CN101985626B

CN101985626B - Component flavin dependent monooxygenase gene in prokaryote and application thereof

Info

Publication number: CN101985626B
Application number: CN2010105431621A
Authority: CN
Inventors: 许平; 盖忠辉; 王镇轩; 王晓玉
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2010-11-13
Filing date: 2010-11-13
Publication date: 2012-04-25
Anticipated expiration: 2030-11-13
Also published as: CN101985626A

Abstract

The invention relates to a component flavin dependent monooxygenase gene in prokaryote and application thereof, which belong to the technical field of biological engineering. The coded flavin dependent monooxygenase can be used as a catalyst for use in generation of indigos from indoles, the nucleotide sequence of the gene has 1,371 basic groups and can code 456 amino acids. The enzyme expressed by the gene has the capability of catalyzing in the generation of the indigos from indoles, and Escherichia coli transformant containing the gene can efficiently convent the indoles in a culture medium into indigos. The component flavin dependent monooxygenase gene can be obtained by extracting total deoxyribose nucleic acid (DNA) from a soil sample, building a metagenomic library, screening functions and analyzing sequencing. The enzyme has the potentiality for catalyzing the indoles to generate the indigos. The component flavin dependent monooxygenase gene is the first monooxygenase gene separated from the prokaryote, provides new enzyme having a greater application prospect for biological synthetic dyes and provides a more bright application prospect for the biological production of the dye indigos at the same time.

Description

Single-component flavine dependent form monooxygenase gene and purposes in the prokaryotic organism

Technical field

What the present invention relates to is a kind of gene of technical field of bioengineering, specifically, is single-component flavine dependent form monooxygenase gene and purposes in a kind of prokaryotic organism.

Background technology

Indigo is a kind of bright-colored and competent blue dyes, is one of natural dyestuff of finding the earliest, is widely used in industry such as printing and dyeing, medicine and food.Along with the raising of expanding economy and people's level of understanding, " natural, pollution-free " idea is rooted in the hearts of the people.Microorganism growth is fast, cultivation is easy, is easy to industriallization, utilizes the technology of microorganisms producing natural pigment to have vast potential for future development.Genetically engineered, enzyme engineering, medium engineering and genetic continuous development have promoted the development of the indigo research of biosynthesizing.To this century, the indigo attention that begins to cause the scientific worker of bio-transformation synthesis of natural.The harmful waste that indigo generation is produced in bio-transformation lacks than chemical process, and is not only energy-conservation but also cheap, to the conservation of nature environment, to keep aspects such as the eubiosis all be significant undoubtedly.

The flavine monooxygenase (FMO) of single-component generally all is present in the eukaryote, because of selecting and chiral catalysis in its zone, and characteristics such as good stability, has received widely in fields such as the metabolism of allos biomass, pharmacokinetics and biocatalysis are synthetic and to have paid close attention to.FMO comes to light in hepatomicrosome at first, and in all Mammalss and other most eukaryotes, discovery is arranged all.In many hypotypes of the Mammals and the mankind, all exist, and every kind all has its substrate specificity and organizes limitation.Five kinds of FMO genes and six kinds of FMO pseudogenes are being separated to from human genome, and wherein FMO3 is a most important hypotype in the human liver.FMO cytochrome p450 system active collaborative down, the detoxifcation of the intravital poisonous substance of people and other xenobiotics is played crucial effect.The great-hearted heteroatoms that this monooxygenase can catalysis links to each other with carbon bond comprises nitrogen, sulphur, phosphorus, selenium or iodine.Different with mammiferous FMO, the FMO in the yeast can not the nitrogenous compound of oxidation, and only biological thiol is had vigor.In endoplasmic reticulum, zymic FMO need be through providing the environment of an oxidation, thereby the albumen that contains disulfide linkage is carried out effective folding.In plant, FMO also can be to the hormone that in plant-growth, plays an important role, and plant hormone carries out biosynthesizing.Genome sequence analysis revealed, FMO gene homologue frequently occur in Plant Genome, for example in Arabidopis thaliana, have 29 gene homologues.

2003, people such as Choi found the FMO gene in first bacterium in Korea S in biting methyl bacterium SK1 (Methylophaga sp strain SK1), crossing of its gene were expressed and partly identified show that this kind of enzyme can the many aminated compoundss of oxidation.The FMO gene that obtains in this bacterium is carried out at Bacillus coli cells recombinant expressed, find its stably catalyzing indole generate a large amount of indigo.This FMO gene number of including in the GenBank DB is AF494423; Relevant achievement is published on the Biochem Biophys ResCommun magazine; Bite methyl bacterium SK1 and be preserved in Japan Collection of Microorganisms, be numbered JCM number:14647.

