CN101497888A - Monascus purpureus orotic acid-5'-phosphate decarboxylase gene and uses thereof - Google Patents

Abstract

The invention relates to monascus orotic acid-5'-phosphoric acid decarboxylase genes and application thereof, belonging to the technical field of microbe gene. The invention comprises the monascus orotic acid-5'-phosphoric acid decarboxylase genes with an SEQ ID NO.1 sequence or homologous compounds which have at least 80 percent homology with the SEQ ID NO.1 sequence and amino acid sequences coded by the genes or the homologous compounds; in a gene construction, the genes or the homologous compounds are effectively connected with one or more regulating signals; and the invention also comprises the application of the genes or the homologous compounds in monascuses as a selective marker. The invention constructs uracil auxotrophs monascuses with stable inheritance by the way of genetic engineering, and the bacterial strains not only are convenient for the genetic transformation and the screening of recombination bacterial strain but also maintain the physiological-biochemical characteristics of wild type bacterial strains, are favorable for the culture of the recombination bacterial strains and the effective expression of foreign proteins, and have relatively high industrial application value.

Description

Monascus purpureus orotic-5 '-phosphate decarboxylase gene and uses thereof

Technical field

The invention belongs to the microbial gene technical field, relate to vitamin B13-5 with SEQ ID NO.1 sequence '-phosphate decarboxylase gene or its homologue and uses thereof.

Technical background

The history in existing thousand of the application of Monascus anka Nakazawa et sato (Monascus claims monascus again) on food and Chinese materia medica, modern study find that Monascus anka Nakazawa et sato can produce several physiological active substances.These physiologically active substances are mainly the meta-bolites of Monascus anka Nakazawa et sato, comprise antibacterial substance (for example: monascorubrin), material for lowering blood pressure (for example: γ-An Jidingsuan, vagusstoff), hypoglycemic material, anti-cancer material, antioxidant, ergosterol, longer chain fatty acid, cholesterol synthesis inhibitor (for example: monacolin) etc.Therefore, the attention that is subjected to height is used in the research and development of Monascus anka Nakazawa et sato in recent years.

In order to improve meta-bolites such as monascorubin and the monacolinK output that using value is arranged, improve feed stock conversion, reduce or eliminate the Citrinin in the monascus product simultaneously, people have done a large amount of work.But discover, control by bacterial screening or to technological condition for fermentation that to regulate the synthetic of product obviously be not enough.Utilizing genetic engineering technique to transform Monascus anka Nakazawa et sato may be the key point that fundamentally addresses these problems.

The conversion system of the relevant Monascus anka Nakazawa et sato of having reported is all with the pressure of antibiotics resistance as the allogenic gene stably express.The tame gene expression system that with the antibiotics resistance is selective pressure exists following defective: 1, need add microbiotic aborning, to guarantee the stably express of allogenic gene, make production cost increase; 2, antibiotic use is the important source of causing potential trace allergic phenomena to take place in the gene engineering product preparation process; 3, clinically,,, might cause the diffusion of drug resistance gene, increase its possibility to environmental dissemination if contain drug resistance gene in particular for agricultural, food and industrial bacterial strain.Red colouring agent for food, also used as a Chinese medicine is the traditional zymotic product of China, and it is raw material with the rice, forms through the Monascus anka Nakazawa et sato fermentation, and is mainly directly edible as distiller's yeast, Chinese medicine, food dye and fodder additives etc.So the structure of Monascus anka Nakazawa et sato antibiotic-free conversion system has important significance for theories and practical value.

With vitamin B13-5 '-serve as a mark gene and be proved to be a kind of very effective gene transformation system as the conversion system of recipient bacterium of phosphate decarboxylase gene (pyrG gene) with the defective fungal strain of this gene.If the complete pyrG gene of Monascus anka Nakazawa et sato can be used as the marker gene of transformation and selection, the strain of Monascus anka Nakazawa et sato uridylic auxotrophy promptly can be used as the required F-strain of conversion, thereby transforms Monascus anka Nakazawa et sato by gene transformation.But the pyrG gene of Monascus anka Nakazawa et sato is still without open at present.

Summary of the invention

The objective of the invention is to clone the pyrG gene of Monascus anka Nakazawa et sato, being used to make up with the pyrG gene is the conversion system of selective marker.

The invention provides the nucleotide sequence (sequence 1) or the complementary sequence of the pyrG gene of Monascus anka Nakazawa et sato, make it can be used as the selectable marker gene of uracil auxotrophy bacterial strain conversion system, the use of this mark need not to add microbiotic, oppositely select easily, this makes it gene can be inserted in the microorganism.

