CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Application No. PCT/CN2018/081272, filed Mar. 30, 2018, which was published in the Chinese language on Oct. 4, 2018, under International Publication No. WO 2018/177402 A1, which claims priority under 35 U.S.C. § 119(b) to Chinese Application Nos. 201710203473.5, filed Mar. 30, 2017 and 201710741963.0, filed Aug. 25, 2017, the disclosures of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “065824_4US1_Sequence_Listing” having a creating date of Jul. 27, 2021 and having a size of 69.8 kb. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the field of genetic engineering, and to the high-efficiency expression of phytase derived from Gram-negative bacteria, particularlyEscherichia coliphytase, in filamentous fungi, especiallyAspergillus niger.

BACKGROUND

Phytase, that is, myo-Inositol hexakisphosphate phosphohydrolase, is an orthophosphoric-monoester phosphohydrolase, which catalyzes the hydrolysis of phytic acid to produce lower inositol phosphate derivatives and inorganic phosphoric acid. In some instances, phytic acid can be hydrolyzed into free inositol. Phytic acid is the most abundant in the seeds of crops such as grains, beans and oil crops, and is present in an amount of up to 1% to 3%, accounting for 60% to 80% of the total phosphorus content in the plants. However, the phosphorus in physic acid cannot be directly absorbed and utilized, and needs to be hydrolyzed into inorganic phosphate in the digestive tract. Studies have shown that monogastric animals (for example, pigs, chickens, ducks, and gooses, etc.) have low utilization of phosphorus in phytic acid due to the lack of phytase. Meanwhile, the strong electronegativity of phytic acid causes it to form insoluble salts with a divalent or trivalent cation such as Ca2+, Zn2+, and Fe2++, hindering the absorption of minerals in the small intestine. It also forms complexes with proteins, amino acids and fatty acids, affecting their absorption and utilization. Phytic acid also binds to pepsin, chymotrypsin, trypsin, and others to reduce the digestive enzyme activity. Therefore, the addition of phytase to feed for monogastric animals can increase the utilization of phosphorus in the feed, reduce the phosphorus content in the animal excrements, and increase the energy utilization rate of the proteins and the feed.

Commercial phytase is primarily derived fromAspergillus niger(as described in U.S. Pat. No. 5,436,156),Escherichia coli(as described in U.S. Pat. No. 7,432,098),Citrobactergenus (such as theCitrobacter braakiistrain described in US 20100261259), andBrucella(such asButtiauxellasp. described in U.S. Pat. No. 8,143,046) and so on. These phytases have different acid and heat resistances due to their different origins. Nielsen et al. (J Agric Food Chem. 2015, 63(3): 943-50) compared the performances of commercial phytases and showed thatEscherichia coliphytase exhibits the best performances. CommercialEscherichia coliphytase products are all expressed in yeasts, such as schizosaccharomyces andPichia pastoris, and most of the phytase products on the Chinese market are produced byPichia pastoris. Since phytase is mainly used in the areas of feed and food, andPichia pastorisneeds to use methanol as a carbon source to induce protein expression when a protein is expressed inPichia pastoris, it is difficult to completely remove methanol as a raw material from commercial phytase, thus causing potential safety hazards. In addition, due to the flammable and explosive nature of methanol, special safety protection is required during transportation and production, which increases the production costs. Moreover, higher requirements are imposed on the production environment, and there are certain potential hazards to the production workers. It is therefore not preferred to usePichia pastoristo produce food and feed additives. Filamentous fungi are well known as cell factories for producing valuable products (such as enzymes). Among them,Aspergillus nigerandAspergillus oryzaeare widely used as expression hosts because of their “Generally Recognized As Safe (GRAS)” characteristics. During the fermentation process, no toxic substances are produced, and the raw materials for fermentation are all cereals and by-products thereof (such as soybean meal, corn syrup, etc.). Therefore,Aspergillus nigerandAspergillus oryzaeare more favored in the industry for the production of enzymes. It has been found through studies that the use ofAspergillus nigerto express phytase derived from Gram-negative bacteria is difficult. For example, US 20100261259 describes the use ofAspergillus oryzae, Aspergillus nigerand yeast to expressCitrobacter braakii(a Gram-negative bacterium) phytase. The results show thatAspergillus oryzaeand yeast can well secrete and express the phytase, whileAspergillus nigercan hardly express the phytase. As described above, the phytase derived from the Gram-negative bacterium (Escherichia coli) and its mutant have better characteristics, and therefore the expression inAspergillus nigerthat is GRAS is expected.

