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Photosynthetic microorganism as well as application and plasmid thereof

A technology of photosynthetic microorganisms and plasmids, applied in the biological field, can solve the problems of high production costs and achieve the effect of reducing the cost of substrates

Active Publication Date: 2022-07-05
上海光玥生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these studies have successfully introduced the synthesis pathway of PLA or its copolymers in microorganisms, the production process will emit greenhouse gases, and need to add a large amount of carbohydrates or expensive precursor substances, the production cost is high, and there is still a long way to go before industrial applications. a long distance
Therefore, it remains a great challenge to seek economically sustainable green production methods of PLA and its copolymers.

Method used

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  • Photosynthetic microorganism as well as application and plasmid thereof
  • Photosynthetic microorganism as well as application and plasmid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Construction of expression plasmid pSyn- pct - phaC - ldh :

[0046] (1) Extract the pSyn_6 plasmid:

[0047] Escherichia coli DH5α containing the pSyn_6 plasmid was inoculated into 5 mL of LB liquid medium containing 100 mg / l spectinomycin according to 2% inoculum, at 37 ° C incubator for 12 h. The cultured cells were extracted with a common plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) according to the method described in the manual.

[0048] (2) Synthesis of propionyl-CoA transferase gene ( pct ) fragment, polyhydroxyalkanoate synthase gene ( phaC ) fragment and the d-lactate dehydrogenase gene ( ldh ) fragment:

[0049] Using the online software JCat, Clostridium propionicum ( Clostridium propionicum ) encoding genes for PCT pct Perform codon optimization, after optimization pct The nucleotide sequence of Pseudomonas is shown in SEQ ID NO: 1; Pseudomonas ( Pseudomonas sp.) encoding gene for PhaC phaC Perform codon optimiza...

Embodiment 2

[0063] Construction of expression plasmid pSyn- pct - phaC - ldh - pdhA - acs :

[0064] (1) Amplification of the pyruvate dehydrogenase gene ( pdhA ) fragment and the acetyl-CoA synthase gene ( acs ) fragment:

[0065]The genomic DNA of Synechococcus PCC7942 was prepared by a conventional method. For this process, please refer to the micro-preparation method of bacterial genome in "Guide to Molecular Biology" published by Science Press;

[0066] Using the Synechococcus PCC7942 genome as a template, PCR amplification was performed according to the method described in the "Experimental Guide for Molecular Cloning (Third Edition)".

[0067] PCR primers: pdhA Gene upstream primer pdhA -F: 5'-gagcgtcagatctcatatggttcaggaacgtacactgc-3', downstream primer pdhA -R:5'-cattctagattctcctctttaatttagtcttctgcccagatgtagc-3'; acs Gene upstream primer acs -F: 5′-taaattaaagaggagaatctagaatgagccagccaacgatcg-3′, downstream primer acs -R: 5'-gggtttgcctggtaccgcggttaagtgccttg...

Embodiment 3

[0078] Construction of expression plasmid pSyn- pct - phaC - ldh - phaAB and pSyn- pct - phaC - ldh - phaAB - pdhA - acs :

[0079] (1) Synthesis of acetoacetyl-CoA reductase gene and β-ketothiolase gene fragments ( phaAB ):

[0080] Using the online software JCat, according to the codon preference of Synechococcus PCC7942, the hookworm copper greedy bacteria ( Cupriavidus necator ) encoding gene for PhaB phaB Perform codon optimization, after optimization phaB The nucleotide sequence is shown in SEQ ID NO: 6; Cupriavidus necator )middle phaA the coding gene phaA Perform codon optimization, after optimization phaA The nucleotide sequence is shown in SEQ ID NO:7. Will phaA and phaB 序列相连,并在前端加上psbA2序列5′-ttaaggttggctaaaagcttggctggctatttagcgtcttctaatccagtgtagacagtagttttggctccgttgagcactgtagccttgggcgatcgctctaaacattacataaattcacaaaagttttcgttacataaaaatagtgtctacttagctaaaaattaagggttttttacacctttttgacagttaatctcctagcctaaaaagcaagagtttttaactaagactcttgccc...

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Abstract

The invention discloses a photosynthetic microorganism and application and plasmid thereof, the photosynthetic microorganism contains a plurality of exogenous genes, and the exogenous genes comprise a coding gene of propionyl-coenzyme A transferase, a coding gene of polyhydroxyalkanoate synthase and a coding gene of d-lactic dehydrogenase. The genetic engineering blue algae provided by the invention can be used for efficiently producing polylactic acid (PLA) and lactic acid copolymers by directly utilizing greenhouse gas CO2 through photosynthesis, so that the consumption of carbohydrates and expensive precursors and the use of inducers are avoided. The invention also provides application of the genetic engineering cyanobacteria, especially application of the genetic engineering cyanobacteria in production of PLA and copolymers, a new technology is developed for production of degradable plastics, the production cost is reduced, and the genetic engineering cyanobacteria has an important application prospect.

Description

technical field [0001] The invention relates to a kind of genetic engineering modification of photosynthetic microorganisms, so as to directly utilize CO through photosynthesis 2 The technology for efficiently producing polylactic acid and lactic acid copolymer belongs to the field of biotechnology. Background technique [0002] Traditional plastics mainly use petroleum as raw materials, and generate polymer state compounds through polymerization reactions, which are widely used in industrial production and daily life. However, traditional plastics are almost difficult to degrade, and their extensive use has caused "white pollution" that is extremely harmful to the ecological environment and the oil crisis. Polylactic acid (PLA) is a degradable plastic made from renewable resources through fermentation, saccharification and polymerization. This "originated from nature, attributed to nature" material can meet the needs of sustainable development, can replace traditional pet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/13C12N15/74C12N15/54C12N15/53C12N15/52C12P7/625C12R1/01
CPCC12N15/52C12N9/0006C12N9/0008C12N9/1029C12N9/93C12N9/13C12N15/74C12P7/625C12Y101/01028C12Y101/01036C12Y102/01075C12Y203/01009C12Y602/01017C12Y208/03001C12Y102/01051C12Y102/04001C12Y102/05001C12Y602/01001C12N2800/22
Inventor 邵慧阎冬郑集杨
Owner 上海光玥生物科技有限公司
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