Plant Physiological Adaptations to the Massive Foreign Protein Synthesis Occurring in Recombinant Chloroplasts

Abstract

Genetically engineered chloroplasts have an extraordinary capacity to accumulate recombinant proteins. We have investigated in tobacco (Nicotiana tabacum) the possible consequences of such additional products on several parameters of plant development and composition. Plastid transformants were analyzed that express abundantly either bacterial enzymes, alkaline phosphatase (PhoA-S and PhoA-L) and 4-hydroxyphenyl pyruvate dioxygenase (HPPD), or a green fluorescent protein (GFP). In leaves, the HPPD and GFP recombinant proteins are the major polypeptides and accumulate to higher levels than Rubisco. Nevertheless, these engineered metabolic sinks do not cause a measurable difference in growth rate or photosynthetic parameters. The total amino acid content of transgenic leaves is also not significantly affected, showing that plant cells have a limited protein biosynthetic capacity. Recombinant products are made at the expense of resident proteins. Rubisco, which constitutes the major leaf amino acid store, is the most clearly and strongly down-regulated plant protein. This reduction is even more dramatic under conditions of limited nitrogen supply, whereas recombinant proteins accumulate to even higher relative levels. These changes are regulated posttranscriptionally since transcript levels of resident plastid genes are not affected. Our results show that plants are able to produce massive amounts of recombinant proteins in chloroplasts without profound metabolic perturbation and that Rubisco, acting as a nitrogen buffer, is a key player in maintaining homeostasis and limiting pleiotropic effects.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression

“…We estimated that in Nt-Nt-CP lines, CTB-Pins accumulation exceeds that of Rubisco by a factor of up to 2 (Fig. 5C), yet no deleterious phenotype was observed, likely as this level of accumulation was attained over time (Bally et al, 2009;Oey et al, 2009). It appeared that the observed reduction in Rubisco large subunit was concomitant with increased levels of CTB-Pins accumulation, as wild-type tissue sampled at the same light and developmental stages showed no such reduction in Rubisco.…”

The Penicillium Chrysogenum Extracellular Proteome. Conversion from a Food-rotting Strain to a Versatile Cell Factory for White Biotechnology

“…Eukaryotic systems, such as insect cells, yeasts, or mammalian host organisms, are capable of a variety of post-translational modifications, but are expensive to operate and have a longer growth period. On the other hand, large amounts of recombinant proteins can be produced at low cost in plants using genetically engineered chloroplasts (104).…”

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

“…biogenesis of the thylakoid membrane; (Hennig et al 2007)) or the severe metabolic burden imposed on the chloroplast due to extreme recombinant protein expression levels (Oey et al 2009a;Scotti and Cardi 2014). Expression levels above 40 % of the total soluble protein can exhaust the gene expression capacity of the chloroplast resulting in Rubisco depletion and a general decrease in plastid-encoded proteins (Bally et al 2009;Zhou et al 2008). The rather low expression levels of our recombinant proteins and the fact that no Rubisco depletion was visible on the Coomassie brilliant blue-stained gels suggests that in our case, the observed phenotypic alterations are more likely due to the recombinant protein interfering with chloroplast biogenesis, metabolism or gene expression.…”