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Recombinant filamentous fungus for producing ethanol from phosphofructokinase 2 mutant, construction of recombinant filamentous fungus and application of recombinant filamentous fungus in ethanol production

A phosphofructokinase, filamentous fungus technology, applied in the fields of genetic engineering and biology, can solve the problems of low ethanol yield and slow metabolism rate of filamentous fungi, and achieve the effects of wide practical application prospects, reduction of production costs, and outstanding application potential.

Pending Publication Date: 2022-03-01
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] To realize the one-step fermentation of filamentous fungal cellulose to ethanol, it is necessary to overcome the characteristics of low ethanol production and slow metabolic rate of filamentous fungi

Method used

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  • Recombinant filamentous fungus for producing ethanol from phosphofructokinase 2 mutant, construction of recombinant filamentous fungus and application of recombinant filamentous fungus in ethanol production
  • Recombinant filamentous fungus for producing ethanol from phosphofructokinase 2 mutant, construction of recombinant filamentous fungus and application of recombinant filamentous fungus in ethanol production
  • Recombinant filamentous fungus for producing ethanol from phosphofructokinase 2 mutant, construction of recombinant filamentous fungus and application of recombinant filamentous fungus in ethanol production

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Construction of sgRNA expression cassette and donor DNA for knocking out phosphofructokinase 2

[0032] First, in this example, the gene encoding phosphofructokinase 2 (phosphofructokinase2, PFK2, Mycth_71484) was knocked out, using the genome editing technology based on CRISPR / Cas9 (Qian Liu, et al. Development of a genome-editing CRISPR / Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase productionstrain engineering. Biotechnology for Biofuels, volume 10, Article number: 1 (2017)) to inactivate target genes. Wherein, the coding nucleotide sequence of PFK2 protein is shown in SEQ ID No.1. The amino acid sequence of the PFK2 protein is shown in SEQ ID No.2.

[0033] 1. Construction of sgRNA expression cassette

[0034] The protospacer of the target gene pfk2 (Mycth_71484) is designed by the software sgRNACas9 tool, which is the target site. The sequence sgRNA promoter, protospacer and scaffold were connected toge...

Embodiment 2

[0044] Construction of the phosphofructokinase 2 expression vector of embodiment 2 mutation

[0045]In this example, the mutation sites of phosphofructokinase 2 (PFK2, Mycth_71484) are: H233G, E306G, H371G. The mutation sites are selected based on their structural information. These three mutation sites are the key amino acids for the phosphatase activity of the enzyme. Through mutations, the phosphatase activity of phosphofructokinase 2 can be inactivated, while only the kinase activity is retained. The mutated nucleotide and amino acid sequences are shown in SEQ ID NO 6 and SEQ ID NO 7, respectively. Using the Myceliophthora thermophila genomic DNA as a template, the mutant pfk2 gene was amplified by fusion PCR. The specific operation is as follows: first, using genomic DNA as a template, four pairs of primers 71484mut-a and 71484mut-b, 71484mut-c and 71484mut-d, 71484mut-e and 71484mut-f, 71484mut-g and 71484mut-h were used to amplify The four fragments of the mutated pfk...

Embodiment 3

[0048] Example 3 Transformation DNA is introduced into Myceliophthora thermophila

[0049] Myceliophthora thermophila ATCC 42464 was inoculated in MM slant medium, and cultured at 45°C for 10 days before use. The formula of MM medium is: [50×Vogel’s salt 20mL, sucrose 20g, agar 15g, histidine (50mg / mL) 20mL, constant volume to 1L, autoclaved]. 50× Vogel’s salt (1L) recipe is: trisodium citrate (1 / 2H 2 O) 150g, anhydrous KH 2 PO 4 250g, anhydrous NH 4 NO 3 100g, MgSO 4 ·7H 2 O 10g, CaCl 2 2H 2 O 5g, trace element solution 5mL, biotin (0.1mg / mL) 2.5mL, constant volume to 1L. Trace element solution formula (100mL): 5g C 6 h 8 O·7H 2 O, 5g ZnSO 4 ·7H 2 O, 1g Fe(NH 4 ) 2 (SO 4 )·6H 2 O, 0.25g CuSO 4 ·5H 2 O, 0.05g MnSO 4 ·H 2 O, 0.05g H 3 BO 3 , 0.05g NaMoO 4 2H 2 O, dissolved in water, dilute to 100mL,

[0050] 1. Transformation of Myceliophthora thermophila protoplasts

[0051] 1) Mycelium preparation

[0052] Mature Myceliophthora thermophila spore...

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Abstract

The invention discloses a phosphofructokinase 2 mutation and / or knockout recombinant filamentous fungus for ethanol production and construction and ethanol production application thereof, which is characterized in that an endogenous phosphofructokinase 2 gene is knocked out and / or a mutated phosphofructokinase 2 gene is expressed in the filamentous fungus; wherein the mutated phosphofructokinase 2 means that only kinase activity is retained and phosphatase activity is lost or reduced after mutation. Compared with the original strain, the obtained genetic engineering strain has the advantages that the ethanol synthesis capability is improved, and the glucose metabolism rate is accelerated.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biotechnology. Specifically, the present invention relates to a recombinant filamentous fungus capable of producing ethanol by knocking out or mutating the phosphofructokinase 2 gene and its construction and ethanol-producing application. The invention also relates to a method for producing ethanol by said recombinant engineered bacterium, and an application for accelerating glucose metabolism rate. Background technique [0002] With the rapid development of the world economy, human beings' demand for energy is increasing day by day. On the one hand, fossil energy such as oil currently used has limited reserves and produces a lot of pollution during the combustion process. Energy and environmental issues have become a major challenge to human development. The transportation industry is a major sector of energy consumption and an important source of greenhouse gas emissions, so there is an...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/90C12N9/12C12P7/06C12P7/10C12R1/645
CPCC12N15/80C12N15/902C12N9/1205C12Y207/01105C12P7/065C12P7/10Y02E50/10
Inventor 田朝光张永利李金根
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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