[국내논문]대장균에서 이소프레노이드 생합성 경로의 대사공학적 개량에 의한 아스타잔틴의 생산성 향상 Enhanced Production of Astaxanthin by Metabolic Engineered Isoprenoid Pathway in Escherichia coli원문보기
이 연구의 목적은 생물공학적으로 이소프레노이드 생합성 유전자를 클로닝하여 이들을 형질전환시킨 대장균을 제조하여 이들을 숙주로 사용하여 아스타잔틴의 생산을 증가시키는 것이다. 본 연구진은 선행연구에서 Paracoccus haeundaensis로부터 아스타잔틴 생산에 관여하는 6개의 아스타잔틴 생합성 유전자군을 보고하였고, 이들 유전자들을 발현 벡타(pCR-XL-TOPO-Crt)에 재조합한 후 이 벡터를 대장균에 형질 전환시켜서 건조중량으로 400 ${\mu}g$/g의 아스타잔틴을 생산하였다. 아스타잔틴의 생산성을 증가시키기 위해서 대장균으로부터 이소프레노이드 생합성 경로에 관여하는 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), isopentenyl (IPP) diphossphate isomerase (idi) 유전자들을 클로닝하였고, 이들 유전자를 (pCR-XL-TOPOCrt-full)와 같이 대장균에 각각 공발현시켰다. idi 유전자와 아스타잔틴 생산에 관여하는 아스타잔틴 생합성 유전자군이 함께 형질 전환된 BL21(DE3) Codon Plus RIL 대장균를 배양하였을때, 건조중량으로 1,200 ${\mu}g$/g의 아스타잔틴을 생산하였다. 따라서 본 연구 결과, 이소프레노이드 생합성 유전자와 아스타잔틴 생합성 유전자군을 공발현 시킬 때 아스타잔틴의 생산이 3배 증가하였다.
이 연구의 목적은 생물공학적으로 이소프레노이드 생합성 유전자를 클로닝하여 이들을 형질전환시킨 대장균을 제조하여 이들을 숙주로 사용하여 아스타잔틴의 생산을 증가시키는 것이다. 본 연구진은 선행연구에서 Paracoccus haeundaensis로부터 아스타잔틴 생산에 관여하는 6개의 아스타잔틴 생합성 유전자군을 보고하였고, 이들 유전자들을 발현 벡타(pCR-XL-TOPO-Crt)에 재조합한 후 이 벡터를 대장균에 형질 전환시켜서 건조중량으로 400 ${\mu}g$/g의 아스타잔틴을 생산하였다. 아스타잔틴의 생산성을 증가시키기 위해서 대장균으로부터 이소프레노이드 생합성 경로에 관여하는 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), isopentenyl (IPP) diphossphate isomerase (idi) 유전자들을 클로닝하였고, 이들 유전자를 (pCR-XL-TOPOCrt-full)와 같이 대장균에 각각 공발현시켰다. idi 유전자와 아스타잔틴 생산에 관여하는 아스타잔틴 생합성 유전자군이 함께 형질 전환된 BL21(DE3) Codon Plus RIL 대장균를 배양하였을때, 건조중량으로 1,200 ${\mu}g$/g의 아스타잔틴을 생산하였다. 따라서 본 연구 결과, 이소프레노이드 생합성 유전자와 아스타잔틴 생합성 유전자군을 공발현 시킬 때 아스타잔틴의 생산이 3배 증가하였다.
The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid bios...
The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ${\mu}g$/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ${\mu}g$/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.
The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ${\mu}g$/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ${\mu}g$/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.
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대상 데이터
coli strain XL1-blue [F'::proA+B+lacIq Δ(lacZ) M15/Tn10 (Tetr)/recA1 endA1 gyrA96(Nalr) thi hsdR17(rk-mk+) supE44 relA1 lac] was used for gene cloning experiments. BL21(DE3) Codon Plus RIL (Staratagen, U.S.A.) was used for the production of astaxanthin. E.
, Milford, MA, USA). The guard column was a Pelliguard LC-18 cartridge (20 mm; SUPELCO, Bellefonte, PA, USA). The mobile phase was a methanol/methyl tert-butyl ether (A/B) gradient having the following parameters (all percentages expressed as v/v) start, 80% A/20% B; 10 min, 65% A/35% B; 20 min, 10% A/90% B.
The injection volume and column temperature were 10 ml and 15℃, respectively. Astaxanthin was detected by absorbance at 470 nm (for phytoene, at 286 nm) and was purchased from Sigma (U.S.A.).
성능/효과
coli strains for comparisons of the common precursors. The results showed the efficient production of IPP, DMAPP and FPP. In order to increase production of astaxanthin, we have used these strains for co-expression of both the astaxanthin biosynthesis gene cluster (pCR-XL-TOPO-Crt-full containing crtW, crtZ, crtY, crtI, crtB, and crtE genes) from P.
coli BL21(DE3) Codon Plus RIL [15]. With comparisons of produced astaxanthin contents from those data with this study, the amounts of accumulated astaxanthin by the co-expression system of both the lytB, idi, and ispA genes in the isoprenoid pathway and the astaxanthin biosynthesis gene cluster (pCR-XL-TOPO-Crt-full containing crtW, crtZ, crtY, crtI, crtB, and crtE genes) have higher values than those of the expression system using only astaxanthin biosynthesis genes. This engineered E.
coli. Consequently, the results of this study can be used to enhance the production of astaxanthin through the manipulation of carotenoid biosynthesis genes in P. haeundaensis and isoprenoid biosynthesis genes from E. coli, and an important application since astaxanthin is a pigment of high economic value. These data will provide a wider base of knowledge on the gene expression of the astaxanthin biosynthesis gene cluster at the molecular level as well as further the biotechnological applications of carotenoids.
후속연구
coli, and an important application since astaxanthin is a pigment of high economic value. These data will provide a wider base of knowledge on the gene expression of the astaxanthin biosynthesis gene cluster at the molecular level as well as further the biotechnological applications of carotenoids. Moreover, the possibility to control separately the expression levels of the lytB, idi, and ispA genes in E.
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