The establishment of new protein expression system using N starvation inducible promoters in Chlorella

Abstract

Chlorella is a unicellular green microalga that has been used in fields such as bioenergy production and food supplementation. In this study, two promoters of N (nitrogen) deficiency-inducible Chlorella vulgaris N Deficiency Inducible (CvNDI) genes were isolated from Chlorella vulgaris UTEX 395. These promoters were used for the production of a recombinant protein, human granulocyte-colony stimulating factor (hG-cSf) in Chlorella vulgaris UTEX 395 and Chlorella sp. ArM0029B. To efficiently secrete the hG-cSf, the protein expression vectors incorporated novel signal peptides obtained from a secretomics analysis of Chlorella spp. After a stable transformation of those vectors with a codonoptimized hG-CSF sequence, hG-cSf polypeptides were successfully produced in the spent media of the transgenic Chlorella. To our knowledge, this is the first report of recombinant protein expression using endogenous gene components of Chlorella. Chlorella is a single-celled eukaryote belonging to the green algae. It can grow phototrophically, heterotrophically, or mixotrophically to high biomass, producing different amounts of proteins and lipids 1. It grows rapidly, dividing into four cells in about 6 h. Because Chlorella synthesizes large amounts of proteins and lipids, it has been used as a raw material for biodiesel and also as a food additive 2-4. Chlorella undergoes posttranslational modifications such as protein N-glycosylation 5. The use of Chlorella for the production of valuable proteins has therefore been attempted for more than two decades 6-9. Chen et al. 10 showed expression of rabbit neutrophil peptide using a translational enhancer omega in Chlorella ellipsoidea. Kim et al. 11 produced flounder growth hormone using a 35S cauliflower mosaic virus (35S) promoter in C. ellipsoidea and showed a 25% growth increase of flounder fry. No recombinant proteins produced from Chlorella species have yet been commercialized, although the species has been studied intensively for the potential production of biodiesel. An optimized expression system for producing proteins in Chlorella is urgently needed. Recombinant protein production studies require a highly efficient method for the transformation of Chlorella. Various transformation techniques, including electroporation, PEG transformation, particle bombardment, and Agrobacterium cocultivation, have been used for the efficient transformation of Chlorella spp. 10-14. Recently, Kumar et al. 15 reported an effective method for enhancing transformation efficiency by more than 100-fold using electroporation combined with efficient protoplasting of Chlorella. Besides efficient transformation, an optimized system for the endogenous expression of proteins is indispensable. An expression system involving appropriate promoters is one of the most important factors for highefficiency production of recombinant proteins. In Chlamydomonas reinhardtii, the promoter of the heat shock protein gene HSP70a has been widely used for the expression of foreign genes in C. reinhardtii 16,17. In recent yea...

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