公开(公告)号：US20070067869A1

公开(公告)日：2007-03-22

申请号：US11376831

申请日：2006-03-16

申请人： Lauralynn Kourtz ,  Oliver Peoples ,  Kristi Snell

发明人： Lauralynn Kourtz ,  Oliver Peoples ,  Kristi Snell

IPC分类号： A01H1/00 ,  C12N15/82 ,  C12N5/04

CPC分类号： C12N15/8247 ,  C12N15/8237 ,  C12N15/8243

摘要： Methods and constructs for the introduction of multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway are provided. In one embodiment, the constructs contain two or more enzyme-encoding genes, each under the control of an inducible promoter and each with a polyadenylation signal. The constructs are used to produce transgenic plants, in which the expression of the enzymes are increased when a chemical inducing agent is applied, and a biosynthetic product of the series of enzymes encoded by the transgenes is produced. Constructs may be used which contain two or more enzyme-encoding genes under the control of one or more promoters activated by activator molecules or complexes expressed from a transgene or transgenes, which are themselves under the control of one or more inducible promoters and switched on following the external application of a chemical. The transgene or transgenes expressing the activator molecules or complexes may be included in the same construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. Alternatively, the transgene or transgenes expressing the activator molecules or complexes may be on a different construct from the construct containing multiple genes encoding enzymes in a multi-enzyme biosynthetic pathway. The activator molecule can be expressed using a constitutive promoter in an inactive form which is converted to the active form following application of the chemical inducing agent.

摘要翻译： 提供了在多酶生物合成途径中引入编码酶的多个基因的方法和构建体。 在一个实施方案中，构建体含有两个或更多个酶编码基因，每个基因在诱导型启动子的控制下并且各自具有多聚腺苷酸化信号。 构建体用于产生转基因植物，其中当施用化学诱导剂时酶的表达增加，并且产生由转基因编码的一系列酶的生物合成产物。 可以使用含有两个或更多个酶编码基因的构建体，所述两个或多个酶编码基因在一个或多个由活化剂分子或由转基因或转基因表达的复合物所激活的启动子的控制下，所述启动子本身处于一个或多个诱导型启动子的控制下， 化学品的外部应用。 表达活化剂分子或复合物的转基因或转基因可以包含在含有多酶生物合成途径中的酶的多个基因的相同构建体中。 或者，表达活化剂分子或复合物的转基因或转基因可以与含有多酶生物合成途径中的酶的多个基因的构建体在不同的构建体上。 可以使用非活性形式的组成型启动子来表达活化剂分子，其在施用化学诱导剂后转化为活性形式。

公开(公告)号：US20060084155A1

公开(公告)日：2006-04-20

申请号：US11245891

申请日：2005-10-07

申请人： Gjalt Huisman ,  Frank Skraly ,  David Martin ,  Oliver Peoples

发明人： Gjalt Huisman ,  Frank Skraly ,  David Martin ,  Oliver Peoples

IPC分类号： C12P7/62 ,  C07H21/04 ,  C12N9/10 ,  C12N1/21 ,  C08G63/82 ,  C12N15/74

CPC分类号： C12N9/0008 ,  C08G63/06 ,  C12N9/1029 ,  C12N9/1096 ,  C12N15/8243 ,  C12P7/625

摘要： The gene encoding a 4-hydroxybutyryl-Co A transferase has been isolated from bacteria and integrated into the genome of bacteria also expressing a polyhydroxyalkanoate synthase, to yield an improved production process for 4HB-containing polyhydroxyalkanoates using transgenic organisms, including both bacteria and plants. The new pathways provide means for producing 4HB containing PHAs from cheap carbon sources such as sugars and fatty acids, in high yields, which are stable. Useful strains are obtaining by screening strains having integrated into their genomes a gene encoding a 4HB-CoA transferase and/or PHA synthase, for polymer production. Processes for polymer production use recombinant systems that can utilize cheap substrates. Systems are provided which can utilize amino acid degradation pathways, α-ketoglutarate, or succinate as substrate.

