Synechocystis genetically engineered bacterium for biosynthesizing inositol as well as construction method and application thereof

A genetically engineered bacteria and biosynthetic technology, applied in the field of Synechocystis genetically engineered bacteria and its construction for biosynthesizing inositol

Active Publication Date: 2020-08-21
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Synechocystis genetically engineered bacterium for biosynthesizing inositol as well as construction method and application thereof
  • Synechocystis genetically engineered bacterium for biosynthesizing inositol as well as construction method and application thereof
  • Synechocystis genetically engineered bacterium for biosynthesizing inositol as well as construction method and application thereof

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Experimental program
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Embodiment 1

[0037] Construction of strain WT-INO1SS:

[0038] (1) Using the whole genome of Saccharomyces cerevisiae BY4741 as a template, using the nucleotide sequences shown in SEQ ID NO.10 and SEQ ID NO.11 in the sequence listing as upstream and downstream primers, amplified to obtain inositol- 1-monophosphatase gene INO1, the nucleotide sequence of the INO1 gene is shown in SEQ ID NO.1;

[0039] (2) Using the nucleotide sequences shown in SEQ ID NO.12 and SEQ ID NO.13 in the sequence listing as upstream and downstream primers, using the plasmid pCP3031 containing the cpc560 promoter and rbcL terminator as a template, amplified to obtain the third Backbone fragment, the third skeleton fragment and the INO1 gene fragment obtained in step (1) are bluntly connected to construct the recombinant expression vector p3031I, with the nucleotides shown in SEQ ID NO.14 and SEQ ID NO.9 in the sequence listing Sequences were verified for upstream and downstream primers.

[0040] (3) Use the nucle...

Embodiment 2

[0048] Construction of strain WT-INO1SSZP.

[0049] (1) Using the nucleotide sequences shown in SEQ ID NO.23 and SEQ ID NO.24 in the sequence listing as upstream and downstream primers, and using the plasmid pBA3031-HM(the*) as a template, perform PCR reverse amplification to obtain The first skeleton fragment, the first skeleton fragment is self-ligated to obtain the recombinant expression vector P3;

[0050] Using the nucleotide sequences shown in SEQ ID NO.25 and SEQ ID NO.24 as upstream and downstream primers, and using the plasmid pBA3031-HM(the*) as a template, PCR reverse amplification was performed to obtain the second backbone fragment, the The two backbone fragments were self-ligated to obtain the recombinant expression vector P4.

[0051] (2) Use the nucleotide sequences shown in SEQ ID NO.8 and SEQ ID NO.9 in the sequence table as upstream and downstream primers, and use the recombinant expression vector P3 as a template to perform PCR amplification to obtain Ppsb...

Embodiment 3

[0058] Optimization of Glucose Supplement in Culture Conditions of Genetically Engineered Bacteria WT-INO1SSZP

[0059] Inoculate the genetically engineered bacteria WT-INO1SSZP in a 100mL shake flask (initial OD of bacterial solution 730 nm=0.1, the medium volume after inoculation is 25mL), at 150rpm, 30℃, 50μmol photons m -2 the s -1 Under the conditions, 5mM glucose was added to the medium every 24h. After culturing the WT-INO1SSZP strain for three days, all the cells were centrifuged at 4°C and 3000×g for 10 min to collect the cells, and the collected cells were resuspended in fresh BG-11 liquid medium with a final concentration of 2 mM theophylline. The resuspended cell culture was continued for 5 days, and then the inositol production was measured. After testing, it was found that WT-INO1SSZP was optimized by adding glucose, and the production of inositol reached 21.74mg L after 8 days of culture -1 .

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Abstract

The invention discloses synechocystis genetically engineered bacterium for biosynthesizing inositol as well as a construction method and application of the synechocystis genetically engineered bacterium. The construction method comprises the following steps: obtaining an inositol-1-phosphate synthase gene from saccharomyces cerevisiae, integrating the inositol-1-phosphate synthase gene and an inositol-1-monophosphase gene obtained from synechocystis 6803 onto a plasmid pCP3031, and constructing p3031S to obtain the recombinant synechocystis 6803 engineering bacterium WT-INO1SS. The recombinantsynechocystis 6803 engineering bacterium WT-INO1SSZP is obtained by taking plasmid pBA3031-HM (the *) as a template, constructing P0168-ZP, and transferring the P0168-ZP into the engineering bacterium WT-INO1SS. Experiments prove that the yield of final inositol of the genetically engineered bacterium is 21.74 mg/L <-1 >. Biosynthesis of inositol is successfully realized in synechocystis 6803, and the blank that no cyanobacteria is used as a chassis for producing inositol at present is filled up. The important significance is realized on the production of inositol by utilizing photosyntheticmicroorganisms.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to a Synechocystis genetically engineered bacterium for biosynthesizing inositol, a construction method and application thereof. Background technique [0002] Inositol (cis-1, 2, 3, 5-trans-4, 6-cyclohexanehexol), also known as cyclohexanehexanol, is an indispensable growth factor, which has so far been found in mammalian cells, higher Found in plants, fungi and some bacteria. It has nine isomers, of which 7 are non-optical and 2 are optically active. Among them, myo-inositol (hereinafter referred to as MI) and its derivatives are the most abundant in nature, and thus have attracted widespread attention. The molecular formula of inositol is C 6 h 12 o 6 , the molecular weight is 180.16, easily soluble in water. Inositol plays an important role in the regulation of ion channel permeability, phosphate levels, cellular metabolic flux, transcription and transla...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12P7/18C12R1/01
CPCC12N9/16C12N15/74C12P7/18
Inventor 孙韬王晓帅陈磊张卫文
Owner TIANJIN UNIV
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