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A method for increasing the density of fermented cells by using hemoglobin

A hemoglobin and cell technology, applied in the biological field, can solve the problems that are not conducive to the high-density growth of cells and the decrease of oxygen utilization efficiency, so as to achieve the effect of increasing the growth quantity and density, increasing the utilization rate, and reducing losses

Active Publication Date: 2019-02-05
北京微构工场生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because hemoglobin is in the cytoplasm, the outer membrane, cell wall and cell membrane fully separate hemoglobin from oxygen, which limits the contact between hemoglobin and oxygen, and the oxygen utilization efficiency decreases, which is not conducive to the high-density growth of cells.

Method used

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  • A method for increasing the density of fermented cells by using hemoglobin
  • A method for increasing the density of fermented cells by using hemoglobin
  • A method for increasing the density of fermented cells by using hemoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Embodiment 1, increase fermentation unit cell density by intracellular expression of VHb hemoglobin gene

[0091] 1. Construction of engineering bacteria

[0092] 1. Construction of expression vector pSEVA331-Pporin+vgb

[0093] 1), the acquisition of the target promoter Pporin

[0094] Extract the genome of Halomonas TD01 (Halomonas TD01, now preserved in the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, preservation number CGMCC4353; patent publication number CN102816729A) as a template, with G-Pporin-1TF and G-Pporin-2TR as Primers, PCR amplification with pfu enzyme.

[0095] Primers are:

[0096] G-Pporin-1TF: 5'gataacaatttcacacaggacggccgctgagacctgccag 3'

[0097] G-Pporin-2TR: 5' ctcctctttctctagtaaagtctgcagctggcttgc 3'

[0098] PCR reaction conditions:

[0099] Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and ...

Embodiment 2

[0159] Example 2, through the expression of the VHb hemoglobin gene in the periplasmic space to increase the cell density of the fermentation unit

[0160] 1. Construction of engineering bacteria

[0161] 1. Construction of expression vector pSEVA331-Pporin+Tat-vgb

[0162] 1), the acquisition of the target gene Pporin

[0163] The genome of Halomonas TD01 (Halomonas TD01) was extracted as a template, G-Pporin-1TF and G-Pporin-2TR were used as primers, and PCR amplification was performed with Pfu enzyme.

[0164] Primers are:

[0165] G-Pporin-1TF: 5'gataacaatttcacacaggacggccgctgagacctgccag 3'

[0166] G-Pporin-2TR: 5' ctcctctttctctagtaaagtctgcagctggcttgc 3'

[0167] The PCR product of 218bp was obtained, which was the target gene Pporin, and its nucleotide sequence was the 33rd-250th position from the 5' end of sequence 2 in the sequence listing.

[0168] 2), the acquisition of the target gene tat

[0169] The genome of Escherichia coli K-12 line JM109SG was extracted a...

Embodiment 3

[0357] Example 3, Increase the cell density of the fermentation unit by displaying hemoglobin on the cell surface

[0358] 1. Construction of engineering bacteria

[0359] 1. Construction of expression vector pSEVA331-Pporin Tat-vgb-Ag43

[0360] 1), the acquisition of the target gene Pporin

[0361] The genome of Halomonas TD01 (Halomonas TD01) was extracted as a template, G-Pporin-1TF and G-Pporin-2TR were used as primers, and pfu enzyme was used for PCR amplification.

[0362] Primers are:

[0363] G-Pporin-1TF: 5'gataacaatttcacacaggacggccgctgagacctgccag 3'

[0364] G-Pporin-2TR: 5' ctcctctttctctagtaaagtctgcagctggcttgc 3'

[0365] The PCR product of 218bp was obtained, which was the target gene Pporin, and its nucleotide sequence was the 33rd-250th position from the 5' end of sequence 7 in the sequence listing.

[0366] 2), the acquisition of the target gene tat

[0367] The genome of Escherichia coli K-12 line JM109SG was extracted as a template, G-tat-1TF and G-tat-...

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Abstract

The invention discloses a method of improving fermentation cell density using hemoglobin and provides a method of improving microorganism fermentation cell density. According to the method, by expressing hemoglobin in periplasmic space or displaying it by cell surface display, and by rebuilding cell outer membranes, contact of the hemoglobin with oxygen is facilitated, thus improving oxygen utilization and improving microorganism fermentation density. Experiments show that an engineering bacterium built by the invention can increase microorganism fermentation cell density and content of intracellular accumulated cells PHB.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for increasing the density of fermented cells by using hemoglobin. Background technique [0002] High-density fermentation technology has always been an efficient technology required in industrial production. For many aerobic microorganisms, the most important factor limiting cell density during fermentation is the availability of oxygen. Therefore, it is necessary to develop a variety of technologies aimed at improving the oxygen utilization rate of aerobic microorganisms. [0003] Currently known methods for increasing cell density are mainly divided into two categories, one of which is to change the fermentation conditions of microorganisms to improve the utilization of oxygen. For example, during the fermentation process, increase the ventilation in the fermenter, increase the pressure of the fermenter, increase the rotation speed of the stirring paddle in the fermenter, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/75C12N15/78C12N15/74C12P7/62C12P21/02C12P13/00C12R1/19C12R1/125
Inventor 陈国强欧阳鹏飞郭瑛瑛毅万李天兰陆红
Owner 北京微构工场生物技术有限公司
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