公开(公告)号：US20040172675A1

公开(公告)日：2004-09-02

申请号：US10703906

申请日：2003-11-07

Applicant: Metabolix, Inc.

Inventor： Lara Madison ,  Gjalt W. Huisman ,  Oliver P. Peoples

IPC: C12N001/20 ,  A01H001/00 ,  C12N015/82 ,  C12P007/62

CPC classification number: C12N9/88 ,  C12N9/0008 ,  C12P7/625

Abstract: Methods for engineering transgenic organisms that synthesize polyhydroxyalkanoates (PHAs) containing 3-hydroxyhexanoate as comonomer have been developed. These processes are based on genetically engineered bacteria such as Escherichia coli or in plant crops as production systems which include PHA biosynthetic genes from PHA producers. In a preferred embodiment of the method, additional genes are introduced in wild type or transgenic polyhydroxybutyrate (PHB) producers, thereby creating new strains that synthesize 3HH monomers which are incorporated into PHAs. The 3HH monomer preferably is derived in microbial systems using butanol or butyrate as feedstocks, which are precursors of 3-hydroxyhexanoyl-CoA. Pathways for in vivo production of butyrol-CoA specifically encompassing butyryl-CoA dehydrogenase activity are provided.

Abstract translation: 已经开发了合成含有3-羟基己酸作为共聚单体的聚羟基链烷酸酯（PHA）的转基因生物的工程方法。 这些方法基于遗传工程细菌如大肠杆菌或植物作物，作为包括PHA生物合成基因的生产系统。 在该方法的优选实施方案中，在野生型或转基因聚羟基丁酸酯（PHB）生产者中引入另外的基因，从而产生合成并入PHA的3HH单体的新菌株。 3HH单体优选在使用丁醇或丁酸盐作为原料的微生物系统中衍生，其是3-羟基己酰基-CoA的前体。 提供了具体包含丁酰辅酶A脱氢酶活性的丁酰-coA的体内生产途径。

公开(公告)号：US20040033572A1

公开(公告)日：2004-02-19

申请号：US10600480

申请日：2003-06-20

Applicant: Metabolix, Inc.

Inventor： Frank A. Skraly ,  Oliver P. Peoples

IPC: C12P007/62 ,  C08G063/78 ,  C08G063/82 ,  C08G063/87

CPC classification number: C12N9/93 ,  C08G63/06 ,  C12N9/1029 ,  C12P7/625

Abstract: Several novel PHA polymer compositions produced using biological systems include monomers such as 3-hydroxybutyrate, 3-hydroxypropionate, 2-hydroxybutyrate, 3-hydroxyvalerate, 4-hydroxybutyrate, 4-hydroxyvalerate and 5-hydroxyvalerate. These PHA compositions can readily be extended to incorporate additional monomers including, for example, 3-hydroxyhexanoate, 4-hydroxyhexanoate, 6-hydroxyhexanoate or other longer chain 3-hydroxyacids containing seven or more carbons. This can be accomplished by taking natural PHA producers and mutating through chemical or transposon mutagenesis to delete or inactivate genes encoding undesirable activities. Alternatively, the strains can be genetically engineered to express only those enzymes required for the production of the desired polymer composition. Methods for genetically engineering PHA producing microbes are widely known in the art (Huisman and Madison, 1998, Microbiology and Molecular Biology Reviews, 63: 21-53). These polymers have a variety of uses in medical, industrial and other commercial areas.

Abstract translation: 使用生物系统制备的几种新型PHA聚合物组合物包括单体如3-羟基丁酸酯，3-羟基丙酸酯，2-羟基丁酸酯，3-羟基戊酸酯，4-羟基丁酸酯，4-羟基戊酸酯和5-羟基戊酸酯。 这些PHA组合物可以容易地延伸以加入另外的单体，包括例如3-羟基己酸酯，4-羟基己酸酯，6-羟基己酸酯或其它含有7个或更多个碳的长链3-羟基酸。 这可以通过使用天然PHA生产者和通过化学或转座子诱变进行突变来删除或灭活编码不期望的活性的基因来实现。 或者，菌株可以被遗传工程化以仅表达生产所需聚合物组合物所需的那些酶。 基因工程生产PHA的微生物的方法在本领域是众所周知的（Huisman and Madison，1998，Microbiology and Molecular Biology Reviews，63：21-53）。 这些聚合物在医疗，工业和其他商业领域具有多种用途。

公开(公告)号：US20040137586A1

公开(公告)日：2004-07-15

申请号：US10773916

申请日：2004-02-06

Applicant: Metabolix, Inc.