SUMMARY

An object of the present invention is to provide a signal peptide which enhances the secretory expression ofEscherichia coliphytase or a mutant thereof in a filamentous fungus.

Another object of the present invention is to provide a codon-optimized gene encoding theEscherichia coliphytase or a mutant thereof.

Another object of the present invention is to provide a codon-optimized DNA sequence encoding theEscherichia coliphytase or a mutant mature peptide thereof.

A further object of the present invention is to provide a method for enhancing the secretory expression of theEscherichia coliphytase or a mutant thereof in a filamentous fungus.

The objects of the present invention can be accomplished through the following technical solutions:

In one aspect, the present invention provides a signal peptide for enhancing the secretory expression ofEscherichia coliphytase or a mutant thereof in a filamentous fungus, where the signal peptide is selected from theAspergillus oryzaeTAKA amylase signal peptide having an amino acid sequence as shown in SEQ ID NO. 13.

In one embodiment of the present invention, the nucleotide sequence of the signal peptide is as shown in SEQ ID NO. 12.

In one embodiment of the present invention, the filamentous fungus is selected fromAspergillus niger.

In one embodiment of the present invention, theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 4.

In another embodiment of the present invention, the mutant of theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

In another aspect, the present invention provides a codon-optimized gene encodingEscherichia coliphytase or a mutant thereof, which has a nucleotide sequence as shown in SEQ ID NO. 7; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 7, and encodes a protein having the phytase activity.

In one embodiment of the present invention, theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 4.

In another embodiment of the present invention, the mutant of theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

In still another aspect, the present invention provides a codon-optimized DNA sequence encodingEscherichia coliphytase or a mutant mature peptide thereof, which has a nucleotide sequence as shown in SEQ ID NO. 8, or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8, and encodes a protein having the phytase activity.

In one embodiment of the present invention, the codon-optimized DNA sequence encodingEscherichia coliphytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 14.

In another embodiment of the present invention, the codon-optimized DNA sequence encodingEscherichia coliphytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 16.

In still another aspect, the present invention provides a method for enhancing the secretory expression ofEscherichia coliphytase or a mutant thereof in a filamentous fungus. The method comprises linking theAspergillus oryzaeTAKA amylase signal peptide to a DNA sequence encodingEscherichia coliphytase or a mutant mature peptide thereof, and inserting an expression cassette comprising the sequence into a filamentous fungus for expression, where the nucleotide sequence of theAspergillus oryzaeTAKA amylase is as shown in SEQ ID NO. 12.

In one embodiment of the present invention, the amino acid sequence of theAspergillus oryzaeTAKA amylase is as shown in SEQ ID NO. 13.

In one embodiment of the present invention, the filamentous fungus is selected fromAspergillus niger.

In one embodiment of the invention, theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 4, and the mutant of theEscherichia coliphytase has an amino acid sequence as shown in SEQ ID NO. 15 or SEQ ID NO. 17.

In one embodiment of the present invention, the DNA sequence encoding theEscherichia coliphytase or a mutant mature peptide thereof is not codon optimized.

In another embodiment of the present invention, the DNA sequence encodingEscherichia coliphytase or a mutant mature peptide thereof is codon optimized.

In another embodiment of the present invention, the codon-optimized DNA sequence encoding theEscherichia coliphytase or a mutant mature peptide thereof has a nucleotide sequence as shown in SEQ ID NO. 8; or has a nucleotide sequence that is at least 95%, 96%, 97%, 98% or 99% homologous to the nucleotide sequence as shown in SEQ ID NO. 8, and encodes a protein having the phytase activity.

In a preferred embodiment of the present invention, the codon-optimized DNA sequence encoding theEscherichia coliphytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 14.

In a preferred embodiment of the present invention, the codon-optimized DNA sequence encoding theEscherichia coliphytase or a mutant mature peptide thereof is as shown in SEQ ID NO. 16.

