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1.
Microb Cell Fact ; 15(1): 155, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27624332

RESUMO

BACKGROUND: Red yeast, Xanthophyllomyces dendrorhous is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) widely used in the aquaculture, food, pharmaceutical and cosmetic industries. The potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Recently, we developed a multiple gene expression system in X. dendrorhous and enhanced the mevalonate synthetic pathway to increase astaxanthin production. In contrast, the mevalonate synthetic pathway is suppressed by ergosterol through feedback inhibition. Therefore, releasing the mevalonate synthetic pathway from this inhibition through the deletion of genes involved in ergosterol synthesis is a promising strategy to improve isoprenoid production. An efficient method for deleting diploid genes in X. dendrorhous, however, has not yet been developed. RESULTS: Xanthophyllomyces dendrorhous was cultivated under gradually increasing concentrations of antibiotics following the introduction of antibiotic resistant genes to be replaced with target genes. Using this method, double CYP61 genes encoding C-22 sterol desaturases relating to ergosterol biosynthesis were deleted sequentially. This double CYP61 deleted strain showed decreased ergosterol biosynthesis compared with the parental strain and single CYP61 disrupted strain. Additionally, this double deletion of CYP61 genes showed increased astaxanthin production compared with the parental strain and the single CYP61 knockout strain. Finally, astaxanthin production was enhanced by 1.4-fold compared with the parental strain, although astaxanthin production was not affected in the single CYP61 knockout strain. CONCLUSIONS: In this study, we developed a system to completely delete target diploid genes in X. dendrorhous. Using this method, we deleted diploid CYP61 genes involved in the synthesis of ergosterol that inhibits the pathway for mevalonate, which is a common substrate for isoprenoid biosynthesis. The resulting decrease in ergosterol biosynthesis increased astaxanthin production. The efficient method for deleting diploid genes developed in this study has the potential to improve industrial production of various isoprenoids in X. dendrorhous.


Assuntos
Basidiomycota/genética , Vias Biossintéticas/genética , Deleção de Genes , Engenharia Metabólica/métodos , Sequência de Bases , Basidiomycota/metabolismo , Carotenoides/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Farmacorresistência Fúngica/genética , Ergosterol/biossíntese , Fermentação , Expressão Gênica , Genes Fúngicos , Ácido Mevalônico/metabolismo , Reação em Cadeia da Polimerase , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Xantofilas/biossíntese , Xantofilas/metabolismo
2.
Microb Cell Fact ; 13: 175, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25471659

RESUMO

BACKGROUND: Red yeast, Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast known to produce astaxanthin, an anti-oxidant isoprenoid (carotenoid) that is widely used in the aquaculture, food, pharmaceutical and cosmetic industries. Recently, the potential of this microorganism as a platform cell factory for isoprenoid production has been recognized because of high flux through its native terpene pathway. Addition of mevalonate, the common precursor for isoprenoid biosynthesis, has been shown to be critical to enhance the astaxanthin content in X. dendrorhous. However, addition of mevalonate is unrealistic during industrial isoprenoid production because it is an unstable and costly chemical. Therefore, up-regulating the intracellular mevalonate supply by enhancing the mevalonate synthetic pathway though genetic engineering is a promising strategy to improve isoprenoid production in X. dendrorhous. However, a system to strongly express multiple genes has been poorly developed for X. dendrorhous. RESULTS: Here, we developed a multiple gene expression system using plasmids containing three strong promoters in X. dendrorhous (actin, alcohol dehydrogenase and triose-phosphate isomerase) and their terminators. Using this system, three mevalonate synthetic pathway genes encoding acetoacetyl-CoA thiolase, HMG-CoA synthase and HMG-CoA reductase were overexpressed at the same time. This triple overexpressing strain showed an increase in astaxanthin production compared with each single overexpressing strain. Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in ß-carotene and astaxanthin synthesis showed a synergetic effect on increasing astaxanthin production. Finally, astaxanthin production was enhanced by 2.1-fold compared with the parental strain without a reduction of cell growth. CONCLUSIONS: We developed a system to strongly overexpress multiple genes in X. dendrorhous. Using this system, the synthetic pathway of mevalonate, a common substrate for isoprenoid biosynthesis, was enhanced, causing an increase in astaxanthin production. Combining this multiple gene overexpression system with a platform strain that overproduces mevalonate has the potential to improve industrial production of various isoprenoids in X. dendrorhous.


Assuntos
Basidiomycota , Expressão Gênica , Engenharia Metabólica/métodos , Leveduras , Basidiomycota/genética , Basidiomycota/metabolismo , Leveduras/genética , Leveduras/metabolismo
3.
Appl Microbiol Biotechnol ; 98(15): 6787-93, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24737060

RESUMO

Astaxanthin is a valuable carotenoid that is widely used in the aquaculture, food, pharmaceutical, and cosmetic industries. Xanthophyllomyces dendrorhous is a carotenoid-synthesizing yeast strain that produces astaxanthin as its main pigment. Although metabolic engineering using gene manipulation is a valuable way to improve astaxanthin production, a gene expression system for X. dendrorhous has been poorly developed. In this study, three known promoters of X. dendrorhous, glycerol-3-phosphate dehydrogenase (gpd) promoter (Pgpd), glucose dehydrogenase (gdh) promoter (Pgdh), and actin (act) promoter (Pact), were evaluated for use in the overexpression of target proteins using green fluorescence protein (GFP) as an expression level indicator protein. The actin promoter, Pact, showed the highest expression level of GFP when compared with Pgpd and Pgdh. Additionally, to obtain new promoters for higher expression of target protein in X. dendrorhous, intracellular GFP intensity was evaluated for 13 candidate promoters. An alcohol dehydrogenase promoter, Padh4, showed more efficient expression of GFP rather than Pact. Overexpression of crtE gene encoding rate-limiting enzyme of carotenoid synthesis under the adh4 promoter yielded an increase in intracellular astaxanthin content of about 1.7-fold compared with the control strain. The promoters identified in this study must be useful for improving carotenoids production in X. dendrorhous.


Assuntos
Proteínas Fúngicas/genética , Regiões Promotoras Genéticas , Leveduras/metabolismo , Actinas/genética , Regulação Fúngica da Expressão Gênica , Glucose 1-Desidrogenase/genética , Glicerolfosfato Desidrogenase/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Xantofilas/biossíntese , Leveduras/enzimologia , Leveduras/genética
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