Heterologous Leader Sequences in Fusion Constructs Enhance Expression of Geranyl Diphosphate Synthase and Yield of β-Phellandrene Production in Cyanobacteria (Synechocystis)
| Autor: | Nico Betterle, Anastasios Melis |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
cyanobacteria Substrate Specificity Biomass Transgenes Photosynthesis Recombination Genetic biology ATP synthase Nucleotides Chemistry Synechocystis Bacterial Kanamycin General Medicine Erythritol Biochemistry β-phellandrene metabolic engineering Biotechnology Plasmids medicine.drug DNA Bacterial Biomedical Engineering Heterologous Cyclohexane Monoterpenes Protein Sorting Signals Biochemistry Genetics and Molecular Biology (miscellaneous) terpene synthesis Transformation Metabolic engineering Medicinal and Biomolecular Chemistry 03 medical and health sciences Transformation Genetic Bacterial Proteins Genetic Cyclohexenes Operon Genetics fusion protein medicine Gene Alkyl and Aryl Transferases beta-phellandrene DNA GPP synthase biology.organism_classification Fusion protein Recombination 030104 developmental biology Monoterpenes biology.protein Biochemistry and Cell Biology Heterologous expression |
| Zdroj: | ACS synthetic biology, vol 7, iss 3 |
| ISSN: | 2161-5063 |
| Popis: | Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-phellandrene synthase (β- PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C10H16 β-phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase ( GPPS), promoting its expression, while at the same time serving as a selectable marker for the screening of transformants. The work further showed that enhanced GPPS expression increased the yield of β-phellandrene in Synechocystis transformants harboring the β- PHLS gene. Moreover, the research evaluated the expression efficacy of a DNA fragment comprising 87 nucleotides from the 5' end of the cmR gene in fusion with the GPPS gene. This short fusion construct substantially increased the intracellular geranyl-diphosphate synthase level, suggesting that "short-stretch" cmR leader sequences can be used to drive a higher expression level of heterologous biosynthetic genes, while avoiding undesirable internal recombinations, as these sequences are shorter than the threshold of 200 bp, commonly assumed to be the threshold of high efficiency recombinations. |
| Databáze: | OpenAIRE |
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