CN103388003A

CN103388003A - A method for constructing a synthesis route for 3-hydroxybutanone in mitochondria and an application

Info

Publication number: CN103388003A
Application number: CN201310309932XA
Authority: CN
Inventors: 刘立明; 李树波; 陈坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2013-07-23
Filing date: 2013-07-23
Publication date: 2013-11-13

Abstract

The invention discloses a method for constructing a synthesis route for 3-hydroxybutanone in mitochondria and an application, and belongs to the field of genetic engineering. According to the method, by expression of mitochondrion signal peptide (COX4) mediated target genes, over-expression of exogenous acetolactate synthetase (ALS) genes and acetolactate decarboxylase (ALDC) genes in Torulopsis glabrata CCTCC M202019 mitochondria is achieved, and the synthesis route for the 3-hydroxybutanone is constructed successfully, thereby unchoking a carbon metabolic flow from pyruvic acid to the 3-hydroxybutanone and finally achieving accumulation of the 3-hydroxybutanone. Under conditions of multiple vitamin limitation, a Torulopsis glabrata genetically engineered bacterium disclosed by the invention can achieve the accumulation of the 3-hydroxybutanone, and the yield can reach 750 mg/L, thus laying foundations for studying achievement of metabolic engineering transformation in subcellular organelles.

Description

A kind of method and application that builds the 3-Hydroxybutanone route of synthesis in plastosome

Technical field

The present invention relates to a kind of method and application that builds the 3-Hydroxybutanone route of synthesis in plastosome, belong to field of genetic engineering.

Background technology

As a kind of important hardware and software platform compound, 3-Hydroxybutanone is widely used in numerous industry fields such as food, pharmacy and chemical industry.In the 3-Hydroxybutanone synthesis technique, adopt the synthesis technique of microbial fermentation take biomass as raw material,, the advantages such as purity high, low production cost and environmental friendliness high with its product safety performance, and have huge application potential and market outlook.As quadruple vitamin defective type, torulopsis glabrata (T.glabrata CCTCCM 202019) has been realized the suitability for industrialized production of pyruvic acid.Simultaneously, as non-traditional yeast, it also has: (1), as unicellular microorganism, easily utilizes the simple culture media Fast Growth; (2) be easy to genetic engineering modified as monoploid and screening; (3) can grow or the metabolic phenotype transformation by multiple metabolic engineering strategy; (4) has the key precursor material of efficient accumulation 3-Hydroxybutanone---the ability of pyruvic acid; (5) but the distribution by VITAMIN level Effective Regulation carbon metabolic rate; (6) have the advantages such as abundant physiological function information (SGD) and group Epidemiological Analysis technology, for the microorganism of 3-Hydroxybutanone, make the bacterial strain platform is provided.But the genetic characteristics of T.glabrata self determines that it can not accumulate 3-Hydroxybutanone effectively, for this reason need to be by the metabolic engineering strategy.At present, around above-mentioned two key enzymes: acetolactate synthestase (ALS) and acetolactate decarboxylase (ALDC), by introduce simultaneously external source ALS and the ALDC gene that has merged mitochondrial signal peptide sequence (COX4) in the T.glabrata cell mitochondrial, promote the metabolism stream of pyruvic acid node to flow to the synthetic of 3-Hydroxybutanone, realize the accumulation of 3-Hydroxybutanone in T.glabrata, for the carbon metabolism flow with the pyruvic acid node turns to 3-Hydroxybutanone, provide a kind of new strategy and technique means.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of engineering bacteria that builds the 3-Hydroxybutanone route of synthesis in plastosome.

Introduce external source acetolactate synthestase (ALS) and acetolactate decarboxylase (ALDC) gene in described product 3-Hydroxybutanone engineering bacteria.

A technical problem to be solved by this invention is to provide a kind of above-mentioned construction process that builds 3-Hydroxybutanone route of synthesis engineering bacteria in plastosome.

For solving the problems of the technologies described above, technical scheme of the present invention is:

1) clone has been merged the Bacillus subtilis als gene of mitochondrial signal peptide sequence (COX4);

2) synthetic has merged the B.subtilis ALDC gene after mitochondrial signal peptide sequence (COX4) and codon are optimized;

3) amplification is obtained ALS and be connected with expression vector and be connected with the ALDC gene, obtain recombinant expression vector;

4) with step 3) recombinant expression vector that obtains imports recipient bacterium;

5) verify described engineering bacteria.

Another technical problem that the present invention will solve is to provide a kind of described application of 3-Hydroxybutanone route of synthesis engineering bacteria in the fermentative production 3-Hydroxybutanone that build in plastosome.

