An Attempt of Transplantation of Carotenoid Genes from Eukaryote to Prokaryote

Bioproduction of useful chemicals by living organisms would have some problems with productivity. For one of the possible solutions, transplantation of the entire pathway to a well-known host cell, such like Escherichia coli, by gene transfer have been considered. Although the genome projects reveals all of gene set in one organism, however, it is empirically obvious that only gathering and transferring gene set for the relevant metabolic pathway isn’t enough to perform the pathway in heterologous host. To elucidate possible problem with gene expression beyond the barrier of species, especially between eukaryote and prokaryote, a series of astaxanthin, carotenoid, biosynthesis genes from algae were tried to transfer to E. coli as a case study. Haematococcus lacustris (=H. pluvialis) NIES-144, which is one of the famous algae for astaxanthin production, was rendered for a donor. cDNA of the strain sampled at astaxanthin production phase was subjected to Next Generation Sequencer (NGS) not only to determine the sequence of carotenoid genes but also to evaluate relative expression abundance of these genes. Identified 8 genes for astaxanthin were then assembled into a plasmid with designed artificial operon by OGAB (Ordered Gene Assembly by Bacillus subtilis) method according to the gene expression abundance. In parallel with this, we also constructed an allelic carotenoid operon plasmid using bacterial genes obtained from Erwinia. Recombinant E. coli of alga operon didn’t produce any astaxanthin, while that of bacterial one did. To identify possible problematic gene(s) in the algae operon, we performed to construct a series of chimeric operons comprised from gene(s) of algae and bacteria. Finally we identified that only lycopene synthesis step that is comprised from 3 genes have problem, while other 5 reaction steps than lycopene synthesis well work in E. coli. In the presentation, possible reason for defect of lycopene synthesis will be discussed.