CN103232986B

CN103232986B - Method for producing isoprene

Info

Publication number: CN103232986B
Application number: CN201310199919.3A
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: 2014-06-18
Anticipated expiration: 2033-05-27
Also published as: CN103232986A

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, 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 has multiple production isoprene at present, except C ₅cut separate outside isoprene, the industrial synthesis method that also can adopt is produced.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 adopting is now mainly to depend on non-renewable fossil oil oil, although synthetic and constantly maturation of isolation technique, but starting material can become the key factor of restriction isoprene industry development at last, in producing in addition, also exist the problem of high energy consumption, high pollution.Therefore, adopting the cleaning methods such as biology to produce isoprene just becomes study hotspot, but current existing production method still exists the low inferior problem of production efficiency.

Summary of the invention

The object 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.

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, to carry out metabolism and generate more isoprene; Specifically exactly the recombinant expression vector that carries isoprenoid synthase mutant gene is proceeded 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 enzyme before 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 obtaining, and can be widely used in the fermentative production of isoprene, improves the output of isoprene.

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, wherein A is the head space gas of the Pichia yeast that contains wild-type isoprenoid synthase; B is the head space gas of Pichia yeast engineering CCTCC NO:M2013202.

Embodiment

Describe the present invention below in conjunction with specific embodiment.

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

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 LB substratum adds 100ug/mL penbritin.

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, select, 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 aminoacid sequence and molecular structure to this enzyme, find indivedual amino acid in isoprenoid synthase active centre to replace and can effectively improve its enzyme activity, and then increase the output of isoprene.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).

Design primer, utilizes and merges PCR method structure MIsps, and add restriction enzyme site EcoRI and NotI at two ends.

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’

First pcr amplification goes out upstream and downstream fragment MIsps-1 and MIsps-2, respectively.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 PCR product.

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

Reclaim test kit with glue and reclaim 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, obtain positive colony with LB-Amp plate screening, the exactness of definite its gene order of order-checking, obtains 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, be more conducive in karyomit(e) that goal gene homologous recombination enters pichia spp.Yeast cell, after the sorbyl alcohol of 1M is processed, mixes with the linear plasmid that contains MIsps gene after purifying, and electricity consumption conversion instrument transforms.Yeast cell after recovery is coated on MD flat board, cultivates 3-4d for 30 ℃.The single bacterium colony growing on picking MD flat board respectively, is 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, removes supernatant, to OD600=1.0 (approximately 100-200ml), adds 0.5% methyl alcohol with BMMY re-suspended cell, carries out abduction delivering.In 1L shaking flask, add above-mentioned culture, add a cover two-layer sterile gauze or cheese cloth, put into shaking table continued growth.Every 24 hours, add methyl alcohol to final concentration and be 0.5% to continue induction.Check the amount of substratum, guarantee correctly to add methyl alcohol, because Evaporation meeting reduces the volume of substratum.

Recombinant yeast pichia pastoris cell after abduction delivering 72-96h, with sealing plug sealing, continues to cultivate after 30min in shaking flask, and shaking flask is processed to 30min at 60 ℃, extracts 1mL head space gas and carries out gas chromatographic detection, using 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 2.6mg/L. fermentation tail gas, with the control group A (Pichia yeast that contains wild-type isoprenoid synthase, isoprene output is 2mg/L fermentation tail gas) compare, the output of isoprene has improved 30%, illustrates that the vigor of the isoprenoid synthase after sudden change strengthens, thereby isoprene output is increased.The expression product analytical results of multiple recombinant bacteriums has also proved the reliability of the inventive method.

From recombinant bacterium, screen the recombinant yeast pichia pastoris called after pichia pastoris phaff GSMISPS (Pichia pastoris GSMISPS) that isoprene output is the highest, 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. a method for synthetic isoprene, is characterized in that, described method is in pichia spp, to express isoprenoid synthase mutant claimed in claim 1, the synthetic isoprene of pichia spp metabolism of recycling restructuring.

A pichia pastoris engineered strain of producing isoprene ( pichia pastoris) GSMISPS, it carries the expression vector of expressing isoprenoid synthase mutant claimed in claim 1, and its deposit number is CCTCC NO:M2013202.