The technique of fermentation productions of ethylformic acid is: 30 ℃, the described engineering bacteria of 200rpm shake-flask culture, to mid-log phase, are seeded to Medium of shaking flask fermentation with 10% inoculum size; Fermentation parameter is: pH5.5,30 ℃, 200rpm.

The present invention utilizes the method for the peptide-mediated free expression of mitochondrial signal, obtains a strain and produces the 3-Hydroxybutanone engineering bacteria.Produce 3-Hydroxybutanone with engineering bacteria provided by the invention, can break through the bottleneck that yeast does not accumulate, after fermentation 64h, 3-Hydroxybutanone output can reach 750mg/L.

Description of drawings

Fig. 1 pY26-COX4-ALS physical map and bacterium colony PCR checking thereof

The A:pY26-COX4-ALS physical map

B:pY26-COX4-ALS bacterium colony PCR checking

M:Marker1-8:pY26-COX4-ALS transformant the result 9: negative control 10: positive control

Fig. 2 pY26-COX4-ALS-COX4-ALDC physical map and bacterium colony PCR checking thereof

The A:pY26-COX4-ALS-COX4-ALDC physical map

B:pY26-COX4-ALS-COX4-ALDC bacterium colony PCR checking

M:Marker1: positive control 2: negative control 3-7: transformant the result

Build the checking of 3-Hydroxybutanone route of synthesis engineering bacteria in Fig. 3 plastosome

A: utilize primer GPD-F/R to carry out bacterium colony PCR checking primer schematic diagram;

M:Marker1: negative control 2: positive control 3: former bacterium bacterium colony PCR result 4: engineering bacteria bacterium colony PCR result

B: utilize primer TEF-F/R to carry out bacterium colony PCR checking primer schematic diagram;

Figure 43-oxobutanol yield comparison

Embodiment

Carry out by the following examples further to illustrate the present invention, the following example is used for illustration purpose but not is used for the restriction scope of the invention.The experimental technique of unreceipted actual conditions in the following example, all operate according to the common described condition of molecular cloning handbook basically.

Build the construction process of 3-Hydroxybutanone route of synthesis engineering bacteria in embodiment 1 cell mitochondrial.

1, the clone carries the B.subtilis als gene of mitochondrial signal peptide sequence (COX4)

Take the B.subtilis total genomic dna as template, to adopt and to have merged yeast saccharomyces cerevisiae mitochondrial signal peptide sequence (COX4) and with ALS-F and the ALS-R of NotI and BglII restriction enzyme site, be primer, amplification obtains to contain the als gene of COX4 sequence.With corresponding restriction endonuclease, the ALS fragment is carried out double digestion, purifying, connection structure plasmid pY26TEF-ALS (figure lA) with plasmid pY26TEF.After connecting in product conversion JMl09 competent cell, coating is dull and stereotyped with the LB of ammonia benzyl resistance, and through 37 ℃ of incubated overnight, the picking transformant is verified, as figure lB.Positive transformant is inoculated liquid LB substratum, and extraction plasmid, enzyme are cut, are verified and check order and confirm plasmid pY26-ALS _COX4Successfully construct.

2, the clone carries the B.subtilis ALDC gene of mitochondrial signal peptide sequence (COX4)

Take the B.subtilis total genomic dna as template, adopt to merge COX4 and with ALDC-F and the ALDC-R of BamHI and XhoI restriction enzyme site, be primer, amplification obtains to contain the ALDC gene of COX4 sequence.1) the plasmid pY26-ALS that builds _COX4Basis on, with corresponding restriction endonuclease to ALDC fragment and plasmid pY26TEF-ALS _COX4Carry out double digestion, purifying, connection structure plasmid pY26-ALS _COX4-ALDC _COX4(Fig. 2 A).After connecting in product conversion JMl09 competent cell, coating is dull and stereotyped with the LB of ammonia benzyl resistance, and through 37 ℃ of incubated overnight, the picking transformant is verified, as Fig. 2 B.Positive transformant is inoculated liquid LB substratum, and extraction plasmid, enzyme are cut, are verified and check order and confirm plasmid pY26-ALS _COX4-ALDC _COX4Successfully construct.

Table l expressing gene ALS _COX4And ALDC _COX4Required primer and sequence thereof increase

3, build 3-Hydroxybutanone route of synthesis engineering bacteria in plastosome

The recombinant plasmid pY26-ALS that adopts the LiAc conversion method to extract _COX4-ALDC _COX4Be converted into T.glabrata, with goal gene fragment and water, do yin and yang attribute contrast, conversion fluid through after cultivate after, coating MM uridylic defect screening culture medium is dull and stereotyped, is positioned in 30 ℃ of constant incubators, grows transformant after cultivating 3 to 5d, contrasts without transformant.Picking wherein larger transformant lines in new screening flat board with the cultivation of going down to posterity, and repeats same operation 3 times.

