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Phosphoketolases for improved production of acetyl coenzyme a-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids

一种类异戊二烯、异戊二烯合酶的技术，应用在生物化学设备和方法、微生物的测定/检验、酶等方向，能够解决损耗、生产收率降低等问题

Inactive Publication Date: 2016-05-04
DANISCO US INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Loss of one carbon atom results in reduced production yields of acetyl-CoA-derived metabolites, isoprenoid precursors, isoprene and isoprenoid molecules

Method used

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  • Phosphoketolases for improved production of acetyl coenzyme a-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids
  • Phosphoketolases for improved production of acetyl coenzyme a-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids
  • Phosphoketolases for improved production of acetyl coenzyme a-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids

Examples

Experimental program
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Effect test

example 1

[0540] Example 1: Identification of Phosphoketolases

[0541] To identify phosphoketolases useful for improved production of acetyl-CoA-derived (acetyl-CoA-derived) metabolites, isoprenes, isoprenoid precursors, and isoprenoids in recombinant cells, in The CDART program in the NCBI network is used to select all gene products that are consistent with the known phosphoketolase domain structure (GeerL et al. (2002), "CDART: proteinhomology by domain architecture.", GenomeRes.12 (10) 1619- twenty three). Sequences were further refined by selecting RefSeq sequences from the initial domain construction search. Next, the sequences were divided into 22 different groups based on sequence similarity (Clustering by Passing Messages Between Data Points. Brendan J. Frey and Delbert Dueck, University of Toronto Science 315, 972-976, February 2007). Briefly, amino acid sequences were multiple-aligned using ClustalW. Pairwise percent identities (PIDs) were calculated. This is operationa...

example 2

[0556] Example 2: Identification of Phosphoketolases in Bacterial Genomes Deleting Phosphofructokinase

[0557] Bacterial genomes were searched for annotated phosphoketolase (PKL) but not phosphofructokinase (PFK), a key enzyme in carbon flux through glycolysis. Several organisms that fit these criteria were selected for high activity and high yield of glucose to isoprene for a specific PKL from this list of five PKLs from the plant Burkholderiaphytofirmans PsJN (SEQ ID NO: 47), Lactobacillus buchneri NRRLB-30929 (SEQ ID NO: 48), Bifidobacterium gallicum DSM20093 (SEQ ID NO: 49), Bifidobacterium dentium Bd1 (SEQ ID NO: 50 ) and Bifidobacterium bifidum IPLA20015 (SEQ ID NO: 51). Since most PKLs from the full repertoire of organisms have not yet been characterized, the selection of five PKLs was made based on sequence differences and the best environmental evidence of hyperactivity available from the literature. PKL from Bifidobacterium dentium shows a pH optimum of 7 (Sgorb...

example 3

[0558] Example 3: Cloning of identified phosphoketolases

[0559] PKL obtained from Bifidobacterium longum subsp. infantis, Enterococcus gallinarum and Clostridium acetobutylicum were each assayed for their enzymatic activity. Bifidobacterium longum subsp. infantis PKL has a Km of 5.7±1.16mM, 4.56±0.2sec -1 kcat, and 0.79±0.2mM -1 sec -1 kcat / Km, Enterococcus gallinarum (Enterococcus gallinarum) PKL has a Km of 10.4±1.03mM, 1.35±0.04sec -1 kcat, and 0.13±0.1mM -1 sec -1 kcat / Km, and Clostridium acetobutylicum (Clostridium acetobutylicum) PKL was found to have a Km of 10.3±0.67mM, 2.18±0.05sec -1 kcat, and 0.21±0.06mM -1 sec -1 kcat / Km. Constructs encoding Bifidobacterium longum subsp. infantis, Enterococcus gallinarum, or Clostridium acetobutylicum PKL were used as controls to screen candidate PKL enzymes for in vivo and in vitro activity.

[0560]The amino acid sequence (SEQ ID NO: 93) of Enterococcus gallinarum PKL was obtained from GenBank and processed in GeneAr...

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Abstract

The invention provides phosphoketolases for improved production of acetyl coenzyme a-derived metabolites, isoprene, isoprenoid precursors, and isoprenoids. This present invention relates to cultured recombinant cells comprising a heterologous phosphoketolase (PKL) polypeptide that are capable of increased production of acetyl coenzyme A-derived metabolites, as well as methods for producing and using the same. In some embodiments, the recombinant cells further comprise one or more mevalonate (MVA) pathway polypeptides for the production of isoprenoid precursors, isoprene and isoprenoids.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application 61 / 810,696, filed April 10, 2013, and U.S. Provisional Patent Application 61 / 834,359, filed June 12, 2013, the disclosures of each of which are incorporated by reference in their entirety way incorporated into this article. technical field [0003] The present invention relates to cultured recombinant cells comprising heterologous phosphoketolase (PKL) polypeptides, which enhance the production of acetyl-CoA-derived metabolites, and methods of making and using them. In some embodiments, the recombinant cell further comprises one or more polypeptides of the mevalonate (MVA) pathway, which are used to produce isoprenoid precursors, isoprene, and isoprenoid. Background technique [0004] Glycolysis allows the metabolic conversion of carbon sources into intermediate compounds such as acetyl-coenzyme A (acetyl-CoA), which is an important intermediate ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC12Q1/527C12P19/32C12P5/007C12N9/88C12Y401/02009C12P5/026
Inventor Z·Q·贝克J·W·穆诺斯D·H·韦尔斯J·姚
Owner DANISCO US INC
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