Part:BBa_K1959001
Modification of phytoene desaturase (PDS/CrtI)
The BBa_K1959002 contains the coding sequence (CDS) of phytoene desaturase (PDS), which is codon-optimized for rice (Oryza sativa) according to the CrtI sequence of Erwinia uredovora in original part: BBa_K118003 and included a Pea transit peptide of RUBISCO small subunit in its N-terminus. PDS/CrtI (EC 1.3.99.31) catalyzes the conversion of 15-cis-phytoene to all-trans-lycopene.
Usage and Biology
Phytoene desaturase (PDS, EC 1.3.99.31) is an enzyme involved in β-carotene production. β-carotene, one intermediate product of astaxanthin biosynthesis, is converted from all-trans- lycopene by endogenous gene β-LCY in plants. Synthesis of plant lycopene is a four-step reaction regulated by four genes. While in bacteria, Erwinia uredovora, forming of lycopene is a one-step reaction catalyzed by CrtI (Figure. 1). Therefore, we used CrtI gene of Erwinia uredovora to simplify the reconstructed biosynthetic pathway of astaxanthin in our project. CrtI gene had been used and developed to standard Biobrick (BBa_K118003) by previous iGEM team. However, original CrtI gene is not suitable to express in plants. Thus, codon optimization on CrtI is made for rice. In addition, the Pea transit peptide of Rubisco small subunit was fused with the optimized CDS of CrtI, allowing plastid-import of CrtI, which makes it stable and functional. With the help of endogenous gene β-LCY, the β-carotene was synthezed in rice endosperm, stepping forward to astaxanthin biosynthesis.
Figure.1 Phytoene desaturation in plants (left) and bacteria (right).
Synthesis of lycopene in plants is a four-step reaction regulated by four genes, consisting of the two desaturases, phytoene desaturase (PDS) and f-carotene desaturase (ZDS). While in Erwinia uredovora, forming of lycopene is a one-step reaction encompassing all four desaturation steps and one cis-trans isomerization step catalyzed by CrtI.[1]
Codon optimization of BBa_K118003
BBa_K118003 is the coding sequence of CrtI from Erwinia uredovora, which may be unstable in rice. In order to enhance its expression efficiency, we optimized EuCrtI’s codon according to the codon bias of rice. Figure 2 shows the sequence difference between EuCrtI (BBa_K118003) and coCrtI (codon-optimized for rice) (BBa_K1959002). Figure 3 shows the same amino acid sequence of EuCrtI and coCrtI.
Figure 2 Sequence difference between EuCrtI (BBa_K118003 )and coCrtI (BBa_K1959002).
Figure 3 Amino acid sequence of translated DNA of EuCrtI (BBa_K118003) and coCrtI (BBa_K1959002).
Sequence alignment shows that there are no differences between amino acid sequence of original CtrI and codon-optimized CrtI.
Transcriptional Level of CrtI
Semi-quantitative RT-PCR was performed to detect the expression level of CrtI involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and cDNA was synthesized from 1μg DNase-treated RNA.
Expected bands of the CrtI gene were observed on the gel, indicated that CrtI gene was transcribed in endosperm.
aSTRice Phenotype
CrtI is the key enzyme of astaxanthin biosynthesis. Rice without CrtI fails to accumulate astaxanthin, appearing white in “wild type”. aSTARice contains astaxanthin and appears orange-red-color because of the coordinated expression of CrtI gene and other key astaxanthin biosynthetic genes (Figure.5). Therefore, the phenotype of aSTARice indicated that the CrtI gene is a functional gene in rice.
HPLC analysis
To further confirm the synthetic astaxanthin in aSTARice, HPLC was performed to analyze the pigment composition. Astaxanthin is identified on the basis of retention times related to standard sample. According to the retention time of standard astaxanthin sample, astaxanthin compound of extracts from transgenic rice can be confirmed (Figure.6). In addition, astaxanthin possessed the biggest peak area in the carotenoids profile, indicated that astaxanthin was the predominant carotenoid in aSTARice.
The above results demonstrated our part (BBa_K1959002) is a functional part suitable for expression in plants.
Reference
[1]Schaub, P., Yu, Q., Gemmecker, S., Poussincourmontagne, P., Mailliot, J., & Mcewen, A. G., et al. (2012). On the structure and function of the phytoene desaturase crti from pantoea ananatis, a membrane-peripheral and fad-dependent oxidase/isomerase. Plos One, 7(6), e39550-e39550.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 242
Illegal XhoI site found at 1372 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1582
- 1000COMPATIBLE WITH RFC[1000]
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