CN103232986A

CN103232986A - Method for producing isoprene

Info

Publication number: CN103232986A
Application number: CN2013101999193A
Authority: CN
Inventors: 王华明; 黄亦钧; 李宾
Original assignee: Qingdao Vland Biotech Group Co Ltd
Current assignee: Qingdao Vland Biotech Group Co Ltd
Priority date: 2013-05-27
Filing date: 2013-05-27
Publication date: 2013-08-07
Anticipated expiration: 2033-05-27
Also published as: CN103232986B

Abstract

The invention aims at providing a method for producing isoprene. An isoprene synthetase mutant gene is transformed into pichia pastoris to build a pichia pastoris engineering strain capable of recombining and expressing heterogenous isoprene synthetase mutant, so as to produce the isoprene in an efficient fermentation manner. The isoprene synthetase mutant obtained by the method has better activity than that of untransformed enzyme. Furthermore, the method for producing isoprene provided by the invention is simple, reliable, and environmentally-friendly; the built pichia pastoris engineering strain can largely produce the isoprene, and can be widely applied to fermentation production of the isoprene; and the yield of the isoprene is improved.

Description

A kind of isoprene production method

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of production method of isoprene.

Background technology

Isoprene (2-methyl isophthalic acid, the 3-divinyl) be a kind of high-volatile conjugated diene, it is elastomeric principal monomer, its consumption accounts for 95% of isoprene ultimate production, be mainly used in synthetic polyisoprene rubber, its output is only second to styrene-butadiene rubber(SBR) and cis-1,4-polybutadiene rubber and occupies the 3rd of synthetic rubber, also can be used for synthetic resins, liquid polyisoprene rubber etc.In recent years, people are with the synthetic Linalool of isoprene, squalene etc., thus further synthetic perfume, medicine, agricultural chemicals etc.The precursor substance of isoprene or natural product terpenoid.

The industrial method that multiple production isoprene is arranged, remove C at present ₅cut separate outside isoprene, industrially also can adopt synthesis method production.Because various countries' cost of material is different with supply situation, the synthetic method that country variant is taked is also different, but more isoprene still directly separates from C5 fraction.Because the production method of the isoprene adopted now is mainly to depend on non-renewable fossil oil oil, although synthetic constantly ripe with isolation technique, but starting material can become the key factor of restriction isoprene industry development at last, also exist the problem of high energy consumption, high pollution in producing in addition.Therefore, the cleaning methods such as employing biology are produced isoprene just becomes study hotspot, but current existing production method still exists production efficiency to hang down inferior problem.

Summary of the invention

The purpose of this invention is to provide a kind of isoprene production method, that a kind of isoprenoid synthase mutant gene is transformed in pichia spp, structure obtains the engineering strain of the recombinant expressed allos isoprenoid synthase mutant of energy, and then high-efficiency fermenting is produced isoprene.

At first the present invention provides a kind of isoprenoid synthase mutant, and its aminoacid sequence is SEQ ID NO:1; The nucleotides sequence of its encoding gene is classified SEQ ID NO:2 as.

Another aspect of the present invention provides the application of above-mentioned isoprenoid synthase mutant, is transformed in pichia spp and comes metabolism to generate more isoprene; The concrete recombinant expression vector that will carry exactly the isoprenoid synthase mutant gene proceeds in pichia spp.

The present invention provides a kind of pichia pastoris phaff GSMISPS (Pichia pastoris GSMISPS) that produces isoprene on the other hand, it carries the expression vector that can express above-mentioned isoprenoid synthase mutant gene, this recombinant bacterial strain has been preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University on May 14th, 2013, its deposit number is CCTCC NO:M2013202.

The isoprenoid synthase mutant that the present invention obtains, than not having the front enzyme of transformation to have better activity.And isoprene production method provided by the invention is simple, reliable, environmental protection, builds the pichia pastoris phaff engineering bacteria energy high yield isoprene obtained, and can be widely used in the fermentative production of isoprene, improves the output of isoprene.

The accompanying drawing explanation

Fig. 1: the genetic map of the present invention carrier pPIC3.5-MIsps used;

Fig. 2: the gas chromatogram that the synthetic isoprene of metabolism of the present invention detects, the head space gas that wherein A is the Pichia yeast that contains the wild-type isoprenoid synthase; The head space gas that B is Pichia yeast engineering CCTCC NO:M2013202.

Embodiment

Below in conjunction with specific embodiment, describe the present invention.

The present invention's experiment material used and reagent are as follows:

1, bacterial strain and carrier

Coli strain DH5a, pichia spp, carrier pPIC3.5, all purchased from Invitrogen company.

2, enzyme and test kit

The PCR enzyme, plasmid extraction, glue purification, restriction enzyme, test kit etc. are purchased from Shanghai Sheng Gong company.

3, substratum

Escherichia coli culture medium is LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).LB-Amp is that the LB substratum adds the 100ug/mL penbritin.

The yeast culture base is YPD (1% yeast extract, 2% peptone, 2% glucose);

Yeast culture base BMGY (1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V));

Inducing culture BMMY (1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 0.5% methyl alcohol (V/V)).

But for above-mentioned reagent or material, in the product that the function that those skilled in the art can realize according to it is sold on market, selected, and be not limited only to the concrete record of this specification sheets embodiment.

The structure of embodiment 1 isoprenoid synthase mutant MIsps and pPIC3.5-MIsps expression vector

Aminoacid sequence from the isoprenoid synthase (Isps) of kudzu (Pueraria montana var.lobata) is SEQ ID NO:3, and its coding nucleotide sequence is SEQ ID NO:4.By the analysis of the character such as the aminoacid sequence to this enzyme and molecular structure, find indivedual amino acid in isoprenoid synthase active centre are replaced effectively to improve its enzyme activity, and then increase the output of isoprene.The applicant finds that the Ser of 408 is replaced with to Asp can make enzyme activity strengthen, and the aminoacid sequence of the isoprenoid synthase after sudden change is SEQ ID NO:1, and its coding nucleotide sequence is SEQ ID NO:2(MIsps).

