Cyanobacteria are a group of prokaryotes that are capable of direct conversion of carbon dioxide to value added products. These microorganisms have gained attention in the field of biotechnology due to their efficient photoautotrophy, and amenability to genetic engineering. They have already been commercially exploited before, with their potential stemming from their minimal feedstock requirement. These microscopic cell factories only need carbon dioxide, water, sunlight, and low-cost mineral nutrients. Also, most microalgal species are generally regarded as safe microorganisms, therefore, fit for human consumption. There is a need to develop advanced recombinant DNA technologies for the generation of low-cost biopharmaceutical proteins, without relying on animal systems, and without causing depletion of natural resources, emission of greenhouse gases, or other environmental degradation. In this respect, a direct photosynthetic production of such compounds is promising.
Cyanobacteria have been researched in the recent past thanks to the incredible advancements in synthetic and molecular biology. The rapid and low-cost production and synthesis of transgenic and codon-optimized DNA constructs and the natural transformability of the broad majority of cyanobacterial species made phototropic organisms tractable for complex genetic engineering. In addition, several of these photosynthetic microorganisms have shown growth rates comparable to Saccharomyces cerevisiae, thereby solving a long-standing problem of the slow growth rate of the model cyanobacteria.
The expression of heterologous proteins in cyanobacteria has drawn much research interest in recent years. Overexpression of functional enzymes is needed to increase flux and yield if the intended end-product is a specialty or commodity compound. It’s also necessary to create a driving force to direct cellular metabolism towards the manufacture of the intended product. Although the expression of heterologous prokaryotic proteins in cyanobacteria seems achievable after coupling a solid promoter and RBS to the targeted DNA, hindrances at the protein translation level make eukaryotic proteins expression a bit challenging.
The “fusion constructs approach” has previously been implemented for the overexpression of eukaryotic proteins in cyanobacteria. This method entails the fusion of the target heterologous eukaryotic gene to a stable native or non-native gene. However, the functionality of multi-proteins in a single fusion construct hasn’t been validated yet in this regard. Fusion constructs can limit or even impede the catalytic activity of the participating enzymes. In vivo cleavage of the accumulating heterologous fusion proteins is needed to separate the moieties of the fusion construct to yield a pure target protein. In vivo cleavage in E. coli has been achieved previously by coexpressing, along with the target fusion protein, the tobacco etch virus protease. The former has a specific tobacco etch virus protease cleavage site. However, tobacco etch virus protease appeared unstable in bacteria, requiring stabilization by fusing it to the soluble maltose-binding protein. The latter served as the leader sequence in the construction.
In a new study published in the journal ACS Synthetic Biology, University of California at Berkeley researchers Dr. Xianan Zhang, Dr. Nico Betterle, Dr. Diego Hidalgo Martinez, and led by Professor Anastasios Melis developed an innovative in vivo tobacco etch virus protease cleavage system in cyanobacteria that are overexpressing fusion construct proteins, intending to separate the target heterologous proteins from their fusion leader sequences. The study demonstrated the feasibility of overexpression system, cellular stability, and exploitation of transgenes in cyanobacteria.
In the context of recombinant protein stability in Synechocystis sp. PCC 6803, the authors developed an in vivo cellular tobacco etch virus cleavage system to separate the target heterologous proteins from their fusion leader sequence. The authors found that product instability as the third level of difficulty in their attempt to overexpress eukaryotic recombinant proteins in photosynthetic microorganisms. As fusion constructs, various eukaryotic recombinant proteins exemplified by the human α-interferon and plant isoprene synthase could be expressed to 10-20% of the cellular protein. However, when the authors cleaved them from their stabilizing leader sequence and released them in the cyanobacteria cytosol, they were degraded and either couldn’t be detected or could be detected as residual faint protein bands. Among the recombinant proteins analyzed, only the prokaryotic tetanus toxin fragment C protein was stable, whereas the eukaryotic isoprene synthase and interferon were degraded by the cell.
The findings of the study exemplify how the plant isoprene synthase and human interferon are unstable in cyanobacteria and how fusion constructs can help overcome this challenge. They also show that some recombinant bacterial proteins can be heterologously overexpressed in cyanobacteria by using exogenous or endogenous promoters, with or without using the fusion construct approach. The new research work led by Professor Anastasios Melis boost protein production in cyanobacteria and can eventually be a big help in the industrial production of a range of therapeutic proteins.
Researchers Xianan Zhang, Nico Betterle, Diego Hidalgo Martinez, and Anastasios Melis. Recombinant Protein Stability in Cyanobacteria. ACS Synthetic Biology, issue 10 2021, pages 810−825Go To ACS Synthetic Biology