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：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。

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：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。