公开(公告)号：US20050170480A1

公开(公告)日：2005-08-04

申请号：US11053551

申请日：2005-02-08

申请人： Gjalt Huisman ,  Oliver Peoples ,  Frank Skraly

发明人： Gjalt Huisman ,  Oliver Peoples ,  Frank Skraly

IPC分类号： C12N15/09 ,  C12N1/21 ,  C12N9/00 ,  C12N9/02 ,  C12N9/04 ,  C12N9/10 ,  C12N9/88 ,  C12N15/90 ,  C12P7/62 ,  C12R1/19 ,  C12N15/74

CPC分类号： C12N9/1029 ,  C12N9/00 ,  C12N9/0004 ,  C12N9/0006 ,  C12N9/1025 ,  C12N9/13 ,  C12N9/88 ,  C12N15/90 ,  C12P7/625 ,  Y10S435/829 ,  Y10S435/831 ,  Y10S435/877

摘要： Transgenic microbial strains are provided which contain the genes required for PHA formation integrated on the chromosome. The strains are advantageous in PHA production processes, because (1) no plasmids need to be maintained, generally obviating the required use of antibiotics or other stabilizing pressures, and (2) no plasmid loss occurs, thereby stabilizing the number of gene copies per cell throughout the fermentation process, resulting in homogeneous PHA product formation throughout the production process. Genes are integrated using standard techniques, preferably transposon mutagenesis. In a preferred embodiment wherein mutiple genes are incorporated, these are incorporated as an operon. Sequences are used to stabilize mRNA, to induce expression as a function of culture conditions (such as phosphate concentration), temperature, and stress, and to aid in selection, through the incorporation of selection markers such as markers conferring antibiotic resistance.

公开(公告)号：US20060040281A1

公开(公告)日：2006-02-23

申请号：US11086605

申请日：2005-03-22

申请人： Oliver Peoples ,  Anthony Sinskey

发明人： Oliver Peoples ,  Anthony Sinskey

IPC分类号： C12Q1/68 ,  C12P7/62 ,  C12N1/21

CPC分类号： C12N9/0006 ,  C12N9/00 ,  C12N9/1029 ,  C12N15/52 ,  C12P7/625

摘要： A method for controlling and modifying biopolymer synthesis by manipulation of the genetics and enzymology of synthesis of polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA) polyesters at the molecular level in procaryotic and eukaryotic cells, especially plants. Examples demonstrate the isolation, characterization, and expression of the genes involved in the production of PHB and PHA polymers. Genes encoding the enzymes in the PHB and PHA synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB polymerase or PHA polymerase) from Zoogloea ramigera strain I-16-M, Alcaligenes eutrophus, Nocardia salmonicolur, and Psuedomonas olevarans were identified or isolated and expressed in a non-PHB producing organism, E. coli. Specific modifications to the polymers include variation in the chain length of the polymers and incorporation of different monomers into the polymers to produce co-polymers with different physical properties.

公开(公告)号：US20060183209A1

公开(公告)日：2006-08-17

申请号：US11355440

申请日：2006-02-16

申请人： Oliver Peoples ,  Lara Madison ,  Gjalt Huisman

发明人： Oliver Peoples ,  Lara Madison ,  Gjalt Huisman

IPC分类号： C12N9/02 ,  C07H21/04 ,  C12N5/04

CPC分类号： C12N9/0008 ,  C07K2319/00 ,  C12N9/10 ,  C12N9/1025 ,  C12N9/1029 ,  C12N9/88 ,  C12N15/62 ,  C12N15/82 ,  C12N15/8243 ,  C12P7/625

摘要： In order to optimize the flux or flow of carbon intermediates from normal cellular metabolism into PHAs it is desirable to optimize the expression of the enzymes of the PHA biosynthetic pathway. Gene fusions are genetic constructs where two open reading frames have been fused into one and encode hybrid proteins and in some cases bifunctional hybrid enzymes. Linkers may be added to spatially separate the two domains of the hybrid protein. In the case of enzymes which catalyse successive reactions in a pathway, the fusion of two genes results in bringing two enzymatic activities into close proximity to each other. When the product of the first reaction is a substrate for the second one, this new configuration of active sites may result in a faster transfer of the product of the first reaction to the second active site with a potential for increasing the flux through the pathway.

摘要翻译： 为了优化碳中间体从正常细胞代谢到PHAs的通量或流量，优选优化PHA生物合成途径的酶的表达。 基因融合是遗传构建体，其中两个开放阅读框已融合在一起并编码杂交蛋白质，并且在某些情况下编码双功能杂合酶。 可以加入接头以在空间上分离杂交蛋白的两个结构域。 在催化途径中连续反应的酶的情况下，两个基因的融合导致使两个酶活性彼此靠近。 当第一反应的产物是第二反应的底物时，活性位点的这种新构型可能导致第一反应的产物更快地转移到第二活性位点，具有增加通过该通路的通量的可能性。

公开(公告)号：US20050239179A1

公开(公告)日：2005-10-27

申请号：US11072735

申请日：2005-03-04

申请人： Frank Skraly ,  Oliver Peoples

发明人： Frank Skraly ,  Oliver Peoples

IPC分类号： A01H1/00 ,  C08G8/04 ,  C08G8/26 ,  C12N1/21 ,  C12N15/09 ,  C12N15/52 ,  C12N15/82 ,  C12P7/42 ,  C12P7/62 ,  C12R1/19

