Chemically inducible expression of the PHB biosynthetic pathway in Arabidopsis

Abstract

Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.

Semi‐Synthetic Ecdysteroids as Gene‐Switch Actuators: Synthesis, Structure–Activity Relationships, and Prospective ADME Properties

“…The derivatives of 20E are five monomethyl ethers at the 2-, 3-, 14-, 22-or 25-OH (1-4, 10), four dimethyl ethers at the 22-OH and one each of 2-, 3-, 14-, and 25-OH (11)(12)(13)(14), and one tetramethyl ether (15). PoA derivatives include three mono-O-methyl ethers at the 2-, 14-or 22-OH (16)(17)(18), and three dimethyl ethers (19)(20)(21). Additionally, several 20E 22-O-ether analogues were prepared, including compounds with O-n-alkyl groups up to a four-carbon chain (5, 6 and 8) and the allyl (7), benzyl (8) and 2'-ethyloxiranyl (23) ether groups.…”

“…[18,19] Additionally, gene switches have biopharmaceutical applications in areas such as cell-based assays and animal models for developmental drug testing, as well as biotherapeutics and biomaterials production. [20] Among the various available gene-switch platforms, the insect ecdysteroidregulated switches are refractory to human endogenous steroids, and typically show very low basal transgene expression, high inducibility, and broad dose-response gradation-usually outperforming other systems in each of these aspects. [21][22][23] The EcR gene switch functions through assembly of 1) the nuclear receptor EcR with a heteropartner protein (the vertebrate retinoid X receptor, RXR, its insect orthologue, the ultraspiracle protein, USP, or a variant); 2) a DNA response element complementary to the DNA binding domains of EcR and the hetero-…”

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

“…Polymer content in plants transformed with pCAB was measured by gas chromatography-mass spectrometry (GC-MS) procedures after subjecting tissue to a simultaneous extraction and butanolysis procedure (Kourtz et al, 2007). Tissue from seven independent transplastomic CAB lines was analyzed for PHB content over three regeneration cycles using these procedures, and average polymer levels ranging from 10.0% to 18.8% dry weight PHB were observed (Fig.…”

“…PHB concentrations in plant tissue were measured by GC-MS as described previously (Kourtz et al, 2007) using 30 to 150 mg of lyophilized leaf material.…”

Transformation as a Tool for Genetic Analysis in Populus

“…Photos on left and right panels represent explants and plants before and after induction respectively. Top: Induction of a GUS reporter gene under the control of the ecdysone induction system (construct provided by Metabolix Co. and based on the construct used in (Kourtz et al, 2007)) (a, b). Photo courtesy of Cathleen Ma, Oregon State University.…”