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

Abstract

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5# end by the host plant's psbA coding sequence and at the 3# end by the host plant's 3# psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons

“…Some polycistronic transcripts in plastids clearly remain unprocessed and are translated efficiently [e.g., the psbE operon comprising four small genes for polypeptides of photosystem II and the dicistronic psaA/B mRNA encoding the two reaction center subunits of photosystem I (33,34)], but the rules determining the dependency of translation on intercistronic RNA processing are currently unknown. Thus which (trans)genes in synthetic operons will be expressed efficiently from unprocessed polycistronic transcripts and which transgenes will require intercistronic processing for efficient translation remains unpredictable, and previous attempts to stack transgenes in operons have produced mixed results (e.g., [35][36][37].…”

Synthetic Botany

“…No gene silencing has been observed in chloroplasts despite such high accumulation of foreign transcripts (169 times higher than in nuclear transgenic plants, Lee et al 2003) or foreign protein (46% of total leaf protein [De Cosa et al 2001]). Metabolic engineering for the production of bioplastic monomers (Bohmert-Tatarev et al 2011) and compounds of nutritional relevance (Craig et al 2008;Hasunuma et al 2008;Apel and Bock 2009) has also been applied to chloroplasts. The high biosynthetic capacity of the chloroplast is closely linked to the polyploid nature of the system: At 10 -100 chloroplasts per cell, and 10 -1000 genomes per chloroplast (Bendich 1987), stable integration of a transgene into the chloroplast genome enables a substantial amplification in transgene copy number.…”

Transplastomic plants for innovations in agriculture. A review

“…08-337-105r) in order to prove the concept of producing polyhydroxybutyrate (PHB), a biodegradable polyester plastic, in transplastomic tobacco. According to published results (Bohmert-Tatarev et al 2011), high levels of PHB were accumulated in T1 plants, up to an average of 18 % dry weight in leaves and 9 % in the dry biomass of the entire plant.…”