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Method for producing 1, 4-butanediamine by fermenting xylose and xylose-containing hydrolysate

A technology of agmatase and xylose isomerase, applied in the field of genetic engineering, can solve the problems of low final yield and the like, and achieve the effect of expanding carbon sources

Pending Publication Date: 2022-07-22
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although in existing reports, xylose has been used to produce 1,4-butanediamine, it has the disadvantage of low final yield, for example: in the method disclosed by Volker F.Wendisch et al. Xylose isomerase derived from Xanthomonas rape and xylulokinase derived from Corynebacterium glutamicum construct the xylose metabolic pathway, but the final yield of 1,4-butanediamine is only 15.1 mM (published in "Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine" paper)

Method used

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  • Method for producing 1, 4-butanediamine by fermenting xylose and xylose-containing hydrolysate
  • Method for producing 1, 4-butanediamine by fermenting xylose and xylose-containing hydrolysate
  • Method for producing 1, 4-butanediamine by fermenting xylose and xylose-containing hydrolysate

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Construction of recombinant bacteria PC0 / pEC-XK99E-xylA, PC0 / pEC-XK99E-xylAB and PC0 / pEC-XK99E-P52-xylAB

[0045] (1) Primer design

[0046] According to the gene sequence of xylA (the nucleotide sequence encoding the xylose isomerase is shown in SEQ ID NO 6) and the gene sequence of xylB (the nucleotide sequence encoding the xylulokinase) in the whole genome nucleic acid sequence of Escherichia coli K-12 in NCBI The nucleotide sequence is shown in SEQ ID NO 7), the PCR primer F of the design plasmid pEC-XK99E-xylA 1 and R 1 , PCR primer F of pEC-XK99E-xylAB 2 and R 2 , PCR primer F of pEC-XK99E-P52-xylAB 3 and R 3 and F 4 and R 4 .

[0047] F 1 : 5'-caggaaacagaccatggaattcaaaggaggacaaccatgcaagcctattttgaccagc-3';

[0048] R 1 : 5'-gcaggtcgactctagaggatccttatttgtcgaacagataatggtttaccag-3';

[0049] F 2 : 5'-caggaaacagaccatggaattcaaaggaggacaaccatgcaagcctattttgaccagc-3';

[0050] R 2 :5'-gcaggtcgactctagaggatccttacgccattaatggcagaagttgc-3';

[0051] F...

Embodiment 2

[0066] Example 2: Growth assay of recombinant bacteria PC0 / pEC-XK99E-xylA, PC0 / pEC-XK99E-xylAB, PC0 / pEC-XK99E-P52-xylAB in the medium with only xylose carbon source

[0067] (1) Preparation of culture medium:

[0068] Seed medium (g L -1 ): glucose 50, (NH 4 ) 2 SO 4 20, Yeast Powder 20, KH 2 PO 4 1.5, MgS0 4 ·7H 2 O1.0, MnS0 4 ·H 2 O 0.3, CaCO 3 1.0;

[0069] Fermentation medium (g L -1 ): Xylose 100, (NH 4 ) 2 SO 4 40, Yeast Powder 10, KH 2 PO 4 1.5, KCl 1.0, MgS0 4 ·7H 2 O 0.5, MnS0 4 ·H 2 O 0.02, FeS0 4 ·7H 2 O 0.02, CaCO 3 20.

[0070] (2) The recombinant bacteria PC0 / pEC-XK99E-xylA, PC0 / pEC-XK99E-xylAB, PC0 / pEC-XK99E-P52-xylAB constructed in Example 1 and the starting strain PC0 were respectively mixed with a concentration of 10 μg·mL -1 Chloramphenicol or 10μg·mL -1 Chloramphenicol and 50 μg·mL -1 After the kanamycin-resistant BHI solid medium is streaked and activated, a single colony is picked and inoculated into the seed medium prepar...

Embodiment 3

[0073] Example 3: Shake flask fermentation production of 1,4-butanediamine by recombinant bacteria PC0 / pEC-XK99E-xylAB and PC0 / pEC-XK99E-P52-xylAB

[0074] (1) Preparation of culture medium:

[0075] Seed medium (g L -1 ): glucose 50, (NH 4 ) 2 SO 4 20, Yeast Powder 20, KH 2 PO 4 1.5, MgS0 4 ·7H 2 O1.0, MnS0 4 ·H 2 O 0.3, CaCO 3 1.0;

[0076] Fermentation medium (g L -1 ): carbon source 150, (NH 4 ) 2 SO 4 40, Yeast Powder 10, KH 2 PO 4 1.5, KCl 1.0, MgS0 4 ·7H 2 O 0.5, MnS0 4 ·H 2 O 0.02, FeS0 4 ·7H 2 O 0.02, CaCO 3 20.

[0077] Wherein: the carbon source is prepared by mixing xylose and glucose in a mass ratio of 1:1, 1:2, 2:1, 3:1, 3:2, 3:4, and 3:5, respectively. The concentration is 150g·L -1 mixed sugar as a carbon source.

[0078] (2) The recombinant bacteria PC0 / pEC-XK99E-xylAB and PC0 / pEC-XK99E-P52-xylAB constructed in Example 1 were respectively mixed with a concentration of 10 μg·mL -1 Chloramphenicol and 50 μg·mL -1 After the kana...

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Abstract

The invention discloses a method for producing 1, 4-butanediamine by fermenting xylose and xylose-containing hydrolysate, and belongs to the technical field of genetic engineering. According to the invention, a shuttle plasmid pEC-XK99E between Escherichia coli and Corynebacterium crenatum is adopted, and a xylose isomerase and xylulokinase coding gene cluster derived from Escherichia coli K-12 is expressed in a previously constructed 1, 4-butanediamine high-yield strain PC0. A shake flask fermentation result shows that the original bacterium PC0 does not have the capability of metabolizing and utilizing the xylose, but the recombinant corynebacterium crenatum has the capability, so that the 1, 4-butanediamine can be directly produced by directly utilizing the xylose and xylose-containing hydrolysate. And finally, fermenting in a 5-L fermentation tank for 72 hours to accumulate 33.4 g.L <-1 >, 4-butanediamine. The method for producing the 1, 4-butanediamine has the advantages of high efficiency and low content of byproducts of L-arginine, agmatine and other heteroacids, and the separation and purification of the product 1, 4-butanediamine in the later stage are easier.

Description

technical field [0001] The invention relates to a method for producing 1,4-butanediamine by utilizing xylose and a xylose-containing hydrolyzate by fermentation, and belongs to the technical field of genetic engineering. Background technique [0002] 1,4-Butanediamine (also known as 1,4-diaminobutane or putrescine) is a four-carbon compound containing two amino groups and is a kind of biological polyamine in nature. As a compound with various applications, 1,4-butanediamine plays an important role in agricultural and industrial fields such as polymers and medicines. [0003] At present, the industrial fermentation production of amino acids and derivatives mainly uses glucose as the substrate, but in order to reduce competition with other industrial applications, it is a promising production method to replace glucose with lower-value biomass raw materials for fermentation. However, in addition to being rich in glucose, various biomass hydrolyzates also contain a large amount...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12N15/61C12N15/60C12N15/54C12N15/55C12N9/12C12N9/78C12N9/88C12N9/90C12P13/00C12R1/15
CPCC12N9/90C12N9/1205C12N9/88C12N9/78C12N15/52C12N15/77C12P13/001C12Y503/01005C12Y207/01017C12Y401/01019C12Y305/03011Y02E50/10
Inventor 徐美娟饶志明杨凤玉宋云海杨套伟张显
Owner JIANGNAN UNIV
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