The present invention also comprise have sequence 1 vitamin B13-5 '-homologue of phosphate decarboxylase, the homology of the derivative amino level of itself and SEQ ID NO.1 sequence is at least 80%.

The homologue of sequence 1 of the present invention comprises allelic variant, specifically comprises the functional variant of the Nucleotide acquisition that can pass through sequence shown in disappearance, insertion or the constant series 1.

The homologue of sequence 1 of the present invention also comprises the single stranded DNA or the RNA of fungi for example or animals and plants homologue, truncated sequence, coding and noncoding DNA sequence.

The homologue of sequence 1 of the present invention also comprises for example derivative of promoter variants.The promotor of sequence 1 upstream can by the exchange of one or more Nucleotide, insert and (or) disappearance modifies, and do not influence the functionally active of promotor.Also can modify promoter sequence and make its active raising, or replace fully, also can from the promotor of allos biology with active higher promotor.

The gene construct that contains pyrG gene order 1 or its homologue, wherein pyrG gene order or its homologue effectively are connected with one or more conditioning signals, make genetic expression comparatively favourable.Gene construct can comprise with constitutive promoter and effectively being connected, making nucleotide sequence express strengthens, gene construct also can have better simply structure, does not promptly insert other conditioning signal before sequence 1 or its homologue, and the natural promoter with regulating effect does not lack.Can also insert other favourable sequence at dna sequence dna 3 ' end, as terminator.The pyrG gene can exist by one or more copies in gene construct, and gene also can be inactivated, and produces the uracil auxotrophy mutant by this deactivation gene.

In principle, new gene construct can use all natural promoters and regulate sequence, also can use synthetic promoter.

The invention still further relates to and produce the uracil auxotrophy method of microorganism.In order to produce the uracil auxotrophy mutant, by modifications such as mutagenesis, large fragment deletions, make the albumen of this genes encoding be inactivated to pyrG gene with sequence 1 or its homologue.The gene of this deactivation method such as protoplast transformation by PEG mediation or electroporation subsequently imports microorganism, and last, homologous recombination produces the auxotrophic mutation body in microorganism, and they can be by (5 '-FOA) resistance is screened to the 5-fluororotic acid.

The invention still further relates to the method for DNA being inserted organism, comprise a kind of carrier that contains complete pyrG gene with sequence 1 or its homologue is inserted in the defective a kind of organism of pyrG gene, and in the substratum that does not contain uridylic, cultivate this organism, only obtained the biological physical efficiency of foreign vector and in this substratum, grown.The microorganism of Shi Yonging fungi preferably in the method, the microorganism of preferred especially Aspergillus.

When having the gene of sequence 1 or its homologue to transform Monascus anka Nakazawa et sato, also can insert by apparatus other any gene.This makes to be structured in and carries the individual gene of a plurality of copies or the bacterial strain of a plurality of genes in plasmid or its genome.

The present invention passes through genetic engineering means, made up the Monascus anka Nakazawa et sato of inheritance stability, uracil auxotrophy, this bacterial strain not only is convenient to carry out the screening of genetic transformation and recombinant bacterial strain, and the physio-biochemical characteristics of wild type strain have been kept, help the cultivation of recombinant bacterial strain and efficiently expressing of foreign protein, have higher industrial application value.

Embodiment

Below in conjunction with laboratory concrete testing data and specific embodiment, further illustrate the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The test method of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in the molecular cloning experiment guide (the yellow training hall of J. Sa nurse Brooker D.W. Russell work waits and translates: Science Press, 2002) for example, or the condition of being advised according to test kit manufacturer.

Application example 1:

The preparation of Monascus anka Nakazawa et sato genomic library.

Select EpiFOS for use ^TMFosmid Library Production Kit (EPICENTRE) makes up the Monascus anka Nakazawa et sato genomic library, according to the described method preparation of its specification sheets.

Application example 2:

The clone of Monascus anka Nakazawa et sato pyrG gene.

The design degenerated primer, the pyrG gene of pcr amplification Monascus anka Nakazawa et sato.

The present invention be used for the fungi vitamin B13-5 of design of primers '-phosphate decarboxylase and relevant gene sequence number (GenBank call number) following (table 1):

Table 1 be used for the fungi of degenerated primer design and vitamin B13-5 '-the phosphate decarboxylase encoding gene

Utilize bioanalysis software BioEdit to the vitamin B13s-5 of these fungies '-the phosphate decarboxylase encoding gene compares, and designed pair of degenerate primers according to their conserved sequence:

OMP-F：5′-GCACAACTTCCTGATCTTCgargaymgnaa-3′

OMP-R：5′-GAGGCGGGGGTCTGGtaytgytgncc-3′

In degenerated primer, R represents G or A, and Y represents C or T, and W represents A or T, and M represents A or C, and K represents G or T, and S represents C or G, and H represents A, C or T, and V represents A, C or G, and B represents C, G or T, and D represents A, G or T, and N represents G, A or C.