An expression cassette, a recombinant expression vector, a recombinant strain, a transgenic cell line or a recombinant strain comprising the gene encoding theEscherichia coliphytase is also provided.

To construct anEscherichia coliphytase expression cassette, a specific promoter, terminator, signal peptide sequence, and regulatory sequence are required. e.g., 5′ UTR, 3′ UTR, and the like.

The promoter may be an endogenous promoter fromAspergillus niger, such as the glycosylase gene promoter, neutral amylase gene promoter, acid amylase gene promoter, and α-glucosidase gene promoter, etc. fromAspergillus niger; or an exogenous promoter, such as the neutral amylase gene promoter fromAspergillus oryzae, glycosylase gene promoter fromRhizopus oryzae; or a promoter variant, such as theAspergillus nigerneutral amylase gene promoter variant. In the present invention, theAspergillus nigerglycosylase gene promoter orAspergillus nigerneutral amylase gene promoter variant is preferred.

A regulatory sequence may be linked to the 3′ end of the promoter, for example, a suitable leader sequence (5′ UTR), that is, an untranslated region of mRNA important for the translation of the host cell, such as the leader sequence ofAspergillus oryzaeneutral amylase andAspergillus nidulanstriose-phosphateisomerase;

For the secretory expression of a specific protein, a signal peptide sequence is required, and in the present invention, theAspergillus oryzaeTAKA amylase signal peptide is preferred for theEscherichia coliphytase.

Preferred terminators are obtained from the genes ofAspergillus nigerglycosylase,Aspergillus oryzaeTAKA amylase,Aspergillus nidulansanthranilate synthase,Aspergillus nigeralpha-glucosidase andFusarium oxysporumtrypsin-like protease.

A specific gene is linked to the promoter, the regulatory sequence, the signal peptide sequence, and the terminator to form an expression cassette. It can be inserted into the genome ofAspergillus nigerby conventional methods, and can be randomly inserted into the genome or integrated into one or more loci. Optional loci include gla (glycosylase), amya (neutral amylase), amyb (neutral amylase), aa (acid amylase), agda (alpha glucosidase), and agdb (alpha glucosidase).

The expression cassette can preferably be linked with one or more selectable markers, which allow(s) for simple selection of cells or strains that have been transformed, transfected, and transduced. The selectable marker is a gene whose product provides biocidal or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hyg (hygromycin phosphofransferose), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulphate adenylyltransferase) and trpC (anthranilate synthase) and their equivalents. Preferred for use inAspergilluscells are amdS and hyg ofAspergillus nidulansorAspergillus oryzae.

The expression cassette is preferably linked to one or more counter-selectable markers (negative selection markers). Selectable markers for filamentous fungal host cells include, but are not limited to, amdS (acetamidase), pyrG (orotidine-5′-phosphate decarboxylase), and hsvTK (herpes simplex virus thymidine kinase).

A recombinant expression vector is obtained by inserting theEscherichia coliphytase gene or an expression cassette containing the gene into an expression vector.

A recombinant strain is obtained by introducing the recombinant expression vector into a host strain of interest which is preferablyAspergillus niger.

Use of theEscherichia coliphytase gene in increasing the expression level ofEscherichia coliphytase is provided.

Use of the expression cassette, the recombinant expression vector, and the recombinant strain in increasing the expression level ofEscherichia coliphytase is also provided.

A method for producingEscherichia coliphytase is provided, which comprises fermenting and culturing the transgenic recombinant strain to obtainEscherichia coliphytase.

The method for introducing a DNA fragment intoAspergillus nigerin the present invention is a conventional method in the art.

Codon optimization refers to the redesign of genes by using preferred codons instead of low-utilization or rare codons. For a detailed description of codon optimization, see the article of Joshua B. Plotkin and Grzegorz Kudla (Nat Rev Genet. 2011; 12(1): 32-42.). Codon optimization has been widely used in heterologous expression systems.

Beneficial Effects of the Present Invention:

The present inventors have found that for the gene encodingEscherichia coliphytase or a mutant thereof, it is preferred to synthesize an artificial gene after codon optimization, and after being linked with theAspergillus oryzaeTAKA amylase signal peptide, the constructed expression cassette is introduced intoAspergillus nigerfor expression. In this way, a large amount of secreted and expressed phytase can be obtained in the culture supernatant.

