Page 5 - C. Biotechnology and molecular imaging

Antibiotic-free system for phytoene production from recombinant E. coli
Seung-Hye Lee , Seon-Won Kim *
1
1
1 Division of Applied Life Science (BK21 plus Program), PMBBRC, Gyeongsang National University, Jinju 52828,
Republic of Korea
*Corresponding author: Prof. Seon-Won Kim, Tel.: 82-55-772-1362; Fax: 82-55-759-9363; E-mail address: swkim@gnu.ac.kr
BACKGROUND AIM

Phytoene is colorless and absorbs UV-B light from 260 to In this study, for the improved production of phytoene,
320 nm which can be used as a photo-protective material we applied an antibiotic-free expression system. The
to prevent urban skin aging. In order to synthesize system is based on the isoprenoid pathway such as
phytoene in E. coli, the phytoene biosynthesis pathway is the MEP pathway and the MVA pathway. These
introduced by expression vectors. Expression vectors are pathways are required for the production of IPP and
maintained by antibiotics. However, when antibiotics are DMAPP which are essential metabolites for microbial
used in the fermentation process, it causes increase in growth. The genes consisting of the pathways are
production costs. Moreover, as the culture progresses for used instead of the antibiotic marker gene applying
a long time, the antibiotics are decomposing and the selection pressure to maintain the plasmids..
plasmid disappears, leading to a decrease in production.
METHODS

Culture was carried out for 72 hours in 2YT medium containing 3.0%(w/v) glycerol, 0.2%(w/v) arabinose at 30℃,
+with antibiotics, - without antibiotics. Ampicillin and chloramphenicol were added as needed as antibiotics used, and
were used at concentrations of 100µg/mL and 50µg/mL respectively. Phytoene was extracted using acetone. The
extract was centrifuged at 14,000 rpm for 10 minutes, and only the top solution was taken to conduct a quantitative
HPLC analysis.
RESULTS

Table 1. Phytoene production strain. Figure 2. Plasmid stability test of Antibiotic-free

Host Strain 1 Plasmid 2 nd Plasmid phytoene production system.
st
MG1655(DE3) pSNA(-E) pT-PHYm3coE
C-AF pla
AF Host MG1655(DE3)ΔdxrΔadhE::MVAbottom pSNA pT-PHYm3co
AF MG1655(DE3)ΔdxrΔadhE::MVAbottom pSNA(-E) pT-PHYm3coE
dxr is one of the genes in the MEP pathways of E. coli.. pSNA(-E), MVA
pathway expression vector except mvaE; pT-Phym3coE, phytoene Plasmid Stability (%) =
production plasmid containing mvaE.
on Antibiotics containing plate X 100(%)
Figure 1. Phytoene production in antibiotic-free on Antibiotics containing plate
recombinant E. coli.
Antibiotics + - + - + -
1200 24 Hr 100
48 Hr
72 Hr
1000 80
Phytoene (mg/L) 600 Plasmid Stability (%) 60
800
40
400
200 20
0 0 0 24 48 72
C-AF pla AF Host AF Time(hr)
CONCLUSION REFERENCES ACKNOWLEDGEMENTS

As a result, it is possible to maintain the [1] Antonio J.Meléndez-MartínezPaulaMapelli- This work was supported by a grant
plasmid without antibiotics and obtain BrahmCarla M.Stinco 2018. The colourless from the Next-Generation BioGreen 21
improved production of phytoene With carotenoids phytoene and phytofluene: From Program (SSAC, grant#: PJ01326501),
or without antibiotic, the plasmid dietary sources to their usefulness for the Rural Development Administration,
remained at 100% for up to 72 hours. functional foods and nutricosmetics industries. Republic of Korea.
J. Food Anal., 67 (2018), pp. 91-103
This confirmed the establishment of an [2] Peubez., I., Chaudet, N., Mignon, C., Hild,
antibiotic-free phytoene production G., Husson, S., Courtois, V., et al. Antibiotic- Contact information
system and it was confirmed that even free selection in E. coli: new considerations for
when the incubation time is prolonged optimal design and improved production. Micrb. Tel.: 82-55-772-1362
the plasmid can be maintained stably. Cell Fact. 9 2010, 65. doi: 10.1186/1475-2859- Fax: 82-55-759-9363
9-65 E-mail address: swkim@gnu.ac.kr