Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide

Abstract

The purpose of this study was to express an antimicrobial peptide in the chloroplast to further develop the plastid engineering of H. pluvialis. Homologous targeting of the 16S-trnI/trnA-23S region and four endogenous regulatory elements, including the psbA promoter, rbcL promoter, rbcL terminator, and psbA terminator in H. pluvialis, were performed to construct a chloroplast transformation vector for H. pluvialis. The expression of codon-optimized antimicrobial peptide piscidin-4 gene (ant1) and selection marker gene (bar, biolaphos resistance gene) in the chloroplast of H. pluvialis was controlled by the rbcL promoter and psbA promoter, respectively. Upon biolistic transformation and selection with phosphinothricin, integration and expression of ant1 in the chloroplast genome were detected using polymerase chain reaction (PCR), southern blotting, and western blotting. Using this method, we successfully expressed antimicrobial peptide piscidin-4 in H. pluvialis. Hence, our results showed H. pluvialis promises as a platform for expressing recombinant proteins for biotechnological applications, which will further contribute to promoting genetic engineering improvement of this strain.

Two Foreign Antimicrobial Peptides Expressed in the Chloroplast of Porphyridium purpureum Possessed Antibacterial Properties

“…It is generally accepted that a lack of homology in the flanking sequences may result in lower recombination rates [ 27 ]. In this study, 16S rDNA/ trnA -23S was selected as the homologous insertion site, which is widely used for gene insertion in chloroplast transformation, such as in Haematococcus pluvialis [ 28 ], Tisochrysis lutea [ 29 ], Platymonas Subcordiformis [ 30 ], Momordica charantia L [ 31 ]. Some studies have shown that trnI / trnA is an effective gene insertion locus in the chloroplast genome [ 32 ].…”

Advanced Applications for Protein and Compounds from Microalgae

“…Microalgae represent an important biotechnology resource for the production of recombinant proteins for pharmacological applications. Successful results were reported by the genetic manipulation of microalgae for the production of antigens for vaccines, antibodies, immunotoxins, hormones, and antimicrobial agents [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 ]. Strategies and techniques have been developed for the exploitation of microalgae as expression systems for a number of advanced genetic toolkits [ 62 , 63 , 64 , 65 ].…”

“…An interesting example of antimicrobial peptide against both fish and human pathogens is piscidin-4. This was recently produced in H. pluvialis by biolistic manipulation of chloroplast of this microalga [ 81 ].…”

Grand Challenges in Microalgae Domestication

“…Transformation of S. obliquus via A. tumefasciens is feasible (Suttangkakul et al, 2019 ). Both nuclear and chloroplastic transformations have been described for H. pluvialis (Yuan et al, 2019 ; Wang et al, 2020 ; Cui et al, 2021 ). Finally, Charophyta are Green Algae close to Embryophyta, propagating by haploid asexual division; sexual reproduction is known.…”