2008, Italian scholar such as Alfieri was this FMO albumen crystallization, the position from crystalline structure, nicotine ring and its contiguous NADP that contains ⁺Ribose be proved to be to constitute the essential integral part of catalytic site, the midbody that it can stablize oxidized effectively.NADP (H) has been explained in this discovery, and another is special playing a part.The correlative study result has given explanation on Proc Natl Acad Sci magazine.Special sequential analysis through to the FMO of existing bacterial genomes shows, compares with the genome of eucaryon, and the FMO in the bacterium is rare relatively, only can identify some typical FMO.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art; Single-component flavine dependent form monooxygenase gene and purposes in a kind of prokaryotic organism are provided; The present invention is a kind of prokaryotic organism origin; The gene FMO of flavine dependent form monooxygenase that can catalyzing indole to generate indigo blue can be applicable to fields such as biocatalysis is synthetic.

The present invention realizes through following technical scheme:

The present invention relates to single-component flavine dependent form monooxygenase gene in the prokaryotic organism; The flavine dependent form monooxygenase ability catalyzing indole to generate indigo blue of its coding; The nucleotide sequence of this gene is shown in Seq Id No.1, and its aminoacid sequence is shown in Seq Id No.2.

The invention still further relates to the purposes of single-component flavine dependent form monooxygenase gene in the aforesaid prokaryotic organism; The expressed enzyme of described gene has the ability of catalyzing indole to generate indigo blue, and the Escherichia coli transformant that contains described gene can be converted into the indoles in the substratum indigo efficiently.

The present invention makes up grand genomic library from being extracted total DNA the soil of crude oil pollution.Therefrom screen a kind of substrate indoles that can make and become blue activity clone, obtain the FMO gene in a kind of new bacterium after the order-checking, its base sequence is shown in sequence table.This gene does not have similarity with the single-component flavine dependent form monooxygenase base sequence of the bacterium Methylophaga sp.SK1 that has reported, and the similarity of aminoacid sequence is merely 73%.

FMO in the eukaryote; FMO of the present invention is more suitable for carrying out the structure of engineering strain; Be applied among the biocatalysis production; This is because still there are a lot of problems so far in the expression of Eukaryotic gene in prokaryotic organism such as bacterium, as: the appearance of rare codon causes and can not normally translate; Eukaryotic protein can not be modified to cause in bacterium does not have activity; The difference of the intron of eukaryotic gene, promotor, SD sequence and bacterium etc.These a series of problems have caused the difficulty of eukaryote FMO genetic manipulation, have limited its widespread use aborning.Then there is not this type of problem in FMO of the present invention, and is more suitable in practical application.

Embodiment

Below embodiments of the invention are elaborated: following examples provided detailed embodiment and process, but protection scope of the present invention are not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.In following examples, the experimental technique of unreceipted actual conditions is usually according to the normal condition or the condition of advising according to manufacturer.

Following examples are from being extracted total DNA the soil of crude oil pollution; Product interrupts through behind the purifying at random; Then the 6-8kb dna fragmentation is connected on the pUC118 Hinc II/BAP carrier, makes up grand genomic library behind the transformed host cell intestinal bacteria E.coli B5.

The grand genomic library clone that will from soil, separate structure is applied to and contains 100 μ g ml ^-1Penbritin, 20 μ g ml ^-1On the IPTG LB agar plate, be inverted and cultivate after 12 hours, in the petridish bottom, add an amount of indoles solid, 37 ℃ are continued to cultivate.After for some time, picking turns blue single bacterium colony, extracts recombinant plasmid dna, checks order behind the purifying.Insert fragment called after pBY12-20, behind sequence alignment, obtain the FMO gene.1371 bases of mrna length, 456 aminoacid sequences of encoding.

The amplification of FMO gene:

Upstream primer: 5 '-CGACGGATCCATGACCACACGCATCGCCATCATCG-3 '

Downstream primer: 5 '-CAGCCTCGAGTCAGCCCACGGCCTTGATG-3 '

Reaction parameter:

94 ℃ of sex change 5 minutes, then 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 1.5 minutes, after 35 circulations, 72 ℃ were extended 10 minutes, and obtained the PCR product.

The subclone of FMO gene:

Above-mentioned PCR product is reclaimed the goal gene segment after agarose gel electrophoresis is cut glue, be connected on the pMD18-T carrier, obtain plasmid pFMO2-5, be transformed in the competence bacillus coli DH 5 alpha.5ml LB _AmpLiquid shakes pipe and cultivates, and every liter of IPTG of 1 mmole (sec.-propyl-β-D-sulfo-galactopyranoside) induces, and 37 ℃ of incubated overnight have blue material to generate.

