Heparosan-Producing Bacterium and Heparosan Manufacturing Method

Patent metadata

Field	Value
Publication number	US-2016201103-A1
Application number	US-201615082464-A
Country	US
Kind code	A1
Filing date	Mar 28, 2016
Priority date	Oct 2, 2013
Publication date	Jul 14, 2016
Grant date	—

Abstract

A method for producing heparosan is provided. Heparosan is produced by culturing an Escherichia bacterium having a heparosan-producing ability and modified so that expression of one or more genes, such as rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA, gcvR, bcp, hyfA, rpoE, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, and efeO, is/are increased in a medium, and collecting heparosan from the medium.

First claim

1 . An Escherichia bacterium having a heparosan-producing ability, wherein:
the bacterium has been modified so that expression is increased of a gene selected from the group consisting of rpoE, rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, rfaH, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, lepB, rnc, era, dapA, gcvR, bcp, hyfA, nadB, yfiC, srmB, g1414, g1413, nuoE, nuoF, nuoG, glmZ, hemY, hemX, hemD, rlmL, artQ, artM, artJ, rlmC, ybjO, yejO, yejM, yejL, rpoS, ygbN, ygbM, ygbL, g3798, g3797, g3796, g3795, g3794, g3793, g3792, ryjA, soxR, soxS, yjcC, yjcB, efeU, efeO, and combinations thereof.

2 . The bacterium according to claim 1 , which has been modified so that expression of at least the rpoE gene is increased.

3 . The bacterium according to claim 1 , which has been modified so that expression of at least the rfaH gene is increased.

4 . The bacterium according to claim 3 , which has been further modified so that expression is increased of a gene selected from the group consisting of rbsR, rbsK, rbsB, hsrA, glgB, glgX, micF, rcsD, rcsB, ybiX, ybiI, ybiJ, ybiC, ybiB, nusG, pcoR, pcoS, pcoE, yhcN, yhcO, aaeB, aaeA, aaeX, g1455, alpA, g1453, yrbA, mlaB, mlaC, mlaD, mlaE, mlaF, yrbG, norW, ybjI, ybjJ, ybjK, rybB, yjjY, yjtD, thrL, thrA, thrB, fruA, psuK, ytfT, yjfF, fbp, yagU, paoA, paoB, gsiC, gsiD, yliE, irp2, irp1, bhsA, ycfS, and combinations thereof.

5 . The bacterium according to claim 1 , wherein said expression is increased by increasing the copy number of the gene(s), and/or modifying a gene expression control sequence of the gene(s).

6 . The bacterium according to claim 1 , which is Escherichia coli.

7 . The bacterium according to claim 1 , wherein:
the rbsB gene is a DNA comprising the nucleotide sequence of positions 800 to 1690 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 800 to 1690 of SEQ ID NO: 29 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the rbsK gene is a DNA comprising the nucleotide sequence of positions 1816 to 2745 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 1816 to 2745 of SEQ ID NO: 29 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the rbsR gene is a DNA comprising the nucleotide sequence of positions 2749 to 3741 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 2749 to 3741 of SEQ ID NO: 29 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the hsrA gene is a DNA comprising the complementary sequence of the nucleotide sequence of positions 3707 to 5134 of SEQ ID NO: 29, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the complementary sequence of the nucleotide sequence of positions 3707 to 5134 of SEQ ID NO: 29 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the glgB gene is a DNA comprising the nucleotide sequence of positions 989 to 3175 of SEQ ID NO: 34, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 989 to 3175 of SEQ ID NO: 34 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the glgX gene is a DNA comprising the nucleotide sequence of positions 3172 to 5145 of SEQ ID NO: 34, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 3172 to 5145 of SEQ ID NO: 34 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the rcsB gene is a DNA comprising the nucleotide sequence of positions 3312 to 3962 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 3312 to 3962 of SEQ ID NO: 43 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the rcsD gene is a DNA comprising the nucleotide sequence of positions 623 to 3295 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 623 to 3295 of SEQ ID NO: 43 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the micF gene is a DNA comprising the nucleotide sequence of positions 219 to 311 of SEQ ID NO: 43, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 219 to 311 of SEQ ID NO: 43 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the ybiX gene is a DNA comprising the nucleotide sequence of positions 718 to 1395 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 718 to 1395 of SEQ ID NO: 37 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the ybiI gene is a DNA comprising the nucleotide sequence of positions 1469 to 1735 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 1469 to 1735 of SEQ ID NO: 37 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the ybiJ gene is a DNA comprising the nucleotide sequence of positions 2000 to 2260 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the nucleotide sequence of positions 2000 to 2260 of SEQ ID NO: 37 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the ybiC gene is a DNA comprising the complementary sequence of the nucleotide sequence of positions 2488 to 3574 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having an identity of 90% or higher to the complementary sequence of the nucleotide sequence of positions 2488 to 3574 of SEQ ID NO: 37 and is able to increase heparosan-producing ability of an Escherichia bacterium having heparosan-producing ability when expression amount thereof is increased in the bacterium; the ybiB gene is a DNA comprising the complementary sequence of the nucleotide sequence of positions 3715 to 4677 of SEQ ID NO: 37, or a DNA comprising a nucleotide sequence having an identity of 90

Assignees

• Ajinomoto Kk

Inventors

• Yamazaki Shunsuke
• Shimizu Tomoko
• Mori Kenichi
• Tonouchi Naoto

Classifications

• C12P19/26

  Preparation of nitrogen-containing carbohydrates · CPC title

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C08B37/0075

  Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof · CPC title

• C12P19/04 Primary

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title