Part:BBa_K4182008
AA cluster
AA, aspartic acid, is a novel natural herbicide that can be synthesized by fungi (Yan Y et al, 2018). AA targets dihydroxylation dehydrase (DHAD) in the synthesis pathway branched-chain amino acid and leads to the growth inhibition of plants. Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential nutrients for plant growth, and the key point of their biosynthetic pathways are dihydroxydehydrase (DHAD) which catalyzes αβ-dihydroxylation dehydration reaction to form the precursor α-ketoacid. DHAD is highly conserved in different plant species and DHAD with its BCAA biosynthetic pathway does not exist in mammals, making it an ideal target for herbicides. The biosynthetic pathway of AA includes: the precursor pGPP is synthetized via MVA pathway from glucose, which will be catalyzed by FPPS to generate FPP, and eventually to AA by astABC gene cluster. astABC gene cluster was from the soil fungus Aspergillus terreus, and were codon-optimized based on E. coli, chemically synthesized, and cloned in our study. Our primary work on the novel herbicide aspartic acid and its astABC gene cluster will benefit its wide applications in the future.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1877
Illegal EcoRI site found at 4795
Illegal PstI site found at 126
Illegal PstI site found at 306
Illegal PstI site found at 1413
Illegal PstI site found at 2744
Illegal PstI site found at 4552 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1877
Illegal EcoRI site found at 4795
Illegal PstI site found at 126
Illegal PstI site found at 306
Illegal PstI site found at 1413
Illegal PstI site found at 2744
Illegal PstI site found at 4552 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1877
Illegal EcoRI site found at 4795
Illegal BamHI site found at 184 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1877
Illegal EcoRI site found at 4795
Illegal PstI site found at 126
Illegal PstI site found at 306
Illegal PstI site found at 1413
Illegal PstI site found at 2744
Illegal PstI site found at 4552 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1877
Illegal EcoRI site found at 4795
Illegal PstI site found at 126
Illegal PstI site found at 306
Illegal PstI site found at 1413
Illegal PstI site found at 2744
Illegal PstI site found at 4552
Illegal NgoMIV site found at 2015
Illegal NgoMIV site found at 3345
Illegal AgeI site found at 2170
Illegal AgeI site found at 3970 - 1000COMPATIBLE WITH RFC[1000]
Profile
Base Pairs
Design Notes
This gene cluster has been optimized for E. coli
Source
soil fungus Aspergillus terreus
Usage&Biology
Construction and validation of AA synthetic plasmid (plasmid 3).
FPPS and astABC (from the soil fungus Aspergillus terreus) were codon-optimized and genetically synthesized according to E. coli, respectively. where astAB and astC are present on two separate plasmids, respectively.
The final plasmid III uses the medium-copy plasmid MCS1 as the backbone (to avoid metabolic stress caused by high-copy plasmids), contains the astABC trigene and specific transcription terminator T1 from the E. coli rrnB gene regulated by the lac promoter, and multiple highly active ribosomal binding sites (RBS1-3). The astABC gene, LacI-Plac regulatory sequence, and MCS plasmid skeleton were obtained using PCR technology, respectively, and the final plasmid 3 was obtained by further one-step ligation using the golden gate technique.
Figure 3: The plasmid in which Ast ABC is located
Figure 4: The MCS skeleton used and a brief illustration
Figure 5: Complete plasmid profile finally constructed by the experimental group
Figure 6: Agarose gel electrophoresis of the LacI gene
Figure 7: Colony PCR glue plot of plasmid 3 (the presence of the marker gene is used to prove the presence of the plasmid)
References
[1]Resistance-gene-directed discovery of a natural-product herbicide with a new mode of action
| None |