Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production

Beekwilder, Jules; Rossum, Harmen M. van; Koopman, Frank; Sonntag, Frank; Buchhaupt, Markus; Schrader, Jens; Hall, Robert D.; Bosch, Dirk; Pronk, Jack T.; Maris, Antonius J. A. van; Daran, Jean‐Marc

doi:10.1016/j.jbiotec.2013.12.016

Cited by 107 publications

(100 citation statements)

References 35 publications

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“…2013; Beekwilder et al. 2014) requires introduction of multiple (successive) genetic modifications, including integration of product pathway genes at multiple genetic loci and rewiring central metabolism by modifying properties of specific metabolic reactions (e.g. via gene deletion, changing regulatory properties or replacement of native genes by heterologous counterparts) (Wieczorke et al.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Mans

Rossum

Wijsman

et al. 2015

FEMS Yeast Research

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A variety of techniques for strain engineering in Saccharomyces cerevisiae have recently been developed. However, especially when multiple genetic manipulations are required, strain construction is still a time-consuming process. This study describes new CRISPR/Cas9-based approaches for easy, fast strain construction in yeast and explores their potential for simultaneous introduction of multiple genetic modifications. An open-source tool (http://yeastriction.tnw.tudelft.nl) is presented for identification of suitable Cas9 target sites in S. cerevisiae strains. A transformation strategy, using in vivo assembly of a guideRNA plasmid and subsequent genetic modification, was successfully implemented with high accuracies. An alternative strategy, using in vitro assembled plasmids containing two gRNAs, was used to simultaneously introduce up to six genetic modifications in a single transformation step with high efficiencies. Where previous studies mainly focused on the use of CRISPR/Cas9 for gene inactivation, we demonstrate the versatility of CRISPR/Cas9-based engineering of yeast by achieving simultaneous integration of a multigene construct combined with gene deletion and the simultaneous introduction of two single-nucleotide mutations at different loci. Sets of standardized plasmids, as well as the web-based Yeastriction target-sequence identifier and primer-design tool, are made available to the yeast research community to facilitate fast, standardized and efficient application of the CRISPR/Cas9 system.

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Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Mans

Rossum

Wijsman

et al. 2015

FEMS Yeast Research

Self Cite

378

462

View full text Add to dashboard Cite

show abstract

“…However, the US Food and Drug Administration (FDA) does not categorize E. coli as a GRAS microorganism. Efforts in the GRAS yeast Saccharomyces cerevisiae have resulted in yields too low for viable industrial production (1 mg/g dry cell weight) [17, 18]. One promising microorganism is the non-conventional yeast Yarrowia lipolytica .…”

Section: Introductionmentioning

confidence: 99%

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

et al. 2018

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Backgroundβ-Ionone is a fragrant terpenoid that generates a pleasant floral scent and is used in diverse applications as a cosmetic and flavoring ingredient. A growing consumer desire for natural products has increased the market demand for natural β-ionone. To date, chemical extraction from plants remains the main approach for commercial natural β-ionone production. Unfortunately, changing climate and geopolitical issues can cause instability in the β-ionone supply chain. Microbial fermentation using generally recognized as safe (GRAS) yeast offers an alternative method for producing natural β-ionone. Yarrowia lipolytica is an attractive host due to its oleaginous nature, established genetic tools, and large intercellular pool size of acetyl-CoA (the terpenoid backbone precursor).ResultsA push–pull strategy via genome engineering was applied to a Y. lipolytica PO1f derived strain. Heterologous and native genes in the mevalonate pathway were overexpressed to push production to the terpenoid backbone geranylgeranyl pyrophosphate, while the carB and biofunction carRP genes from Mucor circinelloides were introduced to pull flux towards β-carotene (i.e., ionone precursor). Medium tests combined with machine learning based data analysis and 13C metabolite labeling investigated influential nutrients for the β-carotene strain that achieved > 2.5 g/L β-carotene in a rich medium. Further introduction of the carotenoid cleavage dioxygenase 1 (CCD1) from Osmanthus fragrans resulted in the β-ionone production. Utilization of in situ dodecane trapping avoided ionone loss from vaporization (with recovery efficiencies of ~ 76%) during fermentation operations, which resulted in titers of 68 mg/L β-ionone in shaking flasks and 380 mg/L in a 2 L fermenter. Both β-carotene medium tests and β-ionone fermentation outcomes indicated the last enzymatic step CCD1 (rather than acetyl-CoA supply) as the key bottleneck.ConclusionsWe engineered a GRAS Y. lipolytica platform for sustainable and economical production of the natural aroma β-ionone. Although β-carotene could be produced at high titers by Y. lipolytica, the synthesis of β-ionone was relatively poor, possibly due to low CCD1 activity and non-specific CCD1 cleavage of β-carotene. In addition, both β-carotene and β-ionone strains showed decreased performances after successive sub-cultures. For industrial application, β-ionone fermentation efforts should focus on both CCD enzyme engineering and strain stability improvement.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0984-x) contains supplementary material, which is available to authorized users.

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“…The expression of a carotenoid cleavage oxygenase from Petunia hybrida in a strain already producing β-carotene, led to the release of detectable levels of a compound known as β-ionone, which has a highly desired violet scent [65]. Interestingly, a polycistronic version (genes separated from each other by viral T2A sequences) of the abovementioned carotenogenic genes was successfully expressed in yeast [66].…”

Section: Terpenoidsmentioning

confidence: 99%

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

2018

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Yeast-especially Saccharomyces cerevisiae-have long been a preferred workhorse for the production of numerous recombinant proteins and other metabolites. S. cerevisiae is a noteworthy aroma compound producer and has also been exploited to produce foreign bioflavour compounds. In the past few years, important strides have been made in unlocking the key elements in the biochemical pathways involved in the production of many aroma compounds. The expression of these biochemical pathways in yeast often involves the manipulation of the host strain to direct the flux towards certain precursors needed for the production of the given aroma compound. This review highlights recent advances in the bioengineering of yeast-including S. cerevisiae-to produce aroma compounds and bioflavours. To capitalise on recent advances in synthetic yeast genomics, this review presents yeast as a significant producer of bioflavours in a fresh context and proposes new directions for combining engineering and biology principles to improve the yield of targeted aroma compounds.

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Polycistronic expression of a β-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to β-ionone production

Cited by 107 publications

References 35 publications

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

CRISPR/Cas9: a molecular Swiss army knife for simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae

Engineering the oleaginous yeast Yarrowia lipolytica to produce the aroma compound β-ionone

The Smell of Synthetic Biology: Engineering Strategies for Aroma Compound Production in Yeast

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