Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis

Abstract

Bacterial lipoproteins play crucial roles in host–pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N‐terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten‐fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N‐terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration‐dependent interference with essential functions of chloroplastic membranes required for primary metabolism.

Inducible gene expression from the plastid genome by a synthetic riboswitch

“…The possibility to express transgenes from the plastid genome has stirred considerable excitement among plant biotechnologists (23,27,28), mainly due to the attainable enormous expression levels (29) and the increased transgene containment provided by maternal inheritance of plastids in most crops (30,31). However, constitutive plastid transgene expression can cause severe mutant phenotypes (e.g., 32,33) and, also for this reason, the development of tightly controllable systems for inducible gene expression is of utmost importance. Here we have explored the possibility of engineering riboswitches to function as translational regulators of gene and transgene expression in plastids.…”

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

“…So far, only the expression of a dengue virus serotype 3 premembrane and envelope polyprotein has been reported in plastids (Kanagaraj et al 2011). We demonstrate that dengue virus envelop protein domain III-based antigens can be expressed in tobacco chloroplasts and that the expression of challenging proteins via the ethanol-inducible expression system offers a possibility to avoid deleterious phenotypes associated with overexpression (Hennig et al 2007;Petersen and Bock 2011;Scotti et al 2015). The transplastomic plant lines with the transgene expression cassette controlled by the strong constitutive ribosomal RNA operon promoter showed distinct growth retardations and mild pigment deficiency.…”

“…The two main reasons underlying pigment deficiency or a delay in plant development are toxicity of the transgene product due to interference of the recombinant protein with essential processes in the chloroplast (e.g. 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).…”

“…However, high-level accumulation of recombinant proteins in chloroplasts can also have a negative impact on plant growth (Hennig et al 2007;Petersen and Bock 2011;Scotti et al 2015). Although, most foreign proteins are non-toxic to chloroplasts, in some cases, abnormal phenotypes like chlorosis of the leaves, male sterility and growth retardation have been reported (Lössl et al 2003;Tregoning et al 2003;Waheed et al 2011).…”

A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (HGAD65)

“…RNA was reverse transcribed to cDNA by SuperScript II Reverse Transcriptase (Invitrogen) according to the manufacturer's protocol. Briefly, 5 μg of total RNA, 1 μl Oligo(dT) [12][13][14][15][16][17][18] (500 μg/ml), 1 μl dNTP Mix (10 mM each) and 5 μl sterile distilled water were mixed and incubated at 65°C for 5 min. Following addition of 4 μl First-Strand Buffer and 2 μl 0.1 M DTT, the reaction mixture was further incubated for 2 min at 42°C.…”