Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa

Area of Science:

• * Synthetic biology
• * Algal biotechnology
• * Genetic engineering

Background:

• * Previous development of a self-cloning system using uridine monophosphate synthase gene (cUMPS) in Coccomyxa sp. strain Obi.
• * Need for marker-free transgenic strains in microalgae for various applications.

Purpose of the Study:

• * To develop a Cre/loxP-based system for efficient removal of the cUMPS marker gene from the Coccomyxa genome.
• * To construct marker-free transgenic Coccomyxa strains overexpressing specific genes for enhanced lipid production.
• * To establish a versatile method applicable to other eukaryotic microalgae.

Main Methods:

• * Development of a Cre/loxP recombination system for marker gene excision.
• * Intracellular delivery of purified Cre recombinase.
• * Sequential introduction of expression cassettes for acyl-(acyl-carrier-protein) thioesterase and type-2 diacylglycerol acyltransferase genes.
• * Construction of marker-free transgenic strains.

Main Results:

• * Successful removal of the cUMPS marker gene using the Cre/loxP system, generating uracil-auxotrophic (Ura-) strains.
• * Construction of marker-free transgenic Coccomyxa strains lacking foreign DNA except the loxP sequence.
• * One engineered strain exhibited a 1.4-fold increase in lipid productivity compared to the wild-type strain.

Conclusions:

• * The developed Cre/loxP-based marker-gene-recycling system enables the creation of marker-free transgenic microalgae.
• * This system facilitates sequential gene expression and enhances lipid productivity.
• * The method holds potential for application in diverse eukaryotic microalgal species.