Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

Wriessnegger, Tamara; Augustín, Peter; Engleder, Matthias; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Zellnig, Günther; Schwab, Helmut; Pichler, Harald

doi:10.1016/j.ymben.2014.04.001

Cited by 162 publications

(117 citation statements)

References 78 publications

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“…To the best of our knowledge, the highest titers and volumetric (ϩ)-valencene productivities so far were described for Rhodobacter sphaeroides expressing the mevalonate operon from Paracoccus zeaxanthinifaciens and the codon-optimized (ϩ)-valencene synthase CnVS, which reached a titer of 352 mg · liter Ϫ1 and a productivity of 4.88 mg · liter Ϫ1 · h Ϫ1 (35). The yeasts Saccharomyces cerevisiae (titer, 1.36 mg · liter Ϫ1 ; productivity, 18 g · liter Ϫ1 · h Ϫ1 ), Schizophyllum commune (titer, 16.6 mg · liter Ϫ1 ; productivity, 115 g · liter Ϫ1 · h Ϫ1 ), and Pichia pastoris (titer, 51 mg · liter Ϫ1 ; productivity, 1 mg · liter Ϫ1 · h Ϫ1 ), however, proved suitable for the in situ conversion of (ϩ)-valencene to more valuable products, such as (ϩ)-nootkatone (35,54,55).…”

Section: Discussionmentioning

confidence: 99%

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Binder

Frohwitter

Mahr

et al. 2016

Appl Environ Microbiol

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Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-␤-D-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum. In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (؉)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCEOptogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum. By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications. Corynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1-5), but it has also been engineered, for instance, for the production of lower alcohols (6-9), organic acids (10-13), diamines (14, 15), and carotenoids (16-18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogene...

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Section: Discussionmentioning

confidence: 99%

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Binder

Frohwitter

Mahr

et al. 2016

Appl Environ Microbiol

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“…Other examples include the diterpenes taxadiene Engels et al, 2008), levopimaradiene and sclareol (Ignea et al, 2014b;Schalk et al, 2012), the sesquiterpenes α-santalene (Chen et al, 2013b;Scalcinati et al, 2012), 5-epi-aristolochene (Nguyen et al, 2012), bisabolene (Peralta-Yahya et al, 2012), nootkatone (Wriessnegger et al, 2014), Z,E-farnesol and E-β-caryophyllene (Ignea et al, 2012), the monoterpenes sabinene (Ignea et al, 2014a;Zhang et al, 2014), limonene and perillyl alcohol (Alonso-Gutierrez et al, 2013;Brennan et al, 2012), and the hemiterpene gas isoprene (Lindberg et al, 2010). However, efforts so far have mostly focused on the production of specific, industrially-relevant, terpenes.…”

Section: Introductionmentioning

confidence: 99%

Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast

Ignea

Iοannou

Georgantea

et al. 2015

Metabolic Engineering

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Terpenes are a large class of natural products, many of which are used in cosmetics, pharmaceuticals, or biofuels. However, terpene's industrial application is frequently hindered by limited availability of natural sources or low yields of chemical synthesis. In this report, we developed a modular platform based on standardized and exchangeable parts to reproduce and potentially expand the diversity of terpene structures in Saccharomyces cerevisiae. By combining different module-specific parts, we exploited the substrate promiscuity of class I diterpene synthases to produce an array of labdane-type scaffolds. These were subsequently modified by a scaffold decoration module consisting of a mutant library of a promiscuous cytochrome P450 to afford a range of hydroxylated diterpenes. Further P450 protein engineering yielded dedicated and efficient catalysts for specific products. Terpenes produced include precursors of pharmacologically important compounds, molecules that are difficult to obtain from natural sources, or new natural products. The approach described here provides a platform on which additional gene mining, combinatorial biosynthesis, and protein engineering efforts can be integrated to sustainably explore the terpene chemical space.

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“…Other successful attempts to produce terpenoids in yeast include the highly sought-after nootkatone, which imparts a strong grapefruit aroma [63]. This was achieved in P. pastoris with a similar engineering strategy which has been proven fruitful for S. cerevisiae to overexpress a truncated version of its HMG1 gene to improved levels.…”

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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Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

Cited by 162 publications

References 78 publications

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast

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

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