# Sustainable Microalgae and Cyanobacteria Biotechnology
## 5 New Tools to Improve Microalgal Applications: Recombinant Protein Production in Microalgae  
The recombinant protein industry has developed more than 170 recombinant proteins used in medicine. Third generation has transformed the therapeutic recombinant protein, in which third generation focus on new routes of administration and increasing efficiency and safety [142; 143].  
In the biotechnological industry: medical biotechnology, enzyme technology, biopolymers, bioplastics, biofuels, bioremediation, and agricultural biotechnology have seen an intense productivity improvement by the selection of higher producing microbial strains with the application of recombinant DNA technology. New technologies should search new targets for strain improvement programs [144]. The eukaryotic systems used for pharmaceutical production include mammalian cell cultures, bacteria, insect cells, yeast, and plants, however with some limitations [145; 146; 147; 148]. Some limitations such as complex nutritional, maintenance, and sterile conditions in mammalian expression systems, in plant expression systems have technical and regulatory concerns for full scale production [149; 150; 151; 152].  
In this sense, microalgae and cyanobacteria are diverse organisms with properties relevant for large-scale production systems, making them an attractive host for recombinant protein production [153; 152]. Proteins, peptides, unsaturated fatty acids, vitamins, pigments, and other valuable compounds can be produced at lower producing costs [145; 154].  
Recently, a geminiviral vector has been used for the effective expression of therapeutic proteins in microalgae, with two advantages over other receptors: the innocuity of several strains and an excellent platform for the development of biological products [155]. For example, the SARS-CoV-2 receptor binding domain (RBD) and the basic fibroblast growth factor (bFGF) were subcloned in a geminiviral vector and used for nuclear transformation to temporary express these proteins in C. _reinhardtii_ and _C. vulgaris_, respectively [156]. Some advantages are important for the use of C. _reinhardtii_ and _C. vulgaris_, they are safe to consume as dietary supplements, easy cultivation, lack of pathogens, simple culture media,and growth in sterile conditions; these properties make an attractive heterologous protein production system [151, 155, 157, 158, 159, 160].  
Microalgal metabolism has been manipulated to produce high-valuable compounds according to photobioreactor engineering. In other strategies, transcriptional remodeling of lipid metabolism in _Chlamydomonas_, some regulators have been identified [161]. For example, Jia et al., [162] demonstrated that in _Chlamydomonas_, overexpression of a gene encoding a DofF transcription factor significantly increased intracellular lipid content. Some stress regulators have been identified, TF-encoding genes (an important strategy used by plants to adapt to environmental changes) [163], the Myb transcription factor in response to Pi starvation response [164], and lipid remodeling regulator 1 (LRL1) in response to P-depleted condition [165].  
The green alga _Chlamydomonas reinhardtii_ produces H2 gas in anaerobic conditions, reactions catalyzed by hydrogenase enzymes, another important application possible in microalgae. The expression of the HYDA genes is influenced by anoxia condition. In the presence of O2, transcriptional control of HYDA1 or HYDA2 genes is paralyzed, seems to be regulatory sequences involved in the hypoxia response [164]. It is necessary to isolate mutants that express hydrogenase in the presence of O2 to dismiss the enzymes sensitive to oxygen [166]. By identifying mutant strains with constitutively expressed HYDA gene transcripts would be a very useful tool in assisting with the difficult task of producing O2.tolerant hydrogenase enzymes [165]. These technologies generate a valuable platform to produce relevant industrialize biotechnology with significant yields and financial feasibility.