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Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

ABSTRACT: With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

SUBMITTER: Oey M

PROVIDER: S-EPMC3921121 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

Oey Melanie M Ross Ian L IL Hankamer Ben B

PloS one 20140211 2

With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the ...[more]

PMID: 24523866

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Project description:BackgroundThe global demand for functional proteins is extensive, diverse, and constantly increasing. Medicine, agriculture, and industrial manufacturing all rely on high-quality proteins as major active components or process additives. Historically, these demands have been met by microbial bioreactors that are expensive to operate and maintain, prone to contamination, and relatively inflexible to changing market demands. Well-established crop cultivation techniques coupled with new advancements in genetic engineering may offer a cheaper and more versatile protein production platform. Chloroplast-engineered plants, like tobacco, have the potential to produce large quantities of high-value proteins, but often result in engineered plants with mutant phenotypes. This technology needs to be fine-tuned for commercial applications to maximize target protein yield while maintaining robust plant growth.ResultsHere, we show that a previously developed Nicotiana tabacum line, TetC-cel6A, can produce an industrial cellulase at levels of up to 28% of total soluble protein (TSP) with a slight dwarf phenotype but no loss in biomass. In seedlings, the dwarf phenotype is recovered by exogenous application of gibberellic acid. We also demonstrate that accumulating foreign protein represents an added burden to the plants' metabolism that can make them more sensitive to limiting growth conditions such as low nitrogen. The biomass of nitrogen-limited TetC-cel6A plants was found to be as much as 40% lower than wildtype (WT) tobacco, although heterologous cellulase production was not greatly reduced compared to well-fertilized TetC-cel6A plants. Furthermore, cultivation at elevated carbon dioxide (1600 ppm CO2) restored biomass accumulation in TetC-cel6A plants to that of WT, while also increasing total heterologous protein yield (mg Cel6A plant-1) by 50-70%.ConclusionsThe work reported here demonstrates that well-fertilized tobacco plants have a substantial degree of flexibility in protein metabolism and can accommodate considerable levels of some recombinant proteins without exhibiting deleterious mutant phenotypes. Furthermore, we show that the alterations to protein expression triggered by growth at elevated CO2 can help rebalance endogenous protein expression and/or increase foreign protein production in chloroplast-engineered tobacco.

Project description:BACKGROUND: Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. RESULTS: We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. CONCLUSIONS: We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.

Dataset Information

Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

Publications

Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

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