Inventor： Gjalt W. Huisman ,  Frank Skraly ,  David P. Martin ,  Oliver P. Peoples

IPC: C07H021/04 ,  C12P007/62 ,  C12N009/10 ,  C12N009/16

CPC classification number: C12N9/0008 ,  C08G63/06 ,  C12N9/1029 ,  C12N9/1096 ,  C12N15/8243 ,  C12P7/625

Abstract: 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, null-ketoglutarate, or succinate as substrate.

Abstract translation: 已经从细菌中分离出编码4-羟基丁酰-CoA转移酶的基因，并整合到也表达聚羟基链烷酸酯合酶的细菌的基因组中，以使用包括细菌和植物在内的转基因生物来产生含4HB的聚羟基链烷酸酯的改进的生产方法。 新途径提供了从低廉的碳源（如糖和脂肪酸）生产含有PHB的4HB的方法，其产率高，稳定。 有用的菌株是通过筛选在其基因组中整合了编码4HB-CoA转移酶和/或PHA合成酶的基因的聚合物生产获得的菌株。 用于聚合物生产的方法使用可以利用廉价底物的重组体系。 提供可以利用氨基酸降解途径的系统，α-酮戊二酸或琥珀酸作为底物。

公开(公告)号：US20040023347A1

公开(公告)日：2004-02-05

申请号：US10444031

申请日：2003-05-21

Applicant: Metabolix, Inc.

Inventor： Frank A. Skraly ,  Oliver P. Peoples

IPC: C12P007/62 ,  C12N009/02 ,  C08G063/82 ,  A01H001/00 ,  C12N015/82 ,  C12N005/04 ,  C12N001/21

CPC classification number: C08G8/04 ,  C12N15/52 ,  C12P7/42 ,  C12P7/625

Abstract: Organisms are provided which express enzymes such as glycerol dehydratase, diol dehydratase, acyl-CoA transferase, acyl-CoA synthetase null-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.

Abstract translation: 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：US20040014197A1

公开(公告)日：2004-01-22

申请号：US10607903

申请日：2003-06-27

Applicant: Metabolix, Inc.

Inventor： Gjalt W. Huisman ,  Laura Z. Luo ,  Oliver P. Peoples

IPC: C12N001/21 ,  C12P019/28 ,  C12P019/04 ,  C12P007/62 ,  C12N009/22

CPC classification number: C12N1/08 ,  C12N9/22 ,  C12P7/625

Abstract: DNA constructs and genetically engineered microbial strains constructed using these DNA constructs, which produce a nuclease enzyme with specificity for DNA and/or RNA, are provided. These strains secrete nuclease into the periplasm or growth medium in an amount effective to enhance productivity and/or recovery of polymer, and are particularly suited for use in high cell density fermentation processes. These constructs are useful for modifying microbial strains to improve production and recovery processes for polymers such as intracellular proteins, such as enzymes, growth factors, and cytokines; for producing polyhydroxyalkanoates; and for producing extracellular polysaccharides, such as xanthan gum, alginates, gellan gum, zooglan, hyaluronic acid and microbial cellulose.

