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Fermentative production of carbohydrates by microbial cells utilizing mixed feedstock

A technology of microbial cells and carbohydrates, which is used in the field of fermentation and can solve the problems of offset applicability, high pollution risk of bacterial cell growth, etc.

Pending Publication Date: 2021-10-01
CHR HANSEN HMO GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, heat sterilization of sucrose resulted in considerable hydrolysis, negating the applicability of the method described in WO 2012 / 007481 A2
[0017] As an alternative to heat sterilization, sterile filtration of sucrose solutions can be used, but sterile filtration has a high risk of contamination leading to unwanted bacterial cell growth, especially in industrial-scale fermentations

Method used

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  • Fermentative production of carbohydrates by microbial cells utilizing mixed feedstock
  • Fermentative production of carbohydrates by microbial cells utilizing mixed feedstock
  • Fermentative production of carbohydrates by microbial cells utilizing mixed feedstock

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Embodiment 1-preparation of mixed monosaccharide raw material

[0143] A 50% (w / v) sucrose solution was prepared by dissolving 500 g of sucrose in water. The final volume of the solution was 1 liter. The pH was adjusted by using 96% (v / v) sulfuric acid at a temperature of 30°C to 35°C. Then, the solution was sterilized in a vertical autoclave (Systec VX-65, Linden, Germany) at 121° C. for 45 minutes. Samples were taken before and after heat sterilization and kept frozen until high performance liquid chromatography (HPLC) analysis. HPLC was performed using a RID-IOA Refractive Index Detector (Shimadzu, Germany) and a Waters XBridge Amide column 3.5 μm (250x4.6 mm) (Eschborn, Germany) connected to a Shimadzu HPLC system. With 30% solvent A (50% (v / v) acetonitrile in double distilled water, 0.1% (v / v) NH4OH) and 70% solvent B (80% (v / v) acetonitrile in double distilled water, 0.1 %(v / v) NH4OH) was eluted isocratically at 35°C with a flow rate of 1.4mLmin -1 . Samples...

Embodiment 2

[0146] Example 2 - Feedstock-dependent growth of various gene deletion strains

[0147] The E. coli BL21(DE3) strain (wild type) and the mutant strain E. coli pfkA were compared - (△pfkA), Escherichia coli pfkB - (△pfkB), Escherichia coli pfkA - wxya - Growth behavior of (△pfkA△pfkA). Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). All strains were cultured at 30°C in 100mL shake flasks containing 20mL of mineral salt medium containing 7g·L - 1 NH 4 h 2 PO 4 , 7g·L -1 K 2 HPO 4 , 2g·L -1 KOH, 0.3g·L -1 Citric acid, 2g·L -1 MgSO 4 ×7·H 2 O and 0.015g·L -1 CaCl 2 ×6·H 2 O, add 1mL·L -1 Trace element solution (54.4g L -1 Ferric ammonium citrate, 9.8g·L -1 MnCl 2 ×4·H 2 O, 1.6g·L -1 CoCl 2 ×6·H 2 O, 1g L -1 CuCl 2 ×2·H 2 O, 1.9g·L -1 h 3 BO 3 , 9g·L -1 ZnSO 4 ×7·H 2 O, 1.1g L -1 Na 2 MoO 4 ×2·H 2 O, 1.5g·L -1 Na 2 SeO 3 , 1.5g·L -1 NiSO 4 ×6·H 2 O) with 2...

Embodiment 3

[0148] Example 3 - Production of 2'-fucosyllactose by engineering E. coli strains

[0149] 2'-Fucosyltransferase gene wbgL from Escherichia coli: O126, from Yersinia bokwangii by overexpressing enzymes for de novo synthesis of GDP-fucose (ManB, ManC, Gmd, WcaG) (Yersinia bercovieri) ATCC 43970 sugar efflux transporter yberc0001_9420 and Escherichia coli W csc gene cluster (Accession No. CP002185.1) (including genes for sucrose permease, fructokinase, sucrose hydrolase and transcriptional repressor ( genes cscB, cscK, cscA and cscR)), further genetic engineering revealed genotype lacY + , lacZ - 、fuclK - , wcaJ - , pfkA - coli BL21(DE3) strain, enabling the strain to grow on sucrose as the sole carbon source. Genomic deletions were performed according to the method of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)). Genomic integration of heterologous genes by transposition. Linear DNA fragments were integrated using EZ-Tn5TM transposase (Epicentre, ...

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Abstract

Disclosed are genetically engineered microbial cells for the production of a carbohydrate of interest, wherein the microbial cells possess an increased intracellular availability of at least one sugar phosphate, and produce the carbohydrate of interest when being cultivated in a culture medium containing a mixed monosaccharide feedstock as main carbon and energy source. Also disclosed is a fermentative method of producing a carbohydrate of interest by cultivating said genetically engineered microbial cell in the presence of a mixed monosaccharide feedstock as main carbon and energy source.

Description

[0001] The present invention relates to the fermentative production of target sugars. The present application discloses microbial cells capable of producing sugars of interest, wherein the microbial cells utilize a mixed monosaccharide feedstock as a primary carbon and energy source during fermentation. The present application also discloses a method for producing target sugars by utilizing the microbial cells. Background technique [0002] Human milk contains a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The most important fraction of human milk consists of carbohydrates. The carbohydrate fraction in human milk can be further divided into (i) lactose and (ii) oligosaccharides (human milk oligosaccharides, HMOs). While the disaccharide lactose (galactose-β1,4-glucose) is used by infants as an energy source, oligosaccharides are not metabolized by infants. [0003] The oligosaccharide fraction accounts for up to one-tenth of the t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12P19/04C12P19/18C12N1/21A61K31/7004A61K31/7016A61K31/702
CPCA61K31/7004A61K31/7016A61K31/702C12P19/02C12P19/04C12P19/18C12N15/70C12N9/1051A61K35/74
Inventor S·詹尼温D·瓦滕伯格
Owner CHR HANSEN HMO GMBH
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