CPC分类号： C08G8/04 ,  C12N15/52 ,  C12P7/42 ,  C12P7/625

摘要： Organisms are provided which express enzymes such as glycerol dehydratase, diol dehydratase, acyl-CoA transferase, acyl-CoA synthetase β-ketothiolase, acetoacetyl-CoA reductase, PHA synthase, glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase, which are useful for the production of PHAs. In some cases one or more of these genes are native to the host organism and the remainder are provided from transgenes. These organisms produce poly (3-hydroxyalkanoate) homopolymers or co-polymers incorporating 3-hydroxypropionate or 3-hydroxyvalerate monomers wherein the 3-hydroxypropionate and 3-hydroxyvalreate units are derived from the enzyme catalysed conversion of diols. Suitable diols that can be used include 1,2-propanediol, 1,3 propanediol and glycerol. Biochemical pathways for obtaining the glycerol from normal cellular metabolites are also described. The PHA polymers are readily recovered and industrially useful as polymers or as starting materials for a range of chemical intermediates including 1,3-propanediol, 3-hydroxypropionaldehyde, acrylics, malonic acid, esters and amines.

摘要翻译： 提供了表达酶的生物体，例如甘油脱水酶，二醇脱水酶，酰基辅酶A转移酶，酰基辅酶A合成酶β-酮硫解酶，乙酰乙酰辅酶A还原酶，PHA合成酶，甘油-3-磷酸脱氢酶和甘油-3-磷酸酶， 对PHA的生产有用。 在一些情况下，这些基因中的一种或多种对于宿主生物体是天然的，其余的由转基因提供。 这些生物体产生聚（3-羟基链烷酸酯）均聚物或掺有3-羟基丙酸酯或3-羟基戊酸酯单体的共聚物，其中3-羟基丙酸酯和3-羟基戊酸酯单元衍生自二醇的酶催化转化。 可以使用的合适的二醇包括1,2-丙二醇，1,3-丙二醇和甘油。 还描述了从正常细胞代谢物获得甘油的生化途径。 PHA聚合物容易回收，在工业上可用作聚合物或用作一系列化学中间体（包括1,3-丙二醇，3-羟基丙醛，丙烯酸，丙二酸，酯和胺）的原料。

公开(公告)号：US20050064565A1

公开(公告)日：2005-03-24

申请号：US10341214

申请日：2003-01-13

申请人： Oliver Peoples ,  Anthony Sinskey

发明人： Oliver Peoples ,  Anthony Sinskey

IPC分类号： C12N1/21 ,  C12N9/00 ,  C12N9/04 ,  C12N9/10 ,  C12N15/52 ,  C12N15/60 ,  C12P7/62

CPC分类号： C12N9/0006 ,  C12N9/00 ,  C12N9/1029 ,  C12N15/52 ,  C12P7/625

摘要： A method for controlling and modifying biopolymer synthesis by manipulation of the genetics and enzymology of synthesis of polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA) polyesters at the molecular level in procaryotic and eukaryotic cells, especially plants. Examples demonstrate the isolation, characterization, and expression of the genes involved in the production of PHB and PHA polymers. Genes encoding the enzymes in the PHB and PHA synthetic pathway (beta-ketothiolase, acetoacetyl-CoA reductase and PHB polymerase or PHA polymerase) from Zoogloea ramigera strain I-16-M, Alcaligenes eutrophus, Nocardia salmonicolur, and Psuedomonas olevarans were identified or isolated and expressed in a non-PHB producing organism, E. coli. Specific modifications to the polymers include variation in the chain length of the polymers and incorporation of different monomers into the polymers to produce co-polymers with different physical properties.

摘要翻译： 通过在原核和真核细胞，特别是植物中的分子水平处理聚羟基丁酸酯（PHB）和聚羟基链烷酸酯（PHA）聚酯的遗传学和酶学方法来控制和改变生物聚合物合成的方法。 实施例证明参与PHB和PHA聚合物生产的基因的分离，表征和表达。 鉴定或分离来自Zoogloea苎麻菌株I-16-M，产碱假单胞菌，痢疾杆菌（Nocardia salmonicolur）和橄榄油（Psuedomonas olevarans）的PHB和PHA合成途径（β-酮硫解酶，乙酰乙酰辅酶A还原酶和PHB聚合酶或PHA聚合酶）中的酶编码基因 并在非PHB生物体大肠杆菌中表达。 对聚合物的具体修饰包括聚合物链长度的变化以及将不同单体引入到聚合物中以产生具有不同物理性质的共聚物。