The design primer, utilize and merge PCR method structure MIsps, and add restriction enzyme site EcoRI and NotI at two ends.

The MIsps design of primers is as follows:

MIsps-F1：5’-CCGGAATTCATGTGTGCTACTTCCTCC-3’

MIsps-R1：5’-CAACAAAGCAACACCATCGGAGGAAACGGAAGC-3’

MIsps-F2：5’-GCTTCCGTTTCCTCCGATGGTGTTGCTTTGTTG-3’

MIsps-R2：5’-ATTTGCGGCCGCTTAAACGTACATCAACTG-3’

At first, respectively pcr amplification goes out upstream and downstream fragment MIsps-1 and MIsps-2.PCR reaction system reaction conditions: 94 ℃ of 4min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ are extended 1.5min, 30 circulations, 72 ℃ are extended 7min.Reclaim test kit with glue and reclaim the PCR product.

Adopt two steps to be merged PCR: the first step, PCR reaction system reaction conditions is 94 ℃ of 4min, 94 ℃ of 30s, 72 ℃ are extended 5min, 10 circulations, 72 ℃ are extended 7min.Second step, the PCR product of the first step of take is merged PCR as template, and PCR reaction system reaction conditions is 94 ℃ of 4min, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ are extended 2min, 30 circulations, 72 ℃ are extended 7min.

Reclaim test kit with glue and reclaim the PCR product, with restriction enzyme EcoR1 and Not1 digestion MIsps and carrier pPIC3.5, under the effect of T4 ligase enzyme, 22 ℃ of connections.Transform escherichia coli DH5a, with the LB-Amp plate screening, obtain positive colony, the exactness of its gene order is determined in order-checking, obtains the pPIC3.5-MIsps expression vector, and plasmid map as shown in Figure 1.

Embodiment 2pPIC3.5-MIsps transforms pichia spp and fermentation culture

Utilize restriction enzyme SacI or SalI digestion pPIC3.5-MIsps, make its linearizing, more be conducive in karyomit(e) that the goal gene homologous recombination enters pichia spp.Yeast cell, after the sorbyl alcohol of 1M is processed, mixes with the linear plasmid that contains the MIsps gene after purifying, and the electricity consumption conversion instrument transforms.Yeast cell after recovery is coated on the MD flat board, cultivates 3-4d for 30 ℃.The single bacterium colony grown on picking MD flat board respectively, be seeded to (250ml shaking flask) in 25mL BMGY, and 30 ℃, 250-300rpm shakes to OD600=2-6 (logarithmic growth, approximately 16-18h).Room temperature, the centrifugal 5min of 1500-3000g, collecting cell, remove supernatant, with the BMMY re-suspended cell, to OD600=1.0 (approximately 100-200ml), adds 0.5% methyl alcohol, carries out abduction delivering.Add above-mentioned culture in the 1L shaking flask, add a cover two-layer sterile gauze or cheese cloth, put into the shaking table continued growth.Every 24 hours, add methyl alcohol to final concentration and be 0.5% and induce continuing.Check the amount of substratum, guarantee correctly to add methyl alcohol, because the Evaporation meeting reduces the volume of substratum.

The recombinant yeast pichia pastoris cell after abduction delivering 72-96h, with the sealing plug sealing, after continuing to cultivate 30min, is processed 30min by shaking flask in shaking flask under 60 ℃, extracts 1mL head space gas and carries out gas chromatographic detection, usings the isoprene standard substance as standard.System adopts GC2000 type gas chromatograph, and chromatographic column is CP-Wax58(FFAP) CB(25m * 0.25mm * 0.39mm), detector is flame ionization detector, vaporizer temperature 50 C, column compartment temperature 50 C, 100 ℃ of detector temperatures.Detected result as shown in Figure 2, the pichia spp fermented liquid head space gas of isoprene containing synthase mutant, isoprene output reaches the 2.6mg/L. fermentation tail gas, with the control group A (Pichia yeast that contains the wild-type isoprenoid synthase, isoprene output is the 2mg/L fermentation tail gas) compare, the output of isoprene has improved 30%, illustrates that the vigor of the isoprenoid synthase after suddenling change strengthens, thereby makes isoprene output increase.The expression product analytical results of a plurality of recombinant bacteriums has also proved the reliability of the inventive method.

The highest recombinant yeast pichia pastoris called after pichia pastoris phaff GSMISPS (Pichia pastoris GSMISPS) of screening isoprene output from recombinant bacterium, and being preserved in the Chinese Typical Representative culture collection center of Wuhan, China Wuhan University on May 14th, 2013, deposit number is CCTCC NO:M2013202.

Claims

1. an isoprenoid synthase mutant, the aminoacid sequence of described isoprenoid synthase mutant is SEQ ID NO:1.

2. a gene, described genes encoding isoprenoid synthase mutant claimed in claim 1.

3. gene as claimed in claim 2, its nucleotides sequence is classified SEQ ID NO:2 as.

4. the application of isoprenoid synthase mutant claimed in claim 1 in synthetic isoprene.

5. the method for a synthetic isoprene, is characterized in that, described method is to express isoprenoid synthase mutant claimed in claim 1, the synthetic isoprene of the pichia spp metabolism of recycling restructuring in pichia spp.

6. a pichia pastoris engineered strain of producing isoprene, carry the expression vector of expressing isoprenoid synthase mutant claimed in claim 1, and its deposit number is CCTCC NO:M2013202.