Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

Abstract

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst™ technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when ‘absolute’ containment is required.

Temporary immersion systems in plant biotechnology

“…Recently, RITA TIS have been used to study the expression of a bacterial outer surface protein A from Borrelia burgdorferi by transformed tobacco ( N. tabacum cv. Petit Havana) chloroplasts . After 40 days of cultivation at 4 min immersion and 8 h exposure periods, a maximum yield of outer surface protein A of about 108 mg/L was achieved .…”

Transplastomic plants for innovations in agriculture. A review

“…However, in contrast with some other molecular strategies such as female or male sterility, gene deletor, GeneSafe TM developed to prevent gene flow via pollen and seed from transgenic plants, the seeds of transplastomic plants still carry the transgene (reviewed in Ding et al 2014; see also Section 5.1). Therefore, culturing transplastomic plant cells (and carrot is ideal for this purpose) under aseptic conditions on synthetic media in bioreactors (Michoux et al 2013) is another, reasonably cost-effective alternative to field or greenhouse cultivation. This approach provides fully contained conditions desirable for certain biopharmaceutical products, which may be sufficient to manage public concern or to ensure the deregulation of transplastomic plants (Bock 2014).…”

Translation in Mitochondria and Other Organelles

“…In other studies, the occurrence of mutant phenotypes characterized by chlorotic phenotype and growth retardation seems to be due to interference of the recombinant proteins with the plastid metabolism instead of competition for expression factors ). Temporary immersion bioreactors have been also proposed as an alternative method for the production of proteins toxic for plants (Michoux et al 2013). Temporary immersion bioreactors have been also proposed as an alternative method for the production of proteins toxic for plants (Michoux et al 2013).…”