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Constructs, strains and methods for producing carbohydrates using Synechococcus utex2973

A technology of compounds and microbial strains, applied in the field of biomanufacturing, can solve the problems of high cost of lignin and cellulose degradation and transformation, no cost advantage, etc., and achieve the effect of increasing carbon emissions

Active Publication Date: 2021-02-05
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high cost of degradation and conversion of lignin and cellulose in plant cell walls, fermentation products made from cellulose have no cost advantage compared with petrochemical products (Ziolkowska 2014)

Method used

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  • Constructs, strains and methods for producing carbohydrates using Synechococcus utex2973
  • Constructs, strains and methods for producing carbohydrates using Synechococcus utex2973
  • Constructs, strains and methods for producing carbohydrates using Synechococcus utex2973

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Production of glycogen with Synechococcus UTEX2973

[0060] 1. Experimental steps

[0061] (1) Cultivate Synechococcus sp. UTEX2973 and PCC7942 wild-type algae strains in a column photoreactor: ordinary glass tube, column height 580mm, diameter 30mm, liquid volume 150mL (can hold about 300mL). Initial inoculum concentration OD 730 0.05, the culture medium is BG11 medium (BG11 medium composition is 1.5g / L NaNO 3 ,40mg / L K 2 HPO 4 ·3H 2 O,36mg / L CaCl 2 2H 2 O, 6mg / L citric acid, 6mg / L ferric ammonium citrate, 1mg / L EDTA disodium salt, 20mg / L NaCO 3 ,2.9mg / L H 3 BO 3 ,1.8mg / L MnCl 2 4H 2 O,0.22mg / L ZnSO 4 ·7H 2 O,0.39mg / L NaMoO 4 2H 2 O,0.079mg / LCuSO 4 ·5H 2 O and 0.01mg / L CoCl 2 ·6H 2 O); at 38°C, 250μE / m 2 / s light, 3% CO 2 Air for cultivation. During the incubation, samples were taken to test the OD 730 and glycogen content. And cyanobacteria biomass (Dry cell weight, DCW, cell dry weight) content is by 1ml OD 730 The algal cells of =...

Embodiment 2

[0065] Example 2: Construction of vector pSK015 for introduction into Synechococcus UTEX2973

[0066] Using NS3-F (5'-TCAGCCAGCTCGTCGTGATGTCGAAACCCAAGCC-3') and NS3-R (5'-ACAGTCGGCGTCACGGCAACTTGCTCCGCGTAGG-3') as a primer pair, PCR amplification was performed using plasmid pNS3-3B with cscB (Ducat et al.2012) as a template; And the PCR product was connected with the pBR322 vector that was digested with EcoRI / SalI and filled in with T4 DNA polymerase, thereby obtaining the recombinant plasmid pSK015 ( figure 2 ).

[0067] Conditions and systems for PCR amplification:

[0068] 1. PCR amplification system

[0069]10×amplification buffer: 5 μL, 4 kinds of dNTP mixture (2.5 mM): 4 μL, NS3-F (20 mM): 0.5 μL, NS3-R (20 mM): 0.5 μL, plasmid template DNA (100 ng / μl): 0.5 μL, Fast Pfu Fly DNA polymerization: 1 μL, add double distilled water to 50 μL;

[0070] 2. PCR amplification conditions:

[0071] 95°C for 5min, 95°C for 30s, 55°C for 30s, 72°C for 3min (return to the second st...

Embodiment 3

[0072] Example 3: Conjugative transfer of Synechococcus sp. UTEX2973 and screening of zygotes

[0073] 1. Take 1 mL of the logarithmic growth phase (OD 730 About 1.0) Synechococcus UTEX2973 culture, centrifuge at 5000 rpm for 5 minutes to collect the cells; wash the cells with fresh BG11 medium, and then resuspend the cells in 100 μL of BG11 medium.

[0074] 2. Cultivate Escherichia coli HB101 strains containing pRL443 plasmid and pRL623 plasmid, and Escherichia coli HB101 containing pSK015 plasmid in LB medium (10g / L peptone, 10g / L sodium chloride, 5g / L yeast extract) Two kinds of bacterial strains of bacterial strain, bacterial strain is cultivated to logarithmic growth phase (OD 600 about 1.0); take 1 mL of Escherichia coli culture, and centrifuge at 5000 rpm for 5 minutes to collect the cells; wash the cells with fresh LB medium, and resuspend the cells in 100 μL LB medium respectively.

[0075] 3. Mix the Escherichia coli HB101 strain containing the pRL443 plasmid and t...

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Abstract

The invention relates to the bio-manufacturing field and particularly relates to a construction body for producing carbohydrate compound by virtue of synechococcus UTEX2973, a strain and a method. The construction body contains a promoter, a proton / saccharose synergetic transport protein gene, an antibiotic resistance gene and a homologous arm, wherein the promoter has activity in synechococcus UTEX2973, the proton / saccharose synergetic transport protein gene is controlled by the promoter, the antibiotic resistance gene is used for screening a transformant, and the homologous arm is used for homologously integrating genomes. The carbohydrate compound is synthesized by immobilizing carbon dioxide by virtue of solar energy in a photosynthetic microorganism, namely cyanobacteria, the energy for synthesizing carbohydrates is from the solar energy, and a carbon source is from carbon dioxide. Therefore, a biology base product prepared by virtue of the method is not restricted by insufficient raw materials, the carbon emission is not increased during the use of the biology base product, and the biology base product is a true zero-emission biology base product.

Description

technical field [0001] The present invention relates to the field of biomanufacturing. Specifically, it is a construct, bacterial strain and method for producing carbohydrate compounds using Synechococcus sp. UTEX2973. Background technique [0002] The rapid development of metabolic engineering and synthetic biology has made it possible for metabolically engineered heterotrophic microorganisms, such as E. base chemicals (Hara et al. 2014). Compared with the existing chemical production methods based on petrochemical raw materials, microbial fermentation production methods based on sugar raw materials are more environmentally friendly and sustainable, and have significant application potential. Although the production of chemicals by microbial fermentation has received extensive attention and development, most of the products have high production costs, and only about 30 bio-based chemicals have been commercially produced (E4tech et al. 2015). The economic viability of bio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N1/21C12P19/12C12P19/04C12R1/01
CPCC12N15/74C12N2800/101C12P19/04C12P19/12Y02P20/133Y02P20/59
Inventor 吕雪峰宋阔谈晓明
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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