[논문]Metabolic Engineering of Deinococcus radiodurans for the Production of Phytoene

Jeong, Sun-Wook; Kang, Chang Keun; Choi, Yong Jun

doi:10.4014/jmb.1808.08019

Metabolic Engineering of Deinococcus radiodurans for the Production of Phytoene 원문보기

Journal of microbiology and biotechnology, v.28 no.10, 2018년, pp.1691 - 1699

Jeong, Sun-Wook (School of Environmental Engineering, University of Seoul) , Kang, Chang Keun (School of Environmental Engineering, University of Seoul) , Choi, Yong Jun (School of Environmental Engineering, University of Seoul)

Abstract ▼ AI-Helper

A metabolically-engineered Deinococcus radiodurans R1 strain capable of producing phytoene, a colorless $C_{40}$ carotenoid and a promising antioxidant, has been developed. To make this base strain, first, the crtI gene encoding phytoene desaturase was deleted to block the conversion of phytoene to other carotenoids such as lycopene and ${\gamma}$-carotene. This engineered strain produced $0.413{\pm}0.023mg/l$ of phytoene from 10 g/l of fructose. Further enhanced production of phytoene up to $4.46{\pm}0.19mg/l$ was achieved by overexpressing the crtB gene encoding phytoene synthase and the dxs genes encoding 1-deoxy-$\text\tiny{D}$-xylulose-5-phosphate synthase gene, and by deleting the crtD gene. High cell-density culture of our final engineered strain allowed production of $10.3{\pm}0.85mg/l$ of phytoene with the yield and productivity of $1.04{\pm}0.05mg/g$ and $0.143{\pm}0.012mg/l/h$, respectively, from 10 g/l of fructose. Furthermore, the antioxidant potential of phytoene produced by the final engineered strain was confirmed by in vitro DPPH radical-scavenging assay.

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가설 설정

In spite of a natural carotenoid-producing strain, the titer is still less than that of previous studies reporting phytoene production using eukaryotic cells [5, 8, 29] and non-natural producers [6, 30]. We hypothesized that this is mainly because of low cell density. Previously, the maximum cell density of D.

제안 방법

ND: not detected. All experiments were performed in triplicate; error bars denote standard deviations.
For the construction of plasmids expressing, the crtB , crtE, dxs, and idi genes were amplified using corresponding primer sets (Table 2), and cloned into pRADZ3 at SpeI and NotI sites make the pAM41, pAM42, pAM73, and pAM74, respectively. To construct pAM103, pAM104, and pAM105 expressing the crtE, dxs or idi genes together with the crtB gene, the crtE, dxs or idi genes were amplified using GroE-F and CrtE-R1, Dxs-R1, or Idi-R1 primers, respectively, from pAM42, pAM73, or pAM74 plasmids as templates and subsequently cloned into pAM41, behind the crtB gene, respectively, using NotI and BamHI sites.
The construction of all knockout mutants in this study was carried out using Cre-loxP system as previously described [14]. In brief, the DNA fragment containing lox66-km^r -lox71 cassette flanked by approximately 1 kb of both up and downstream nucleotide sequences of target genes (crtI, dr0810, or crtD) was amplified by PCR from pAM1 plasmid and D.
For the construction of plasmids expressing, the crtB , crtE, dxs, and idi genes were amplified using corresponding primer sets (Table 2), and cloned into pRADZ3 at SpeI and NotI sites make the pAM41, pAM42, pAM73, and pAM74, respectively. To construct pAM103, pAM104, and pAM105 expressing the crtE, dxs or idi genes together with the crtB gene, the crtE, dxs or idi genes were amplified using GroE-F and CrtE-R1, Dxs-R1, or Idi-R1 primers, respectively, from pAM42, pAM73, or pAM74 plasmids as templates and subsequently cloned into pAM41, behind the crtB gene, respectively, using NotI and BamHI sites.

대상 데이터

All experiments were performed in triplicate; AA, ascorbic acid. Error bars denote standard deviations.

이론/모형

The free radical scavenging assay was performed using 2,2- diphenyl-1-picrylhydrazyl (DPPH) to estimate the antioxidant activity of phytoene. Samples were mixed with 1.

성능/효과

Among the four different candidates, the ΔcrtI D. radiodurans expressing the crtB gene was found to be the best for phytoene production (up to 1.85 ± 0.18 mg/l).
3333px;">, 2) engineering of key enzymes to overcome hurdle reactions, and3) in silico simulation for identifying metabolic flux networks, should be carried out to enhance performance for the production of phytoene. In conclusion, the final recombinant D. radiodurans strain we developed is a promising strain for the efficient production of phytoene worthy for use as a potent antioxidant.
Moreover, as can be seen in Fig. 3B, more than 70% increased phytoene production (up to 3.25 ± 0.21 mg/l) was achieved by additional overexpression of the dxs gene in ΔcrtI D. radiodurans strain expressing the crtB gene, whereas phytoene titer was slightly decreased in the crtB expressing ΔcrtI D. radiodurans strain together with the idi gene (1.2 ±0.14 mg/l), or crtE gene (1.5 ± 0.07 mg/l).

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