BIO PRODUCTION
Lycopene is the red pigment that gives tomatoes their color. This pigment is also made by microbes. In fact, transferring a 3-enzyme pathway to E. coli can convert farnesyl diphosphate (FPP) to lycopene. For the laboratory portion of this assignment, you will characterize lycopene production in E. coli. The computational tools and databases presented today can also be used to enhance lycopene production in E. coli or even produce different colors by adding additional genes.
PART I.
Test pAC-LYC plasmid
The pAC-LYC plasmid is available from Addgene, and contains three genes from Erwinia herbicola: CrtE, CrtI, and CrtB. This plasmid is provided by Addgene on an agar stab transformed into Top10 E. coli. That means that it can simply be streaked onto LB Agar with chloramphenicol to make lycopene-producing colonies that should be pink to red in color.
This is how lycopene looks like after cultivating in LB agar with chloramphenicol
PART II
Design strategies for increasing lycopene production
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Through recent advances in molecular genetics, it is possible to eliminate overexpress target genes or to adhere enzymes in the majority of microorganisms. Therefore, a key question is how to identify gene targets or the enzymes that have direct in order to improve the lycopene production or indirect impact on the increase lycopene production first identified a pathway for the production of lycopene.
Using the KEGG tool, we look for lycopene pathway and select enzymes that could be added or knocked out to increase lycopene production in E. coli
PART III:
Design strategies for converting lycopene to beta-carotene
The enzyme that can convert the red pigment lycopene into the orange pigment beta carotene is the Lycopene-beta-cyclase (CrtL, CrtL-b, CrtY). This enzyme catalyses the biosynthesis of β-carotene.
β-Carotene is an organic, strongly colored red-orange pigment abundant in plants and fruits. It is a member of the carotenes, which are terpenoids (isoprenoids), synthesized biochemically from eight isoprene units and thus having 40 carbons. Among the carotenes, β-carotene is distinguished by having beta-rings at both ends of the molecule. β-Carotene is biosynthesized from geranylgeranyl pyrophosphate.
Figure 1. The overview of the Carotenoid pathway
We choose the Carotenoid synthesis Pathway that can easily display different steps in the pathway by different colors.
If E.coli is incubating below 37℃, mRNAs cannot be translated by ribosomes, because temperature-sensitive RBS(ribosome biding site) forms the hairpin. A ribosome cannot bind to the RBS so that it cannot translate CrtY. As a result, the E.coli only can produce CrtE, CrtB and CrtI which convert colorless Farnesyl pyrophosphate to red Lycopene.
When the temperature is at 37℃ or higher, the hairpin is denatured so temperature-sensitive RBS is activated . CrtY is translated, which convert red Lycopene to orange beta-Carotene.