Introduction: The microalga Parachlorella kessleri-I produces high biomass and lipid content
that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing
the complete chloroplast genome is crucial for the construction of a species-specific chloroplast
Methods: In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled;
annotated and genetic transformation of the chloroplast was optimized. For the chloroplast
transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase
(aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively.
Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and
Droplet Digital PCR.
Results: The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat
regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome
encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided
essential information like codons, UTRs and flank sequences for homologous recombination to
make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The
transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but
no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained
the transgene integration into the chloroplast genome, via homologous recombination. The
chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet
Conclusion: The optimization of stable chloroplast transformation in marine alga P. kessleri-I should
open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more
CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.