Screening culture medium (MM) consists of (g/L): glucose 20, and sodium acetate 7, ammonium sulfate 10, potassium primary phosphate 5, sal epsom 0.8, required VITAMIN liquid is appropriate.

4, build 3-Hydroxybutanone route of synthesis engineering bacteria in the checking plastosome

, for the characteristics of expression vector, adopt the checking primer shown in table 2.Take the bacterium liquid of former bacterium and engineering bacteria as template, utilize respectively GPD-F/R and TEF-F/R to carry out bacterium liquid PCR checking, its result should be: can increase in engineering bacteria bacterium liquid and obtain the big or small purpose band that is respectively 800bp and 2000bp, and only take former bacterium bacterium liquid, during as template, can't obtain similar fragment under the same terms.Electrophoresis result as shown in Figure 3.Verify correct engineering bacteria called after: T.glabrata[ALS/ALDC].

Table 2 engineering strain is expressed the checking primer

Embodiment 2 fermentative production 3-Hydroxybutanones

Connect a described product 3-Hydroxybutanone engineering bacteria of ring from fresh inclined-plane to seed culture medium (25mL/250mL Erlenmeyer flask), in 30 ℃, 200r/min shake-flask culture to mid-log phase (24h), with lO% inoculum size (v/v) access fermention medium (50mL/500mL Erlenmeyer flask), in 30 ℃, 200r/min, after fermentation culture 64h, 3-Hydroxybutanone output can build up to 750mg/L.

The seed culture based component is (g/L): glucose 20, and sodium acetate 7, ammonium sulfate 10, potassium primary phosphate 5, sal epsom 0.8, required VITAMIN liquid is appropriate.

Fermention medium consists of (g/L): glucose 100, urea 7, KH ₂PO ₄5, MgSO ₄7H ₂O0.8, CaCO ₃20, VITAMIN liquid and liquid microelement are appropriate.

The detection of 3-Hydroxybutanone, pyruvic acid and glucose in the embodiment fermented liquid

The mensuration of 3-Hydroxybutanone, pyruvic acid and glucose: adopt Agilent A1200 high performance liquid chromatography (HPLC) instrument to measure.

Chromatographic condition

Chromatographic column: Aminex HPX-87H (Bio-Rad)

Moving phase: 5mmol/L H ₂SO ₄

Flow velocity: 0.6mL/min ^-1

Column temperature: 60 ℃

Sample size: 20 μ L

Detector: differential refraction, UV-detector

Detect wavelength: 290nm

Sample preparation: the 5mL fermented liquid is centrifugal 10min under 10000rpm, gets supernatant liquor, and after 0.45 μ m membrane filtration, filtrate is for liquid-phase chromatographic analysis.

Result shows: the engineering bacteria that (1) the present invention builds can accumulate the 3-Hydroxybutanone of 750mg/L, and does not detect Pfansteihl (Fig. 4) in the fermented liquid of starting strain T.glabrata.Show, introduce in the time of due to the foreign gene ALS that carries signal peptide COX4 and ALDC, successfully built the route of synthesis of allos 3-Hydroxybutanone in the T.glabrata cell mitochondrial, realized the metabolic function of accumulation 3-Hydroxybutanone in T.glabrata.

Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.

Claims

1. method and application that builds the 3-Hydroxybutanone route of synthesis in plastosome is characterized by:

1) cloned the Bacillus subtilis als gene of fusion line plastochondria signal peptide COX4;

2) synthetic has merged the B.subtilis ALDC gene after mitochondrial signal PEPC OX4 and codon are optimized;

3) with step 1) and step 2) the PCR product that obtains is connected in expression vector pY26 jointly, obtains expression vector pY26-COX4-ALS-COX4-ALDC;

4) with step 3) the expression vector pY26-COX4-ALS-COX4-ALDC electricity that obtains goes to yeast.

2. construction process according to claim 1, build the Torulopsis glabrata that obtains to produce 3-Hydroxybutanone.

3. the Torulopsis glabrata of product 3-Hydroxybutanone according to claim 2, the output 750mg/L of fermentative production 3-Hydroxybutanone, fermentation condition is: 30 ℃, the described engineering bacteria of 200r/min shake-flask culture are to mid-log phase, inoculate the shaking flask into 500mL with 10% inoculum size, fermentation culture 64 hours; Fermentation parameter is: 200r/min, 30 ℃.