Use this to degenerated primer, carry out pcr amplification with the genomic dna of Monascus anka Nakazawa et sato as template, the PCR product is cloned into carrier pUC18 and order-checking by ordinary method.Obtain with above-mentioned known vitamin B13-5 '-phosphate decarboxylase gene shows the gene fragment of homology.

Application example 3:

The acquisition of pyrG full length gene.

Design a pair of primer according to application example 2 described pyrG gene fragments of being cloned into, from application example 1 described Monascus anka Nakazawa et sato genomic library, filter out the single clone who contains the pyrG gene.In suitable medium, cultivate intestinal bacteria, isolated plasmid dna and order-checking from these thalline by ordinary method.

The primer that uses:

upper：5′-AGATTCACGCCCGTAGTAAACA-3′

lower：5′-AGTTCATCGACATCGGCAACA-3′

Application example 4:

The analysis of nucleotide sequence.

The ClustalW Multiple alignment algorithm that uses Blast algorithm (NCBI) and BioEdit to provide by newer sequence and existing nucleic acid and Protein Data Bank, carries out computer assisted nucleotide sequence analysis.

Application example 5:

The inactivation of Monascus anka Nakazawa et sato pyrG gene.

For the genome copy of the pyrG gene that destroys Monascus anka Nakazawa et sato, pyrG gene all disappearance or excalation also can be able to be used the method for other deactivation gene, produce sudden change or point mutation as inserting, repeat, be inverted, replace several Nucleotide.PyrG fragment with this inactivation transforms Monascus anka Nakazawa et sato, and by the resistance of 5 '-FOA is carried out the screening of transformant, the strain of pyrG genetic flaw has resistance to 1.5mg/mL 5 '-FOA.

The pyrG genetic flaw strain that obtains can copy by inactivation with the genome that confirms Monascus anka Nakazawa et sato pyrG gene by PCR and Southern engram analysis methods analyst.

Application example 6:

With the pyrG gene is the pksCT gene knockout of selective marker with Monascus anka Nakazawa et sato.

Make up the used targeting vector of pksCT gene knockout, with Monascus anka Nakazawa et sato pyrG gene is selective marker, the two ends homology arm is Monascus anka Nakazawa et sato pksCT gene order or its flanking sequence, this targeting vector is transformed into the pyrG genetic flaw strain (seeing application example 5) of Monascus anka Nakazawa et sato, can reaches purpose Monascus anka Nakazawa et sato pksCT gene knockout.

Application example 7:

Use the pyrG gene that other DNA is inserted Monascus anka Nakazawa et sato.

Make up Monascus anka Nakazawa et sato pyrG gene and the placed in-line recombinant plasmid of foreign DNA,, can reach the purpose of foreign DNA being inserted Monascus anka Nakazawa et sato its pyrG genetic flaw strain (seeing application example 5) that is transformed into Monascus anka Nakazawa et sato.

Application example 8:

In same Monascus anka Nakazawa et sato bacterial strain, use the pyrG genetic marker continuously several times.

Upstream and downstream in the pyrG marker gene makes up one section multiple nucleotide sequence in the same way respectively, used as targeting vector Monascus anka Nakazawa et sato engineering strain (seeing application example 7) is transformed, can in the substratum that contains uridylic and 5 '-FOA, screen the bacterial strain that lacks the pyrG gene once more, reuse therefore purpose of pyrG token-based thereby reach.

SEQUENCE?LISTING

＜110〉University Of Nanchang

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Claims (6)

1, monascus purpureus orotic-5 '-phosphate decarboxylase gene, it has SEQ ID NO.1 sequence, or the homologue that has at least 80% homology with SEQ IDNO.1 sequence.

2, gene according to claim 1 or its homologue amino acid sequence coded.

3, contain the vitamin B13-5 with SEQ ID NO.1 sequence of claim 1 or 2 '-gene construct of phosphate decarboxylase gene or its homologue, wherein gene or its homologue effectively are connected with one or more conditioning signals.

4, the carrier that comprises the described gene construct of claim 3.

5, the microorganism that comprises the described gene construct of claim 3.

6, gene order according to claim 1 or its homologue in Monascus anka Nakazawa et sato as the purposes of selective marker.