Production of Polyhydroxybutyrate in Higher Plants

本实验的主要目的是了解影响聚-羟基丁酸酯（PHB）生物合成的碳通量的因素，并探讨提高转基因植物中PHB产量的策略。利用表达PHB生物合成途径的拟南芥细胞的悬浮培养，乙酰辅酶a对脂质、固醇和PHB的通量将被测量。通过添加各种碳源（如醋酸盐和柠檬酸盐）以及脂质和甾醇生物合成抑制剂来操纵生长培养基，将用于控制乙酰辅酶a向各种汇的通量，并增加PHB的产量。此外，我们还将研究添加细菌3-酮硫酶对转基因植物中PHB产生的影响。一个主要的努力将是针对PHB生物合成途径的酶进入质体。其基本原理是，通过将高通量的乙酰辅酶a从种子储存脂质合成转移到PHB合成，可以实现更高量的PHB和对植物生长的最小破坏。将对细菌PHB生物合成基因进行修饰，引入质体靶向序列，并在组成CaMV 35s和种子特异性启动子的控制下指导其表达。这三个PHB基因将分别导入烟草和拟南芥中。将确定蛋白质靶向和信号序列切割的效率，以及质体中细菌酶的活性。不同转基因系之间的异花授粉将产生含有PHB合成所需的所有基因的杂种。将测定杂交转基因植株不同组织中PHB的产量。最后，淀粉生物合成减少对质体中PHB产生的影响将通过回交对淀粉突变体的拟南芥转基因进行检测。第二个目标是纯化足够数量的植物PHB颗粒，用于初步分析其物理和化学性质，并与细菌PHB的性质进行比较。这将通过悬浮培养转基因拟南芥细胞来实现，悬浮培养条件是在PHB产量最大化的条件下生长，这是通过操纵乙酰辅酶a进入各种水槽的通量的实验确定的。在植物中生产生物可降解塑料方面取得了重大突破。这项工作的继续涉及增加每个工厂的塑料产量。分子生物学和代谢抑制剂将尝试将植物的生物合成装置转向合成塑料，而不是需要许多代植物的缓慢选择方法。重要的是，植物的大部分能量用于生长；塑料的生物合成酶将被引导到细胞的特定隔间中。通过将一种负责这一成分的细菌酶引入植物，有望产生更多的塑料最终产品。这些研究如果成功，将开创在植物中生产有用生物材料的努力。＊＊＊

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. ***