Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous

Ledetzky, Nadine; Osawa, Ayako; Iki, Kanoko; Pollmann, Hendrik; Gassel, Sören; Breitenbach, Jürgen; Shindo, Kazutoshi; Sandmann, Gerhard

doi:10.1016/j.abb.2014.01.014

Cited by 22 publications

(15 citation statements)

References 26 publications

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“…5 C, when both crtYB and crtI had a stronger promoter (similar to P GAP ), the β-carotene production of engineered strain was generally higher, and when crtYB had a relatively weaker promoter compared to P GAP , the β-carotene production of engineered strain was decreased. This result was consistent with the previous report that CrtYBp was the rate-limiting enzyme in β-carotene production [ 28 ]. Compared to the promoter combinations in the β-carotene biosynthetic pathway of Caro-13 and Caro-14 strain, all corresponding genes used the same promoter except crtE.…”

Section: Resultssupporting

confidence: 94%

Screening and evaluation of the strong endogenous promoters in Pichia pastoris

et al. 2021

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Background Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. Results Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and β-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P0019, P0107, P0230, P0392, or P0785 was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P0208 or P0627 exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the β-galactosidase activity contributed by P0208, P0627, P0019, P0407, P0392, P0230, P0785, or P0107 was relatively lower than PGAP but higher than PACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous β-carotene biosynthetic pathway in P. pastoris, and the highest yield of β-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. Conclusions The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.

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Section: Resultssupporting

confidence: 94%

Screening and evaluation of the strong endogenous promoters in Pichia pastoris

et al. 2021

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“…The amenability of fungi to genetic manipulation and industrial cultivation makes some of them ideal choices for biotechnological carotenoid production, as that of -carotene or lycopene by B. trispora, or astaxanthin by X. dendrorhous [168]. The availability of genetic engineering tools for X. dendrorhous has allowed its use for the production of alternative xanthophylls such as zeaxanthin [169] or new ketocarotenoids [170].…”

Section: Fungimentioning

confidence: 99%

A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health

Rodríguez‐Concepción

Ávalos

Bonet

et al. 2018

Progress in Lipid Research

753

584

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Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits.

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“…Formation of ketolated β-carotene derivatives echinenone and canthaxanthin demonstrate that the ketolation step is not limited ( Figure 2 E). It has previously been shown that conversion rates of transgenic reactions in X. dendrorhous are dependent on the number of trans gene copies integrated into the genome [ 32 ]. Therefore, either transformation with a plasmid carrying two copies of the 3-hydroxylase gene crtZ as demonstrated by Pollmann et al [ 12 ] or repeated transformation with the crtZ gene is a promising way to improve intermediate conversion to the end product.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial Biosynthesis of Novel Multi-Hydroxy Carotenoids in the Red Yeast Xanthophyllomyces dendrorhous

Pollmann

Breitenbach

Wolff

et al. 2017

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The red yeast Xanthophyllomyces dendrorhous is an established platform for the synthesis of carotenoids. It was used for the generation of novel multi oxygenated carotenoid structures. This was achieved by a combinatorial approach starting with the selection of a β-carotene accumulating mutant, stepwise pathway engineering by integration of three microbial genes into the genome and finally the chemical reduction of the resulting 4,4’-diketo-nostoxanthin (2,3,2’,3’-tetrahydroxy-4,4’-diketo-β-carotene) and 4-keto-nostoxanthin (2,3,2’,3’-tetrahydroxy-4-monoketo-β-carotene). Both keto carotenoids and the resulting 4,4’-dihydroxy-nostoxanthin (2,3,4,2’,3’,4’-hexahydroxy-β-carotene) and 4-hydroxy-nostoxanthin (2,3,4,2’3’-pentahydroxy-β-carotene) were separated by high-performance liquid chromatography (HPLC) and analyzed by mass spectrometry. Their molecular masses and fragmentation patterns allowed the unequivocal identification of all four carotenoids.

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Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous

Cited by 22 publications

References 26 publications

Screening and evaluation of the strong endogenous promoters in Pichia pastoris

Screening and evaluation of the strong endogenous promoters in Pichia pastoris

A global perspective on carotenoids: Metabolism, biotechnology, and benefits for nutrition and health

Combinatorial Biosynthesis of Novel Multi-Hydroxy Carotenoids in the Red Yeast Xanthophyllomyces dendrorhous

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