Enhanced production of taxadiene in<i>Saccharomyces cerevisiae</i>

Nowrouzi, Behnaz; Li, Rachel; Walls, Laura E.; d’Espaux, Leopold; Malcı, Koray; Liu, Liang; Borrego, Nestor Jonguitud; Escalera, Albert I. Lerma; Morones‐Ramírez, José Rubén; Keasling, Jay D.; Rios‐Solis, Leonardo

doi:10.1101/2020.06.08.139600

Cited by 7 publications

(22 citation statements)

References 35 publications

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“…The LRS5 strain produced a maximum taxadiene titer of 137 ± 5 g/L when grown in 4% galactose (100:0) medium. This represented 1.06 and 2.5-fold improvements in taxadiene titer to 137 ± 5m g/L compared with the highest literature titers for the strain at 20 and 30°C (Nowrouzi et al, 2020). Although TASY activity has been found to be enhanced at reduced temperature (Nowrouzi et al, 2020), preliminary studies (results not shown) using LRS6 revealed that CYP725A4 activity was dramatically reduced at 20°C compared with 30°C.…”

Section: Taxane Product Profiles Using Microscale Bioreactorsmentioning

confidence: 80%

“…This represented 1.06 and 2.5-fold improvements in taxadiene titer to 137 ± 5m g/L compared with the highest literature titers for the strain at 20 and 30°C (Nowrouzi et al, 2020). Although TASY activity has been found to be enhanced at reduced temperature (Nowrouzi et al, 2020), preliminary studies (results not shown) using LRS6 revealed that CYP725A4 activity was dramatically reduced at 20°C compared with 30°C. In addition, both growth rate in S. cerevisiae (Nowrouzi et al, 2020) and CYP725A4 activity in E. coli (Biggs et al, 2016) have been found to be reduced at a lower temperature.…”

Section: Taxane Product Profiles Using Microscale Bioreactorsmentioning

confidence: 80%

“…1p-TASY-GFP; ARS1622b::GAL1p-MBP-TASY-ERG20; ARS1114a::TDH-3p-MBP-TASY-ERG20) was originally derived from laboratory strain CEN.PK2-1C (EUROSCARF) and has been described in more detail elsewhere (Nowrouzi et al, 2020;Reider Apel et al, 2016). Strain LRS6 was constructed through the addition of gene sequences encoding CYP725A4, its cognate cytochrome P450 reductase (CPR) enzyme and TAT obtained from Taxus cuspidata.…”

Section: Strain Constructionmentioning

confidence: 99%

“…The organic dodecane layer was separated from the culture medium through centrifugation and a 1-μl sample was injected into a TRACE™ 1300 Gas Chromatograph (Thermo Fisher Scientific) coupled to an ISQ LT single quadrupole mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was achieved using a Trace Gold TG-SQC gas chromatography column using a previously described method (Nowrouzi et al, 2020). To identify and quantify the production of compounds by LRS5 and LRS6 ( Figure S1), pure standards of taxadiene, kindly supplied by Baran Lab (The Scripps Research Institute) and GGOH, obtained from Sigma Aldrich (Gillingham), were used.…”

Section: Taxane and Metabolite Identification And Quantificationmentioning

confidence: 99%

“…On the other hand, the eukaryotic host, S. cerevisiae , possesses the necessary biosynthetic and redox capabilities for the expression of such enzymes (Engels et al, 2008). Despite this, the maximum reported taxadiene titers are eightfold lower in yeast (Nowrouzi et al, 2020). Distributing the early Taxol metabolic pathway among E. coli and S. cerevisiae facilitated a total oxygenated taxane titer of 33 mg/L (Zhou et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Walls

Malcı

Nowrouzi

et al. 2020

Biotech & Bioengineering

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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.

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Section: Taxane Product Profiles Using Microscale Bioreactorsmentioning

confidence: 80%

Section: Taxane Product Profiles Using Microscale Bioreactorsmentioning

confidence: 80%

Section: Strain Constructionmentioning

confidence: 99%

Section: Taxane and Metabolite Identification And Quantificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Walls

Malcı

Nowrouzi

et al. 2020

Biotech & Bioengineering

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Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

Walls

Martínez

Chanona

et al. 2021

Biotechnology Journal

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Background Recent technological advancements in synthetic and systems biology have enabled the construction of microbial cell factories expressing diverse heterologous pathways in unprecedentedly short time scales. However, the translation of such laboratory scale breakthroughs to industrial bioprocesses remains a major bottleneck. Methods and Major Results In this study, an accelerated bioprocess development approach was employed to optimize the biosynthetic pathway of the blockbuster chemotherapy drug, Taxol. Statistical design of experiments approaches were coupled with an industrially relevant high‐throughput microbioreactor system to optimize production of key Taxol intermediates, Taxadien‐5α‐ol and Taxadien‐5α‐yl‐acetate, in engineered yeast cell factories. The optimal factor combination was determined via data driven statistical modelling and validated in 1 L bioreactors leading to a 2.1‐fold improvement in taxane production compared to a typical defined media. Elucidation and mitigation of nutrient limitation enhanced product titers a further two‐fold and titers of the critical Taxol precursors, Taxadien‐5α‐ol and Taxadien‐5α‐yl‐acetate were improved to 34 and 11 mg L‐1, representing a three‐fold improvement compared to the highest literature titers in S. cerevisiae. Comparable titers were obtained when the process was scaled up a further five‐fold using 5 L bioreactors. Conclusions The results of this study highlight the benefits of a holistic design of experiments guided approach to expedite early stage bioprocess development.

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ACtivE: Assembly and CRISPR-targeted in vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

Malcı

Jonguitud‐Borrego

Straten

et al. 2022

Preprint

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Among the numerous genetic tools developed for yeast, CRISPR/Cas system has been a widely used genome editing method thanks to its sophistication. However, CRISPR methods for yeast generally rely on pre-assembled DNAs and extra cloning steps to deliver gRNA, Cas protein, and donor DNA. These laborious steps might hinder its usefulness. Here, we propose a convenient, rapid, standardizable CRISPR method, named Assembly and CRISPR-targeted in vivo Editing (ACtivE), which only relies on in vivo assembly of linear DNA fragments for both 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 ARS-close loci 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 beta-carotene pathway. In only a few days, > 80% integration efficiency for single gene integration and > 50% integration efficiency for triplex integration were achieved 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.

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Enhanced production of taxadiene inSaccharomyces cerevisiae

Cited by 7 publications

References 35 publications

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Definitive screening accelerates Taxol biosynthetic pathway optimization and scale up in Saccharomyces cerevisiae cell factories

ACtivE: Assembly and CRISPR-targeted in vivo Editing for Yeast Genome Engineering Using Minimum Reagents and Time

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