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Development of a multi-gene expression system in Xanthophyllomyces dendrorhous.

Hara KY, Morita T, Mochizuki M, Yamamoto K, Ogino C, Araki M, Kondo A - Microb. Cell Fact. (2014)

Bottom Line: 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.Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in β-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.

View Article: PubMed Central - PubMed

Affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. kiyotaka.hara@dolphin.kobe-u.ac.jp.

ABSTRACT

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 β-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.

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Astaxanthin production by mutant strains overexpressingcrtEandcrtE/crtS. Volumetric astaxanthin concentration (mg/L) of culture fermented for 72 h by the engineered strains overexpressing crtE gene or both crtE and crtS genes (crtE/crtS) with or without acaT/hmgS/hmgR overexpression. The values are means and the error bars show the SD (n = 3). The statistical difference between each gene-expression strain and its host strain analyzed by student’s t-test was shown as P-value.
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Fig4: Astaxanthin production by mutant strains overexpressingcrtEandcrtE/crtS. Volumetric astaxanthin concentration (mg/L) of culture fermented for 72 h by the engineered strains overexpressing crtE gene or both crtE and crtS genes (crtE/crtS) with or without acaT/hmgS/hmgR overexpression. The values are means and the error bars show the SD (n = 3). The statistical difference between each gene-expression strain and its host strain analyzed by student’s t-test was shown as P-value.

Mentions: CrtE catalyzes biosynthesis of geranylgeranyl-pyrophosphate (GGPP) from farnesyl-pyrophosphate (FPP) in the β-carotene synthetic pathway in X. dendrorhous (Figure 1). Breitenbach, et al., reported that self-cloning and overexpression of the crtE gene increased astaxanthin production [13]. To test the synergetic effect on astaxanthin production of enhancing synthetic pathways of mevalonate, β-carotene and astaxanthin, the crtE gene was overexpressed in either the parental strain or the acaT/hmgS/hmgR overexpressing strain using the multiple gene overexpression system. Comparing the volumetric concentration of astaxanthin produced by these strains, crtE overexpression increased the astaxanthin production in both the strains by about 1.3-fold (Figure 4). Additionally, combined overexpression of crtE and crtS, a gene in the astaxanthin synthetic pathway, synergistically increased astaxanthin production in both the parental strain and the acaT/hmgS/hmgR overexpressing strain by about 1.6-fold. The astaxanthin production of the acaT/hmgS/hmgR/crtE/crtS overexpressing strain after 72 h reached 3.0 mg/L, which was 2.1-fold higher compared with the control strain (1.4 mg/L). This result indicates that the increased mevalonate production platform strain overexpressing acaT/hmgS/hmgR has potential to enhance isoprenoids production when combined with additional overexpression of appropriate genes, depending on the target isoprenoid, using the multiple gene overexpression system developed in this study.Figure 4


Development of a multi-gene expression system in Xanthophyllomyces dendrorhous.

Hara KY, Morita T, Mochizuki M, Yamamoto K, Ogino C, Araki M, Kondo A - Microb. Cell Fact. (2014)

Astaxanthin production by mutant strains overexpressingcrtEandcrtE/crtS. Volumetric astaxanthin concentration (mg/L) of culture fermented for 72 h by the engineered strains overexpressing crtE gene or both crtE and crtS genes (crtE/crtS) with or without acaT/hmgS/hmgR overexpression. The values are means and the error bars show the SD (n = 3). The statistical difference between each gene-expression strain and its host strain analyzed by student’s t-test was shown as P-value.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4264253&req=5

Fig4: Astaxanthin production by mutant strains overexpressingcrtEandcrtE/crtS. Volumetric astaxanthin concentration (mg/L) of culture fermented for 72 h by the engineered strains overexpressing crtE gene or both crtE and crtS genes (crtE/crtS) with or without acaT/hmgS/hmgR overexpression. The values are means and the error bars show the SD (n = 3). The statistical difference between each gene-expression strain and its host strain analyzed by student’s t-test was shown as P-value.
Mentions: CrtE catalyzes biosynthesis of geranylgeranyl-pyrophosphate (GGPP) from farnesyl-pyrophosphate (FPP) in the β-carotene synthetic pathway in X. dendrorhous (Figure 1). Breitenbach, et al., reported that self-cloning and overexpression of the crtE gene increased astaxanthin production [13]. To test the synergetic effect on astaxanthin production of enhancing synthetic pathways of mevalonate, β-carotene and astaxanthin, the crtE gene was overexpressed in either the parental strain or the acaT/hmgS/hmgR overexpressing strain using the multiple gene overexpression system. Comparing the volumetric concentration of astaxanthin produced by these strains, crtE overexpression increased the astaxanthin production in both the strains by about 1.3-fold (Figure 4). Additionally, combined overexpression of crtE and crtS, a gene in the astaxanthin synthetic pathway, synergistically increased astaxanthin production in both the parental strain and the acaT/hmgS/hmgR overexpressing strain by about 1.6-fold. The astaxanthin production of the acaT/hmgS/hmgR/crtE/crtS overexpressing strain after 72 h reached 3.0 mg/L, which was 2.1-fold higher compared with the control strain (1.4 mg/L). This result indicates that the increased mevalonate production platform strain overexpressing acaT/hmgS/hmgR has potential to enhance isoprenoids production when combined with additional overexpression of appropriate genes, depending on the target isoprenoid, using the multiple gene overexpression system developed in this study.Figure 4

Bottom Line: 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.Additionally, this triple overexpression of mevalonate synthetic pathway genes together with genes involved in β-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.

View Article: PubMed Central - PubMed

Affiliation: Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodaicho Nada-ku, Kobe, 657-8501, Japan. kiyotaka.hara@dolphin.kobe-u.ac.jp.

ABSTRACT

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 β-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.

Show MeSH
Related in: MedlinePlus