Biocatalytic production of l-fucose

Patent metadata

Field	Value
Publication number	US-10428361-B2
Application number	US-201615559962-A
Country	US
Kind code	B2
Filing date	Feb 19, 2016
Priority date	Mar 26, 2015
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

The present invention relates to biocatalytic methods, comprising purely enzymatic, mixed enzymatic-fermentative and purely fermentative methods, for the direct single-step conversion of L-fucitol to L-fucose, in order to easily obtain L-fucose at high amounts and levels of purity. Suitable recombinant microorganisms and fungi are further disclosed and also the use thereof in said method for the single-step conversion to L-fucose.

First claim

The invention claimed is:

1. A method for producing L-fucose, comprising the following steps:
(a) providing L-fucitol, a galactose oxidase of the enzyme class EC 1.1.3.9, a peroxidase and a catalase,
(b) combining L-fucitol, the galactose oxidase, the peroxidase, and the catalase to form a mixture,
(c) incubating the resulting mixture under conditions permitting the biocatalytic oxidation of L-fucitol to L-fucose, and
(d) optionally isolating the synthesized L-fucose.

2. The method according to claim 1 , wherein step (b) comprises or consists of the following substeps:
(b1) providing a recombinant microorganism or fungus for the recombinant expression of a galactose oxidase of the enzyme class EC 1.1.3.9, wherein the microorganism or fungus comprises a transgene which encodes a galactose oxidase from a fungus of the order of the Hypocreales
(b2) culturing the recombinant microorganism under conditions which permit the synthesis of a galactose oxidase, and
(b3) optionally isolating the synthesized galactose oxidase.

3. The method according to claim 2 , wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., Bacillus spp., Saccharomycetes, Kluyveromyces spp., Aspergillus spp., Trichoderma spp., and a fungus of the phylum Ascomycota.

4. The method according to claim 3 , wherein the recombinant microorganism or fungus is selected from the group consisting of: a Escherichia coli spp. selected from the group consisting of E. coli BL21, E. coli MG1655, E. coli W3110 and descendants thereof; a Bacillus spp, selected from the group consisting of Bacillus licheniformis, Bacillus subitilis, Bacillus amyloliquefaciens , and descendants thereof; a Saccharomycetes selected from the group consisting of a Saccharomycetes selected from the group consisting of the genus Saccharomyces , the genus Komagataella, Pichia pastoris , and descendants thereof; a Kluyveromyces spp. selected from the group consisting K lactis , and descendants thereof; an Aspergillus spp. selected from the group consisting of A. oryzae, A. nidulans, A. niger , and descendants thereof; a Trichoderma spp. selected from the group consisting of T reesei, T harzianum , and descendants thereof; and a fungus of the phylum Ascomycota selected from the group consisting of Myceliopthora thermophila , and a descendant thereof.

5. The method according to claim 4 , wherein the peroxidase is the peroxidase from horseradish ( Armoracia rusticana ).

6. The method according to claim 1 , wherein the catalase is the catalase from cattle ( Bos taurus ).

7. The method according to claim 2 , wherein the transgene encodes a galactose oxidase selected from the genus Fusarium or from the species Fusarium oxysporum.

8. The method according to claim 1 , wherein the peroxidase is selected from a peroxidase of the group consisting of the enzyme class EC 1.11.1.7.

9. The method according to claim 1 , wherein the catalase is selected from a catalase of the group consisting of the enzyme class EC 1.11.1.6.

10. The method according to claim 1 , wherein the concentration of L-fucitol in the mixture at the start of the incubation in step (c) is in the range from 1 to 500 g/L.

11. The method according to claim 1 , wherein the concentration of galactose oxidase in the mixture at the start of the incubation in step (c) is in the range from 0.005 to 6.25 g/L.

12. The method according to claim 1 , wherein the concentration of peroxidase in the mixture at the start of the incubation in step (c) is in the range from 0 to 0.5 g/L.

13. The method according to claim 1 , wherein the concentration of catalase in the mixture at the start of the incubation in step (c) is in the range from 0 to 0.4 g/L.

