9305269 Somerville The main goal of the proposed experiments is to understand the factors influencing the flux of carbon towards poly- - hydroxybutyrate (PHB) biosynthesis and to investigate strategies aimed at increasing the amount of PHB produced in transgenic plants. Using a suspension culture of Arabidopsis cells expressing the PHB biosynthetic pathway, the flux of acetyl-CoA towards lipids, sterols, and PHB, will be measured. Manipulation of the growth media by the addition of various carbon sources (eg acetate and citrate), as well as inhibitors of lipid and sterol biosynthesis, will be used to manipulate the flux of acetyl-CoA towards the various sinks and to increase PHB production. Furthermore, the influence of the addition of the bacterial 3- ketothiolase on the production of PHB in transgenic plants will be examined. A major effort will be directed towards targeting the enzymes of the PHB biosynthetic pathway into the plastids. The rationale is that higher amounts of PHB and minimal disruption of plant growth may be accomplished by diverting the high flux of acetyl-CoA away from seed storage lipid synthesis towards PHB synthesis. The bacterial PHB biosynthetic genes will be modified to introduce a plastid targeting sequence and to direct their expression under the control of the constitutive CaMV 35s and a seed specific promoter. Each of the three PHB genes will be introduced separately into tobacco and Arabidopsis. The efficiency of protein targeting and signal sequences cleavage, as well as the activity of the bacterial enzymes in the plastid, will be determined. Cross-pollination between the various transgenic lines will produce hybrids containing all genes required for PHB synthesis in the plastid. PHB production in different tissues of the hybrid transgenic plants will be measured. Finally, the influence of a reduction in starch biosynthesis on the production of PHB in the plastid will be examined by backcrossing in Arabidopsis transg enics to the starches mutants. A secondary goal is to purify sufficient amounts of plant PHB granules for the initial analysis of their physical and chemical properties, in comparison to the properties of bacterial PHB. This will be accomplished using the suspension culture of transgenic Arabidopsis cells grown under conditions maximizing PHB production, as determined by the experiments on the manipulation of the flux of acetyl-CoA into various sinks. %%% A significant breakthrough in the production of biodegradable plastic in plants has been accomplished from previous work. The continuation of this work involves increasing the amount of plastic production per plant. Instead of a slow selection method which would take many generations of plants, molecular biology and metabolic inhibitors will be tried to divert the biosynthetic apparatus of the plant toward the synthesis of plastic. It is important that the major amount of the energy of the plant be used for growth; the biosynthetic enzymes for the plastic will be directed into a specific compartment in the cell. By introducing a bacterial enzyme responsible for this component into the plant it is hoped that greater amounts of the plastic end product will result. These studies if successful will pioneer the efforts to produce useful biomaterials in plants. ***
