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

Abstract

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5# untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5# UTR and 3# UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5# UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5# UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.Over the expanse of time since the endosymbiotic events that led to the establishment of plant organelles, plant cells have evolved elaborate mechanisms to coordinate the expression of plastid genes with the changing developmental and functional requirements of the cell. In addition to the nucleus-encoded, plastidlocalized RNA polymerase, the nucleus controls the expression of a suite of s-factors required for the active transcription of photosynthetic genes by the plastidencoded RNA polymerase (PEP), with PEP itself being transcribed by nucleus-encoded, plastid-localized RNA polymerase (Allison et al., 1996;Hess and Borner, 1999). Nuclear control over translation of plastid mRNA is exerted through the activities of numerous plastid-localized RNA-binding proteins (RBPs). RBPs appear to have a tight affinity for their cognate sequences in plastid mRNAs, and studies have demonstrated that their interactions are specific for particular genes (Nakamura et al., 1999(Nakamura et al., , 2001Shen et al., 2001;Meierhoff et al., 2003;Schmitz-Linneweber et al., 2005.Plastid mRNAs, expressed as monocistrons or polycistro...

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…This underscores the importance of the species specificity of chloroplast regulatory sequences. Likewise, details of the homologous recombination process and the deletion of mismatched nucleotides were evident using heterologous flanking sequences (Ruhlman et al, 2010). The translation of native polycistrons without the need for processing to monocistrons has been demonstrated (Barkan, 1988;Zoschke and Barkan, 2015), but the similarity of this process using heterologous polycistrons engineered via the chloroplast genome offered even more direct evidence for this process (De Cosa et al, 2001;Quesada-Vargas et al, 2005).…”

“…The expression of precursor proteins via the chloroplast genome demonstrated that cleavage of transit peptides takes place in the stroma and not in the chloroplast envelope (Daniell et al, 1998). Most importantly, the role of nucleus-encoded cytosolic proteins that bind to regulatory sequences and their species specificity were demonstrated using transgenes expressed in chloroplasts (Ruhlman et al, 2010). When the lettuce (Lactuca sativa) psbA regulatory sequence was used to drive transgene expression in tobacco (Nicotiana tabacum) chloroplasts, there was greater than 90% reduction in the accumulation of foreign proteins.…”

“…Therefore, transgenes inserted into chloroplast genomes are expressed at high levels, up to 70% of total leaf protein (De Cosa et al, 2001;Ruhlman et al, 2010). A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014).…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…However, these UTR sequences, as well as any other sequences with significant homology to the host's plastome, must be used with care, since they can cause unwanted rearrangements (Svab and Maliga, 1993;Staub and Maliga, 1994a;Rogalski et al, 2006Rogalski et al, , 2008McCabe et al, 2008;Zhou et al, 2008;Gray et al, 2009). Reducing the use of DNA fragments with homology to endogenous sequences and/or replacing them with heterologous sequences can decrease the occurrence of unexpected rearrangements (Nadai et al, 2009) but may yield lower transgene expression levels (Ruhlman et al, 2010).…”

“…Plastid-encoded gene expression has yielded extremely high levels of protein production, with transformants producing 45% (De Cosa et al, 2001) to 70% (Oey et al, 2009) of the transgeneencoded protein per unit of soluble leaf protein and up to 72% (Ruhlman et al, 2010) of the transgeneencoded protein per unit of total leaf protein in tobacco (Nicotiana tabacum). While these expression levels are too high for most metabolic engineering strategies for the production of chemicals, fuels, or materials without creating unwanted stress on the host plant, expression levels can be somewhat controlled by the choice of regulatory elements flanking the transgenes.…”