A New Strategy to Produce a Defensin: Stable Production of Mutated NP-1 in Nitrate Reductase-Deficient Chlorella ellipsoidea

Abstract

Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5′-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO3. The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.

Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae

“…Microalgae including Chlorella sp. (Trebouxiophyceae) [14,15], Dunaliella salina (Chlorophyceae) [16], Nannochloropsis oculata (Eustigmatophyceae) [17,18] and the diatom Phaeodactylum tricornutum (Bacillariophyceae) [19] have been reported to successfully express biopharmaceuticals.…”

Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective

“…Then, the biologically active defensin was examined by means of in vitro antimicrobial tests. Afterward, the same research group reported to have successfully obtained stable cell lines of transformed C. ellipsoidea expressing bioactive defensin . The recombinant defensin was claimed to be as high as 11.42 mg/L even after 15 generations.…”

“…Although the commercial production of heterologous proteins by Chlorella is currently unachievable, several studies that utilized engineered Chlorella to express heterologous proteins provide much promise (Table ). Various studies have reported that the yields of heterologous proteins by Chlorella vary from 200 ng/L to 11.42 mg/L , which is relatively lower than that for other eukaryotic expression hosts, such as plants (0.1 μg/L to 247 mg/L) , mammalian cells (0.55 to 80 mg/L) , and insect cells (80 to 300 mg/L) . However, low protein yields can be compensated by the short cycle time (typically several days) because of the rapid growth of Chlorella .…”

“…Recently, a series of research reported the expression of rabbit defensin (rabbit neutrophil peptide‐1) in C. ellipsoidea . Similar to lactoferrin, defensins are a family of small cationic peptides constituting the first line of defense against pathogens in the innate immune system of animals and humans . They exhibit broad‐spectrum antimicrobial and cytotoxic properties against many Gram‐positive and Gram‐negative bacteria, fungi, and some enveloped viruses .…”

Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of

“…Transformation of C. ellipsoidea C. ellipsoidea was transformed according to a previously published method [39]. Briefly, cells were cultured to the logarithmic phase in Endo medium (10 mL, containing approximately 10 7 cells/mL), collected by centrifugation and resuspended in 10 mL of a solution of 0.2 M mannitol and 0.2 M sorbitol (Sigma Aldrich, St. Louis, United States).…”