Described LB culture medium prescription is: contain yeast extract paste 10 grams, peptone 10 grams, NaCl 10 grams in per 1,000 ml deionized water, using NaOH solution to regulate the pH value is 7.0,121 degrees centigrade of sterilizations 15 minutes.

LB _AmpLiquid: the LB substratum adds 100 μ g ml ^-1Penbritin.

The mensuration of indigo output:

After will containing the bacillus coli DH 5 alpha process LB liquid nutrient medium activation of plasmid pFMO2-5, be transferred to the LB of 3 100ml _AmpIn the liquid triangular flask, 37 ℃ are cultured to OD ₆₀₀During=0.6 left and right sides, each adds final concentration is that every liter of IPTG of 1 mmole induces, and 37 ℃, 200 rev/mins are continued to cultivate 20 hours.

11,000 rev/mins, the results somatic cells, behind 5 minutes centrifugal supernatants that go, twice of distilled water wash, thalline is resuspended among the 30ml DMSO, and behind 4 times of the diluted samples, ultraviolet/visible spectrophotometer is measured its 620nm place light absorption value.

Prepare indigo standard model, sample concentration is respectively 0.125mg ml ^-1, 0.0625mg ml ^-1, 0.03125mg ml ^-1, 0.015625mgml ^-1, 0.0078125mg ml ^-1, ultraviolet/visible spectrophotometer is measured its 620nm place light absorption value, drawing standard curve respectively.

The making of indigo sample standard curve such as following table:

Indigo standard model concentration (mg ml ^-1)	Indigo standard model 620nm place light absorption value
		0.125	3.362
0.0625	1.756
		0.03125	0.883
0.015625	0.416
		0.0078125	0.209

The typical curve that can get indigo sample from above data is: y=0.03678x, R=0.99963, R ²=0.99926.

Indigo output is confirmed in indigo yield data that 100ml LB liquid nutrient medium obtains and the comparison of the typical curve of indigo sample.

Embodiment 1

It is indigo through the pMD18-T carrier FMO to be expressed production

Method: will carry out amplification PCR products to the FMO gene and after agarose gel electrophoresis is cut glue, reclaim the goal gene segment, and be connected on the pMD18-T carrier, and obtain plasmid and be transformed in the competence bacillus coli DH 5 alpha.5ml LB _AmpLiquid shakes pipe and cultivates, 37 ℃ of incubated overnight.

The bacterium liquid of incubated overnight is transferred to the LB of 3 100ml _AmpIn the liquid triangular flask, 37 ℃ are cultured to OD ₆₀₀During=0.6 left and right sides, each adds final concentration is that every liter of IPTG of 1 mmole (isopropyl ss-D-sulfo-galactopyranoside) induces, and 37 ℃, 200 rev/mins are continued to cultivate 20 hours.

Indigo output is confirmed in indigo yield data that 3 100ml LB liquid nutrient mediums are obtained and the comparison of the typical curve of indigo sample.

LB _AmpLiquid: the LB substratum adds 100 μ g ml ^-1Penbritin.

The result: biological indigo output is about 32mg ml in the 100ml LB substratum ^-1

Embodiment 2

It is indigo through the pET-28a carrier FMO to be expressed production

Method: will carry out amplification PCR products to the FMO gene and after agarose gel electrophoresis is cut glue, reclaim the goal gene segment, and be connected on the pET-28a carrier, and obtain plasmid and be transformed in the competence e. coli bl21 (DE3).5ml LB _KanLiquid shakes pipe and cultivates, 37 ℃ of incubated overnight.

The bacterium liquid of incubated overnight is transferred to the LB of 3 100ml _KanIn the liquid triangular flask, 37 ℃ are cultured to OD ₆₀₀During=0.6 left and right sides, each adds final concentration is that every liter of IPTG of 0.4 mmole induces, and 16 ℃, 130 rev/mins are continued to cultivate 20 hours.

LB _KanLiquid: the LB substratum adds 100 μ g ml ^-1Kantlex.

The result: biological indigo output is about 55mg ml in the 100ml LB substratum ^-1

Claims

1. single-component flavine dependent form monooxygenase gene in the prokaryotic organism is characterized in that, the flavine dependent form monooxygenase of its coding can catalyzing indole to generate indigo blue, and the nucleotide sequence of this gene is shown in Seq ID No.1.

2. the application of single-component flavine dependent form monooxygenase in catalyzing indole to generate indigo blue of the described genes encoding of claim 1.