Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-phellandrene synthase (β- PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C₁₀H₁₆ β-phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase ( GPPS), promoting its expression, while at the same time serving as a selectable marker for the screening of transformants. The work further showed that enhanced GPPS expression increased the yield of β-phellandrene in Synechocystis transformants harboring the β- PHLS gene. Moreover, the research evaluated the expression efficacy of a DNA fragment comprising 87 nucleotides from the 5' end of the cmR gene in fusion with the GPPS gene. This short fusion construct substantially increased the intracellular geranyl-diphosphate synthase level, suggesting that "short-stretch" cmR leader sequences can be used to drive a higher expression level of heterologous biosynthetic genes, while avoiding undesirable internal recombinations, as these sequences are shorter than the threshold of 200 bp, commonly assumed to be the threshold of high efficiency recombinations.

The work aims to convert the secondary slow metabolism of the terpenoid biosynthetic pathway into a primary activity in cyanobacteria and to generate heterologous products using these photosynthetic microorganisms as cell factories. Case study is the production of the 10-carbon monoterpene β-phellandrene (PHL) in Synechocystis sp. PCC 6803 (Synechocystis). Barriers to this objective include the slow catalytic activity of the terpenoid metabolism enzymes that limit rates and yield of product synthesis and accumulation. "Fusion constructs as protein overexpression vectors" were applied in the overexpression of the geranyl diphosphate synthase (GPPS) and β-phellandrene synthase (PHLS) genes, causing accumulation of GPPS up to 4% and PHLS up to 10% of the total cellular protein. Such GPPS and PHLS protein overexpression compensated for their slow catalytic activity and enabled transformant Synechocystis to constitutively generate 24 mg of PHL per g biomass (2.4% PHL:biomass, w-w), a substantial improvement over earlier yields. The work showed that a systematic overexpression, at the protein level, of the terpenoid biosynthetic pathway genes is a promising approach to achieving high yields of prenyl product biosynthesis, on the way to exploiting the cellular terpenoid metabolism for commodity product generation.

Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)₃CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)₃CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)₃CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)₃CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)₃CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)₃ compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest.

Efforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of "fusion constructs as protein overexpression vectors" for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO₂, and H₂O.

Efforts to express human therapeutic proteins in photosynthetic organisms have been described in the literature. Regarding microalgae, most of the research entailed a heterologous transformation of the chloroplast, but transformant cells failed to accumulate the desired recombinant proteins in high quantity. The present work provides methods and DNA construct formulations for over-expressing in photosynthetic cyanobacteria, at the protein level, human-origin bio-pharmaceutical and bio-therapeutic proteins. Proof-of-concept evidence is provided for the design and reduction to practice of "fusion constructs as protein overexpression vectors" for the generation of the bio-therapeutic protein interferon alpha-2 (IFN). IFN is a member of the Type I interferon cytokine family, well-known for its antiviral and anti-proliferative functions. Fusion construct formulations enabled accumulation of IFN up to 12% of total cellular protein in soluble form. In addition, the work reports on the isolation and purification of the fusion IFN protein and preliminary verification of its antiviral activity. Combining the expression and purification protocols developed here, it is possible to produce fairly large quantities of interferon in these photosynthetic microorganisms, generated from sunlight, CO₂, and H₂O.

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.

Main conclusion

Downregulation in the expression of the signal recognition particle 43 (SRP43) gene in tobacco conferred a truncated photosynthetic light-harvesting antenna (TLA property), and resulted in plants with a greater leaf-to-stem ratio, improved photosynthetic productivity and canopy biomass accumulation under high-density cultivation conditions. Evolution of sizable arrays of light-harvesting antennae in all photosynthetic systems confers a survival advantage for the organism in the wild, where sunlight is often the growth-limiting factor. In crop monocultures, however, this property is strongly counterproductive, when growth takes place under direct and excess sunlight. The large arrays of light-harvesting antennae in crop plants cause the surface of the canopies to over-absorb solar irradiance, far in excess of what is needed to saturate photosynthesis and forcing them to engage in wasteful dissipation of the excess energy. Evidence in this work showed that downregulation by RNA-interference approaches of the Nicotiana tabacum signal recognition particle 43 (SRP43), a nuclear gene encoding a chloroplast-localized component of the photosynthetic light-harvesting assembly pathway, caused a decrease in the light-harvesting antenna size of the photosystems, a corresponding increase in the photosynthetic productivity of chlorophyll in the leaves, and improved tobacco plant canopy biomass accumulation under high-density cultivation conditions. Importantly, the resulting TLA transgenic plants had a substantially greater leaf-to-stem biomass ratio, compared to those of the wild type, grown under identical agronomic conditions. The results are discussed in terms of the potential benefit that could accrue to agriculture upon application of the TLA-technology to crop plants, entailing higher density planting with plants having a greater biomass and leaf-to-stem ratio, translating into greater crop yields per plant with canopies in a novel agronomic configuration.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.

Nuclear-encoded light-harvesting chlorophyll- and carotenoid-binding proteins (LHCPs) are imported into the chloroplast and transported across the stroma to thylakoid membrane assembly sites by the chloroplast signal recognition particle (CpSRP) pathway. The LHCP translocation defect (LTD) protein is essential for the delivery of imported LHCPs to the CpSRP pathway in Arabidopsis. However, the function of the LTD protein in Chlamydomonas reinhardtii has not been investigated. Here, we generated a C. reinhardtii ltd (Crltd) knockout mutant by using CRISPR-Cas9, a new target-specific knockout technology. The Crltd1 mutant showed a low chlorophyll content per cell with an unusual increase in appressed thylakoid membranes and enlarged cytosolic vacuoles. Profiling of thylakoid membrane proteins in the Crltd1 mutant showed a more severe reduction in the levels of photosystem I (PSI) core proteins and absence of functional LHCI compared with those of photosystem II, resulting in a much smaller PSI pool size and diminished chlorophyll antenna size. The lack of CrLTD did not prevent photoautotrophic growth of the cells. These results are substantially different from those for Arabidopsis ltd null mutant, indicating LTD function in LHCP delivery and PSI assembly may not be as stringent in C. reinhardtii as it is in higher plants.