Instability of Extrachromosomal DNA Transformed into the Diatom Phaeodactylum Tricornutum

Phaeodactylum tricornutum has been highly studied for its potential as a platform for metabolic engineering. While the possible applications of extrachromosomal expression via an episome have been investigated, there is still a lack of information concerning its efficacy and limitations. Therefore, we studied the episome expression system in P. tricornutum, aiming to elucidate its limitations regarding heterologous protein production and episome rearrangement events. Our objectives were to screen positive transconjugants by fluorescent signal indicating as a proxy for the production of the proteins of interest that could be used for vanillin synthesis, and to characterize the transconjugants by flow cytometry and whole plasmid sequencing. We designed an episome harboring an expression cassette that consisted of the enhanced green-fluorescent-protein (eGFP) linked by Thosea asigna virus 2A self-cleaving peptide (T2A) to a fusion protein of enoyl-CoA hydratase/aldolase (ech) and feruloyl-CoA synthetase (fcs), both from Streptomyces sp. strain V-1. This construction resulted in a percentage of fluorescent transconjugants lower than 10 % and it presented rearranged episomes in the fluorescent and the non-fluorescent transconjugants. The replacement of the fusion protein ech-fcs in the expression cassette with the fluorescent protein mCherry increased the percentage of eGFP fluorescent transconjugants over 80 % suggesting a toxicity of the ech-fcs gene expression and in turn forcing selection for rearranged episomes. A comparison of flow cytometry results and sequencing analysis demonstrated that a successful transformation with an unaltered expression cassette could lead to diatoms that do not produce the protein. On the other hand, transconjugants with mutations or rearrangements in the genes encoding the fusion ech-fcs protein led to fluorescent signal detection. Here, we show that using fluorescent reporters can mislead the selection of positive transconjugants by not being able to identify rearrangements in the genes of interest, and intact cassettes can lack fluorescent signal due to lack of heterologous protein production.