Recombinant Protein Stability in Cyanobacteria

Abstract

The living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in E. coli of recombinant proteins, most of which, however, end-up as "inclusion bodies", i.e., cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10−20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in Synechocystis sp. PCC 6803 by developing and applying an in vivo cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant-and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression.

Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria

“…35 To improve upon this method, we designed a plasmid (pCJ111; Supplemental Figure S3) to deliver CRE recombinase into the rbcLXS locus in PCC 7002 by homologous recombination, in this case as a translational fusion to the rbcL gene. As Nterminal translational fusions have been shown to enhance heterologous gene expression in cyanobacteria 36 as well as increase protein stability, 37 this arrangement delivers CRE in a manner that simultaneously promotes its expression while also ensuring its conditional stability by virtue of the essentiality of rbcLXS. To assay for CRE recombinase activity, a loxP-flanked gentamycin resistance cassette (constructed via Gibson Assembly, 38 Supplemental Figure S4) was integrated into the glpK neutral site in PCC 7002, resulting in the construction of the test strain ΔglpK::loxP-gm-loxP (Figure 3A).…”

Tetanus Toxin Fragment C: Structure, Drug Discovery Research and Production

“…Recently, in 2021, the TTFC protein was expressed in the cyanobacterium Synechocystic sp. PCC6803 (Synechocystis) [115]. This bacterium was initially modified to stably express the tobacco etch virus protease (TEVp).…”

Applications of Synthetic Biotechnology on Carbon Neutrality Research: A Review on Electrically Driven Microbial and Enzyme Engineering

“…Beyond that, recombinant protein stability was also another crucial factor that decided the outcome of the heterologous expression. The recombinant proteins derived from eukaryotic plants and animals are unstable when freely expressed in the cyanobacterial cytosol but stable when fused with a highly expressed cyanobacterial native or heterologous protein, which was demonstrated by expressing the recombinant proteins of the plant origin isoprenoid biosynthetic pathway, human interferon protein, and tetanus toxin fragment C ( Zhang X et al, 2021 ). These fundamental research studies pave the way to construct a robust engineered cyanobacterial cell factory for the large-scale commercial production from CO 2 .…”