Abstract translation: 提供使用这些DNA构建体构建的DNA构建体和遗传工程化的微生物菌株，其产生对DNA和/或RNA具有特异性的核酸酶。 这些菌株以有效提高聚合物的生产力和/或回收率的量将核酸酶分泌到周质或生长培养基中，并且特别适用于高细胞密度发酵过程。 这些构建体可用于修饰微生物菌株以改善聚合物如细胞内蛋白质如酶，生长因子和细胞因子的生产和回收过程; 用于生产聚羟基链烷酸酯; 并且用于生产细胞外多糖，例如黄原胶，藻酸盐，结冷胶，动物胶，透明质酸和微生物纤维素。

公开(公告)号：US20030228669A1

公开(公告)日：2003-12-11

申请号：US10461069

申请日：2003-06-13

Applicant: Metabolix, Inc.

Inventor： Gjalt W. Huisman ,  Oliver P. Peoples ,  Frank A. Skraly

IPC: C12P007/62 ,  C12N001/21 ,  C12P021/02 ,  C08G063/78 ,  C12N015/74

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

Abstract: 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.

Abstract translation: 提供了转基因微生物菌株，其含有整合在染色体上的PHA形成所需的基因。 该菌株在PHA生产过程中是有利的，因为（1）不需要维持质粒，通常不需要使用抗生素或其他稳定压力，和（2）不发生质粒损失，从而稳定每个细胞的基因拷贝数 在整个发酵过程中，在整个生产过程中产生均匀的PHA产物形成。 使用标准技术整合基因，优选转座子诱变。 在其中掺入多个基因的优选实施方案中，将它们作为操纵子并入。 序列用于稳定mRNA，诱导表达作为培养条件（如磷酸盐浓度），温度和应激的函数，并通过引入选择标记（如赋予抗生素抗性的标记）来帮助选择。

公开(公告)号：US20030233677A1

公开(公告)日：2003-12-18

申请号：US10447515

申请日：2003-05-28

Applicant: Metabolix, Inc.

Inventor： Oliver P. Peoples ,  Maurice Moloney ,  Nii Patterson ,  Kristi D. Snell

IPC: A01H001/00 ,  C12N015/82 ,  C07H021/04 ,  C12P007/64 ,  C12N009/02 ,  C12P021/02 ,  C12N005/04

CPC classification number: C12N15/8289 ,  C12N15/52 ,  C12N15/8243 ,  C12N15/8247

Abstract: Methods and systems to modify fatty acid biosynthesis and oxidation in plants to make new polymers are provided. Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained from Aeromonas caviae, and a null-oxidation enzyme system. Some plants have a null-oxidation enzyme system which is sufficient to modify polymer synthesis when the plants are engineered to express the hydratase. Examples demonstrate production of polymer by expression of these enzymes in transgenic plants. Examples also demonstrate that modifications in fatty acid biosynthesis can be used to alter plant phenotypes, decreasing or eliminating seed production and increasing green plant biomass, as well as producing polyhydroxyalkanoates.

公开(公告)号：US20020187530A1

公开(公告)日：2002-12-12

申请号：US10006915

申请日：2001-11-09

Applicant: Metabolix, Inc.

Inventor： Gjalt W. Huisman ,  Frank Skraly ,  David P. Martin ,  Oliver P. Peoples

IPC: C12P007/62 ,  C12N015/74 ,  C12P021/02 ,  C12N001/21

CPC classification number: C12N9/0008 ,  C08G63/06 ,  C12N9/1029 ,  C12N9/1096 ,  C12N15/8243 ,  C12P7/625

Abstract: 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, null-ketoglutarate, or succinate as substrate.

Abstract translation: 已经从细菌中分离出编码4-羟基丁酰-CoA转移酶的基因，并整合到也表达聚羟基链烷酸酯合酶的细菌的基因组中，以使用包括细菌和植物在内的转基因生物来产生含4HB的聚羟基链烷酸酯的改进的生产方法。 新途径提供了从低廉的碳源（如糖和脂肪酸）生产含有PHB的4HB的方法，其产率高，稳定。 有用的菌株是通过筛选在其基因组中整合了编码4HB-CoA转移酶和/或PHA合成酶的基因的聚合物生产获得的菌株。 用于聚合物生产的方法使用可以利用廉价底物的重组体系。 提供可以利用氨基酸降解途径的系统，α-酮戊二酸或琥珀酸作为底物。