility tags improved taxadiene titres 22-fold, up to 57 ± 3 mg/L at 30 °C at shake flask 37 scale. The scalability of the process was highlighted through achieving similar titres during scale 38 up to 25 mL and 250 mL in shake flask and bioreactor cultivations, respectively. Maximum 39 taxadiene titres of 129 ± 15 mg/L and 119 mg/L were achieved through shake flask and bioreactor 40 cultivation, respectively, of the optimal strain at a reduced temperature of 20 °C. The results 41 highlight the positive effect of coupling molecular biology tools with bioprocess variable 42 optimisation on synthetic pathway development. 43 44 Abbreviations 45 46 BTS1, Geranylgeranyl diphosphate synthase; crtE, Geranylgeranyl diphosphate synthase; 47 DMAPP, Dimethylallyl pyrophosphate; ERG8, Phosphomevalonate kinase; ERG9, Farnesyl-48 iv diphosphate farnesyl transferase (squalene synthase); ERG10, 3-hydroxy-3-methylglutaryl-CoA 49 (HMG-CoA) synthase; ERG12, Mevalonate kinase; ERG13, 3-hydroxy-3-methylglutaryl-CoA 50 (HMG-CoA) synthase; ERG19, Mevalonate pyrophosphate decarboxylase; ERG20, Farnesyl 51 pyrophosphate synthetase; FPP, Farnesyl diphosphate; GGOH, (E,E,E)-geranylgeraniol; GGPP, 52 (E,E,E)-Geranylgeranyl diphosphate; GPP, Geranyl diphosphate; HMG1, 3-hydroxy-3-53 methylglutaryl-coenzyme A reductase 1; HMG2, 3-hydroxy-3-methylglutaryl-coenzyme 54 A reductase 2; IDI, Isopentenyl-diphosphate delta-isomerase; IPP, Isopentenyl pyrophosphate; 55 MBP, Maltose binding protein; mvaE, Acetyl-CoA acetyltransferase; mvaS, 56 Hydroxymethylglutaryl-CoA synthase; MVA pathway, Mevalonate pathway; TASY, Taxadiene 57 synthase 58 59 60