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Method for producing fucoxanthin by using mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum

A technology of pyruvate phosphate and Phaeodactylum, applied in the field of bioengineering, can solve the problem of low fucoxanthin production

Active Publication Date: 2021-04-23
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the problem of low fucoxanthin yield in the existing large-scale cultivation of Phaeodactylum tricornutum, the purpose of the present invention is to use genetic transformation technology to convert exogenous C 4 CyPPDK of the pathway was overexpressed in Phaeodactylum tricornutum, and by regulating the phosphorylation site optimization of the enzyme activity, the level of enzyme catalysis was improved, the efficiency of cellular carbon fixation was promoted, and the accumulation of unsaturated fatty acids and fucoxanthin was increased.

Method used

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  • Method for producing fucoxanthin by using mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum
  • Method for producing fucoxanthin by using mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum
  • Method for producing fucoxanthin by using mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Using NetPhos to predict specific phosphorylation sites of exogenous Nannochloropsis sp. PPDK protein (GenBank: EWM23135.1), references Blom N, Gammeltoft S, BrunakS. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. [J]. Journal of Molecular Biology, 1999, 294(5): 1351-62. In the analysis results, 79(T); 93(S); 165(T); 190(S); 193(S); 246(S); 251(T); 256(T); 290 in the protein sequence (T);312(T);334(T);336(T);340(T);386(T);482(T);531(S);550(S);609(T);652 (S); 669(T); 734(T); 741(T); 815(T); 876(T); 880(S) is the main phosphorylation site, 290(T); (T); 334 (T); 531 (S); 550 (S); 652 (S); grooming. We commissioned a gene synthesis company to construct a PPDK phosphorylation site mutant, changed the serine (S) at position 652 to alanine (A), optimized the codon suitable for Phaeodactylum tricornutum, and confirmed the mutation by DNA sequencing base position.

[0061] The construction method of the CyPPDK transformant, and the expr...

Embodiment 2

[0111] The difference from the culture conditions in step (4) in Example 1 is that a high temperature period of 37°C is set for 4 to 6 h / d, and the growth conditions of OP-CyPPDK, UN-CyPPDK and wild-type are determined under the same conditions as other culture conditions. EPA and fucoxanthin content, the results show that the growth of algal strains under high temperature conditions is generally worse than that of algal strains cultivated under normal conditions, and the content of EPA and fucoxanthin is relatively low, as shown in Image 6 , 7 , 8 shown.

[0112] Effect example

[0113] The growth situation of OP-CyPPDK, UN-CyPPDK and wild type measured in Example 1 and Example 2, the detection data of EPA and fucoxanthin content are shown in Table 8 and Table 9 respectively

[0114] Table 8 is each index under the culture condition of embodiment 1

[0115]

[0116] Table 9 is each index under the culture condition of embodiment 2

[0117]

[0118] Codon optimizati...

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Abstract

The invention provides a method for producing fucoxanthin by using a mutant strain for expressing exogenous pyruvate phosphate dikinase in phaeodactylum tricornutum, and particularly relates to algae preference codon optimization on an original CyPPDK gene to obtain a new CyPPDK gene with a nucleotide sequence as shown in SEQ ID NO.7; the constructed shuttle expression vector is named as pPha-fcp-PPDK; and the obtained new gene is introduced into a host algae cell phaeodactylum tricornutum cell in an electric transformation manner, and a transformant is screened to obtain a phaeodactylum tricornutum transgenic algae strain for overexpression of pyruvate phosphate dikinase. Compared with an unoptimized transformant, the optimized transformant for phosphorylation sites of the CyPPDK gene has the advantages that the biomass accumulation is improved by 33.3%, the content of EPA in grease is improved by 11.5%, and the fucoxanthin yield is improved by 21.2%.

Description

technical field [0001] The invention relates to a method for producing fucoxanthin by expressing an overexpression mutant strain of exogenous pyruvate phosphate dikinase in Phaeodactylum tricornutum, belonging to the technical field of bioengineering. Background technique [0002] Phaeodactylum tricornutum belongs to Diatomphyta, Featheria, Phaeodactylum, and Phaeodactylum genus. It is an important aquatic bait, rich in active substances such as EPA (Eicosapentaenoic Acid) and fucoxanthin (Fucoxanthin). Fucoxanthin, also known as fucoxanthin and fucoxanthin, is an oxygen-containing derivative of carotene, which has an allene bond, an epoxide and a conjugated carbonyl group on its molecular polyene chain group, which is a partial polar carotenoid. Fucoxanthin has a variety of biological activities. Taking fucoxanthin can speed up metabolism, increase the metabolism of triglyceride and cholesterol, and has pharmacological effects such as anti-inflammatory, anti-tumor, anti-o...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/82C12N15/65C12N9/12C12N1/13C12P17/02C12P7/64C12R1/89
CPCC12N9/1229C12N15/65C12N15/8243C12N15/8247C12N2800/22C12P7/6427C12P17/02C12Y207/04027
Inventor 范勇李福利姜尔颖丁晓婷师晓艺
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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