Dataset Information

Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production.

ABSTRACT:

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 transformation vector.

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 Digital PCR.

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.

SUBMITTER: Nawkarkar P

PROVIDER: S-EPMC7770631 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Attached culture allows high biomass productivity and is a promising biomass cultivating system because neither a huge facility area nor a large volume of culture medium are needed. This study investigates photosynthetic and transcriptomic behaviors in Parachlorella kessleri cells on a solid surface after their transfer from liquid culture to elucidate the physiological and gene-expression regulatory mechanisms that underlie their vigorous proliferation. The chlorophyll content shows a decrease at 12 h after the transfer; however, it has fully recovered at 24 h, suggesting temporary decreases in the amounts of light harvesting complexes. On PAM analysis, it is demonstrated that the effective quantum yield of PSII decreases at 0 h right after the transfer, followed by its recovery in the next 24 h. A similar changing pattern is observed for the photochemical quenching, with the PSII maximum quantum yield remaining at an almost unaltered level. Non-photochemical quenching was increased at both 0 h and 12 h after the transfer. These observations suggest that electron transfer downstream of PSII but not PSII itself is only temporarily damaged in solid-surface cells just after the transfer, with light energy in excess being dissipated as heat for PSII protection. It thus seems that the photosynthetic machinery acclimates to high-light and/or dehydration stresses through its temporal size-down and functional regulation that start right after the transfer. Meanwhile, transcriptomic analysis by RNA-Seq demonstrates temporary upregulation at 12 h after the transfer as to the expression levels of many genes for photosynthesis, amino acid synthesis, general stress response, and ribosomal subunit proteins. These findings suggest that cells transferred to a solid surface become stressed immediately after transfer but can recover their high photosynthetic activity through adaptation of photosynthetic machinery and metabolic flow as well as induction of general stress response mechanisms within 24 h.

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