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Method for improving transport capacity and utilization capacity of levodextran in saccharomyces cerevisiae strain

A technology of Saccharomyces cerevisiae strain and levoglucosan, which is applied in the field of bioengineering to achieve the effect of increasing growth and levoglucosan utilization and improving transport capacity

Active Publication Date: 2021-09-10
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, after searching, it is found that there are no reports on the method for significantly improving the transport and utilization of levoglucosan in Saccharomyces cerevisiae strains and its special recombinant strains.

Method used

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  • Method for improving transport capacity and utilization capacity of levodextran in saccharomyces cerevisiae strain
  • Method for improving transport capacity and utilization capacity of levodextran in saccharomyces cerevisiae strain
  • Method for improving transport capacity and utilization capacity of levodextran in saccharomyces cerevisiae strain

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Construction of recombinant strain YLGR000 capable of expressing dehydroacetylmuramic acid kinase (AnmK) derived from Rhodotorula toruloides in Saccharomyces cerevisiae

[0032] A DNA sequence synthesis company was commissioned to synthesize a codon-optimized gene encoding AnmK (GenBank: CDR43051.1) derived from Rhodotorula toruloides. The specific sequence is shown in the sequence table SEQ ID NO: 1, and the NotI restriction site was brought into both sides of the gene during synthesis. The plasmid pIYC04 (Chen et al.2013) was linearized with the restriction endonuclease NotI, and then the linearized plasmid and gene fragment PG-Rho were assembled using GIBSON assembly technology to obtain the recombinant plasmid pIYC04-Rho. The structure of plasmid pIYC04-Rho is shown in figure 1 .

[0033] The recombinant plasmid pIYC04-Rho was transformed into Saccharomyces cerevisiae hexose transporter complete strain EBY.VW4000 (Wieczorke et al. 1999) by using lithium ...

Embodiment 2

[0034] Embodiment 2: clone GAL2, GAL2 Q341A , GAL2 W455A genes and construct their expression vectors

[0035] In this example, all the commercially available PCR Mix products are used for PCR, and only template and primer fragments need to be added. The time for DNA synthesis in each cycle of PCR is calculated according to the length of the fragment. For the specific calculation formula, refer to the product manual of PCR Mix. The product used in this example is 1000 bp per minute.

[0036] (1) Obtaining of gene fragment GAL2 and construction of its expression vector pJFE3-GAL2

[0037] The gene fragment GAL2 (Gene ID: 850770) encodes the Saccharomyces cerevisiae hexose transport protein Gal2p (GenBank: CAA97640.1).

[0038] Using the genomic DNA of Saccharomyces cerevisiae strain CEN.PK113-5D as a template, the gene fragment GAL2 was obtained with primers GAL2-1-up (SEQ ID NO: 2) and GAL2-2-dn (SEQ ID NO: 3), wherein the PCR annealing temperature It is 54°C. Wherein, th...

Embodiment 3

[0048] Example 3: Construction of recombinant Saccharomyces cerevisiae introduced into LGK and transporter

[0049] The expression vector pJFE3-GAL2, pJFE3-GAL2 Q341A and pJFE3-GAL2 W455A Saccharomyces cerevisiae strain YLGR000 with levoglucosan metabolic pathway was successively transformed by conventional lithium acetate complete cell transformation method, and cultured in SC-HIS-URA auxotrophic solid medium (1.7g / L yeast basic nitrogen source, 5g / L L ammonium sulfate, 0.77g / L CSM-HIS, 20g / L agar powder, adjust the pH to 6.0-6.5, add 20g / L maltose as carbon source) to screen transformants.

[0050] The correct transformants were named YLGR00G, YLGR341 and YLGR455 respectively.

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Abstract

The invention discloses a method for improving transport capacity and utilization capacity of levoglucosan in a saccharomyces cerevisiae strain. The method comprises the following steps of expressing protein with levoglucosan kinase activity in saccharomyces cerevisiae, and establishing a levoglucosan metabolic pathway in the saccharomyces cerevisiae; the method is realized by utilizing a mutant for expressing the transporter Gal2p in a hexose transporter complete-deficiency saccharomyces cerevisiae recombinant strain in which the levodextran metabolic pathway is established, meanwhile, the invention also provides a recombinant saccharomyces cerevisiae strain YLGR341 and a recombinant saccharomyces cerevisiae strain YLGR455 which are used for related expression of the levodextran transporter Gal2pQ341A or Gal2pW455A and the levodextran kinase, Experiments prove that when the method for improving the transport capacity and the utilization capacity of the levoglucosan in the saccharomyces cerevisiae strain is applied to levodextran fermentation, compared with a non-point-mutation strain, the growth of the Gal2p point-mutation strain has obvious advantages, the levodextran utilization capacity is obviously higher than that of the non-point-mutation strain, wherein the point mutation strains YLGR341 and YLGR455 are 3.70 times and 3.72 times of the non-point-mutation strain YLGR00G correspondingly.

Description

technical field [0001] The invention relates to a method for improving the transport ability and utilization ability of Saccharomyces cerevisiae strain levoglucosan and its special recombinant strain, belonging to the technical field of bioengineering. Background technique [0002] Lignocellulose is a natural and renewable resource with a wide variety of sources. The use of lignocellulose to produce fuels and chemicals can ensure energy security, reduce environmental pollution, and promote carbon neutrality. [0003] Lignocellulose is pretreated to open the cross-links between cellulose, hemicellulose, and lignin. The obtained cellulose fraction is usually hydrolyzed with cellulase to obtain glucose, which is then utilized. Alternatively, cellulose can also be pyrolyzed to obtain Levoglucosan (LG) through a rapid pyrolysis process (Jiang et al. 2019). There is a LG metabolic pathway in some microorganisms. Simply put, LG is catalyzed by levoglucan kinase (LGK) or dehydroac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/31C12N15/54C12N15/81C12R1/865
CPCC12N9/1205C12N15/81C07K14/395C12Y207/0117C12N2800/22Y02E50/10
Inventor 沈煜杨梦丹刘巍峰
Owner SHANDONG UNIV
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