Summary
A bioRxiv preprint reports that two introns from the PRK gene increased reporter expression in Chlamydomonas reinhardtii by up to 35-fold compared with intronless versions. The researchers also used one of the introns to produce detectable patchoulol under conditions where a reference intron was insufficient.
Researchers report that two introns from the phosphoribulokinase (PRK) gene can help increase expression of engineered genes in the green alga Chlamydomonas reinhardtii. In a bioRxiv preprint, the team measured increases of up to 35-fold over matching transgenes without introns, and tested one PRK intron in a construct for producing patchoulol.
Testing PRK introns as expression tools
An intron is a segment within a gene that is transcribed into RNA and then removed as that RNA is processed. Intron-mediated enhancement (IME) describes the increase in gene expression that can occur when introns are included in an engineered gene sequence. The preprint identifies reliable expression of genes inserted into the alga’s nucleus as a continuing challenge for its use in photosynthetic biotechnology.
The researchers assessed PRK introns using two reporter systems and two genetic backgrounds, and varied the promoter and terminator sequences used in the constructs. They compared the results with intronless versions and with the first intron from the RBCS2 gene, described in the preprint as the reference IME element for Chlamydomonas.
The first two PRK introns performed best among the candidates tested. Across the experiments, the authors report expression increases of up to 35-fold relative to intronless counterparts. This is a maximum reported effect, not a single increase that applies to every construct: expression was assessed across different reporters and genetic-control sequences.
A test with patchoulol synthase
To assess whether the approach could support a more demanding engineered gene, the team tested patchoulol synthase, an enzyme used to produce the sesquiterpene patchoulol. The preprint reports detectable patchoulol production when the construct included the first PRK intron, PRKi1. Under the tested conditions, a construct with one copy of the RBCS2 reference intron was insufficient for detectable production.
The authors present PRKi1 and PRKi2 as modular, MoClo-compatible parts. MoClo is a method for assembling DNA constructs from interchangeable components; the team says the introns can be incorporated into a framework intended for biofoundry workflows. The results suggest a way to tune gene expression when designing engineered Chlamydomonas strains, including strains intended for biotechnology applications.
These findings are from a bioRxiv preprint. The reported expression gains and patchoulol result concern experiments in C. reinhardtii and do not by themselves establish performance in other organisms or applications.