14. The method according to claim 1 , wherein, at the start of the incubation in step (c),
the concentration of L-fucitol in the mixture is in the range from 5 to 250 g/L,
the concentration of galactose oxidase in the mixture is in the range from 0.005 to 3.2 g/L,
the concentration of peroxidase in the mixture is in the range from 0 to 0.3 g/L, and
the concentration of catalase in the mixture is in the range from 0 to 0.2 g/L.

15. The method according to claim 1 , wherein, at the start of the incubation in step (c),
the concentration of L-fucitol in the mixture is in the range from 20 to 1.25 g/L,
the concentration of galactose oxidase in the mixture is in the range from 0.005 to 0.5 g/L,
the concentration of peroxidase in the mixture is in the range from 0 to 0.15 g/L, and
the concentration of catalase in the mixture is in the range from 0 to 0.1 g/L.

16. The method according to claim 1 , wherein the concentration of peroxidase in the mixture at the start of the incubation in step (c) is in the range from 0 to 0.3 g/L.

17. The method according to claim 1 , wherein the concentration of peroxidase in the mixture at the start of the incubation in step (c) is in the range from 0 to 0.15 g/L.

18. A method for producing L-fucose, comprising:
adding L-fucitol to a culture medium of a recombinant microorganism or fungus expressing a transgene(s) in functional form, and
culturing the mixture under conditions permitting the biocatalytic oxidation of L-fucitol to L-fucose,
wherein the recombinant microorganism or fungus comprises a transgene which encodes a galactose oxidase of the enzyme class EC 1.1.3.9 from a fungus of the genus Fusarium , and further comprises a transgene selected from a peroxidase selected from a peroxidase of the group consisting of the enzyme class EC 1.11.1.7, and a catalase selected from a catalase of the group consisting of the enzyme class EC 1.11.1.6, wherein the recombinant microorganism or fungus is selected from the group consisting of Escherichia coli spp., Bacillus spp., Saccharomycetes, Kluyveromyces spp., Aspergillus spp., Trichoderma spp., and a fungus of the phylum Ascomycota.

19. The method according to claim 18 , wherein the recombinant microorganism or fungus is selected from the group consisting of: an Escherichia coli spp. selected from the group consisting of E coli BL21, E. coli MG1655, E coli W3110 and descendants thereof, a Bacillus spp, selected from the group consisting of Bacillus licheniformis, Bacillus subitilis, Bacillus amyloliquefaciens , and descendants thereof, a Saccharomycetes selected from the group consisting of the genus Saccharomyces , the genus Komagataella, Pichia pastoris , and descendants thereof, a Kluyveromyces spp. selected from the group consisting of K lactis , and descendants thereof an Aspergillus spp. selected from the group consisting of A. oryzae, A. nidulans, A. niger , and descendants thereof, a Trichoderma spp. selected from the group consisting of T reesei, T harzianum , and descendants thereof, and a fungus of the phylum Ascomycota selected from the group consisting of Myceliopthora thermophila , or a descendant thereof.

20. A method for the biocatalytic conversion of L-fucitol to L-fucose, comprising the following steps:
(a) providing a recombinant microorganism or fungus for the recombinant expression of a galactose oxidase, a peroxidase, and a catalase, wherein the microorganism comprises a transgene which encodes a galactose oxidase from a fungus of the genus Fusarium , a transgene which encodes a peroxidase, and a transgene with encodes a catalase, and wherein the recombinant microorganism or fungus is selected from the group consisting of: an Escherichia coli spp., a Bacillus spp., a

Assignees

• Basf Se

Inventors

• Navickas Vaidotas
• Breuer Michael
• Puhl Michael
• Weingarten Melanie
• Baldenius Kai-Uwe
• Siegel Wolfgang

Classifications

• C12Y101/03009

  Galactose oxidase (1.1.3.9) · CPC title

• C12Y111/01006

  Catalase (1.11.1.6) · CPC title

• C12Y111/01007

  Peroxidase (1.11.1.7), i.e. horseradish-peroxidase · CPC title

• C12N9/0065

  acting on hydrogen peroxide as acceptor (1.11) · CPC title

• C12P19/02 Primary

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title