Fermentation method for improving porcine myoglobin produced by pichia pastoris engineering bacteria

A technology of myoglobin and Pichia pastoris, which is applied in the field of bioengineering, can solve the problems of expensive fermentation substrates and low yield of porcine myoglobin, and achieve the effects of high-efficiency expression and optimization of dissolved oxygen conditions.

Active Publication Date: 2021-07-30
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the production of porcine myoglobin using a constitutive expression system has problems such as low yield and expensive

Method used

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  • Fermentation method for improving porcine myoglobin produced by pichia pastoris engineering bacteria
  • Fermentation method for improving porcine myoglobin produced by pichia pastoris engineering bacteria
  • Fermentation method for improving porcine myoglobin produced by pichia pastoris engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Construction of Pichia pastoris recombinant bacteria expressing porcine myoglobin

[0031] (1) Construction of P. pastoris X33-αGAP-Mb strain

[0032] The porcine myoglobin gene (nucleotide sequence shown in SEQ ID NO: 1) was connected to the multiple cloning site of the integrated expression vector pGAPZαA to construct the recombinant plasmid pGAPZα-A-Mb.

[0033] Transform the constructed recombinant plasmid pGAPZα-A-Mb into Escherichia coli DH5α, spread the transformation solution on an LB plate containing 20 μg / mL Zeocin, and grow a single clone at 37°C, and colonize the single clone by PCR After verification and sequencing, plasmids were extracted from the correct positive clones, and the extracted plasmids were transformed into Pichia pastoris X33 by electroporation to construct recombinant bacteria P. pastoris X33-αGAP-Mb.

[0034] (2) Construction of Pichia pastoris X33-αG1-Mb strain

[0035] Synthesize the G1 promoter sequence (the nucleotide seque...

Embodiment 2

[0037] Embodiment 2 Shake flask horizontal fermentation medium and optimization of fermentation conditions

[0038] 1. Preparation of primary seed liquid: Streak the strain Pichia pastorisX33-αGAP-Mb constructed in Example 1 stored at -80°C on a plate, pick a single colony and inoculate it in a 50 mL sterile shaker containing 5 mL of YPD medium In the bacterial tube, 30°C, 220rpm, shaker culture for 16-18h is the first-class seed solution.

[0039] 2. Preparation of the secondary seed solution: inoculate the primary seed solution into a 250mL shaker flask containing 50mL of YPD medium at an inoculum size of 1%, culture at 30°C, 220rpm, on a shaker for 22h, and cultivate to OD 600 =8~10.

[0040] 3. Fermentation conditions:

[0041] (1) Fermentation of genetically engineered bacteria in different media to produce porcine myoglobin

[0042] Secondary seed liquid is inoculated in the 250mL shaking flask that contains 49mL fermentation medium (respectively YPG, BMGY, YPD medium...

Embodiment 3

[0055] Embodiment 3 produces porcine myoglobin (fermenter level) under different dissolved oxygen conditions

[0056] 1. Preparation of primary seed liquid: Streak the strain Pichia pastorisX33-αGAP-Mb constructed in Example 1 stored at -80°C on a plate, pick a single colony and inoculate it in a 50 mL sterile shaker containing 5 mL of YPD medium In the bacterial tube, 30°C, 220rpm, shaker culture for 16-18h is the first-class seed solution.

[0057] 2. Preparation of the secondary seed solution: inoculate the primary seed solution into a 250mL shake flask containing 50mL of YPD medium at an inoculum size of 1% (1mL / 100mL), culture at 30°C, 220rpm, on a shaking table for 22h, and cultivate to OD 600 =8~10.

[0058] 3. Fermentation conditions: Inoculate the secondary seed liquid into a 5L fermenter containing 1.8L fermentation medium (the fermentation medium is BMGY) with an inoculum size of 10% (10mL / 100mL). The fermentation medium contains 10g / L glycerol and hemoglobin at a...

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Abstract

The invention discloses a fermentation method for improving porcine myoglobin produced by pichia pastoris engineering bacteria, and belongs to the technical field of bioengineering. According to the method disclosed by the invention, porcine myoglobin-producing pichia pastoris engineering Pichia pastoris X33-GAP-Mb is taken as an original strain, and a culture medium and culture conditions are preliminarily optimized at a shake flask level. The Pichia pastoris X33-GAP-Mb and Pichia pastoris X33-G1-Mb are adopted as starting strains, fermentation condition optimization is performed at the fermentation tank level; it is found that when the DO is 30% and the heme fed-batch concentration is 150 mg/L, the pig myoglobin yield can be significantly increased, and can achieve 285.42 mg/L. The biological manufacturing of the porcine myoglobin has the advantages of low pollution, high product quality and the like, has a development prospect, and has important application significance on the industrial production of the porcine myoglobin.

Description

technical field [0001] The invention relates to a fermentation method for improving the production of porcine myoglobin by Pichia pastoris engineering bacteria, and belongs to the technical field of bioengineering. Background technique [0002] Myoglobin is a protein that exists in animal muscle cells. It has important physiological functions such as iron supplementation, oxygen transport, and respiration in organisms, and can give muscle tissue a bright red color. In recent years, with the development of artificial meat technology, the addition of myoglobin can make artificial meat products simulate the color of real meat. [0003] At present, porcine myoglobin is mainly extracted from myocardial tissue. This method has problems such as high cost, long period, low yield, complex process and many by-products which are not conducive to separation. However, the method of heterologously synthesizing porcine myoglobin using microbial cell factories has not yet been reported, a...

Claims

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Application Information

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IPC IPC(8): C12N1/38C12N1/16C12P21/02C12R1/84
CPCC12N1/38C12N1/16C07K14/805
Inventor 赵鑫锐张博涵王紫微周景文堵国成李江华陈坚钱源
Owner JIANGNAN UNIV
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