CN109486835A

CN109486835A - It is a kind of derived from the production alkane key gene muton of cyanobacteria and its application

Info

Publication number: CN109486835A
Application number: CN201811481544.9A
Authority: CN
Inventors: 李鹿之; 陈少鹏; 吴李君
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2018-12-05
Filing date: 2018-12-05
Publication date: 2019-03-19
Anticipated expiration: 2038-12-05
Also published as: CN109486835B

Abstract

The invention discloses a cyanobacteria-derived key gene mutant for alkane production, the gene has the nucleotide sequence shown in SEQ ID NO: 1, or is complementary to the nucleotide sequence shown in SEQ ID NO: 1 nucleotide sequence. The invention also discloses a method and application for obtaining a key gene mutant for hydrocarbon production using the plasmid pEASY-1594-1711 constructed from the wild-type alkane-producing key gene derived from cyanobacteria as a template. The hydrocarbon-producing gene is a mutant of the wild-type hydrocarbon-producing gene derived from cyanobacteria. The total amount of biological alkane production of the strain based on the hydrocarbon-producing gene mutant is increased by 2.9 times compared with the wild type, thereby improving the biological alkane production rate, the Reducing the cost of biological oil production will help accelerate the commercialization of biological oil production.

Description

It is a kind of derived from the production alkane key gene muton of cyanobacteria and its application

Technical field

The present invention relates to the field of biological energy source of genetic engineering, more particularly to are a kind of production alkane key bases derived from cyanobacteria Because of muton and its application.

Background technique

Surge with global economy rapid growth to energy demand, the fossil fuel resources such as petroleum are constantly reduced, and companion It increasingly increases sharply with environmental problem caused by combustion of fossil fuel, the development of recyclable organism fuel gradually causes the attention of people (G.Stephanopoulos,Challenges in enginerrting microbes forbiofuels production, Science 315(2007)801-804).Bioenergy can be relieved its further research as a kind of reproducible energy It is even final to eliminate energy crisis.

Alkane as gasoline, diesel oil, aviation kerosine main ingredient, biological production alkane closer to existing fossil come Source diesel oil is very ideal substitute of diesel fuel.Hydrocarbon is widespread in nature, and many biologies include plant, algae, fungi Hydrocarbon can be produced.For example, induction biosynthesis wax to be to prevent water point evaporation, insect is generated with hydrocarbon pheromones as main component, It generates in photosynthetic cyanobacteria with heptadecane hydrocarbon hydrocarbon (M.Dennis, P.E.Kolattukudy, Acobalt- as main component porphyrin enzyme converts a fatty aldehyde to a hydrocarbon and CO,Proceeding ofthe Natioanl Academy of Sciences 89(1992)5306-5310；Mata TM,Martins AA, Caetano NS.Microalgae for biodiesel production and other applications:A review,Renewable and Sustainable Energy Reviews 14(2010)217-32)。

Cyanobacteria oil-producing has more ideal development prospect compared to the oil plant crops more early studied and forest etc..Cyanobacteria Be one kind be able to carry out plant type produce the photosynthetic prokaryotic micro-organisms of oxygen, as energy microflora of new generation its have with Lower advantage: (1) solar energy, fixed carbon dioxide are absorbed as carbon source for growth, toxigenic capacity is low, vitality is strong；(2) genetic manipulation Simply, genetic modification can be carried out；(3) unit biomass contained energy is higher；(4) it can be grown in fresh water, seawater or sewage, no It fights for soil and water source, effect on environment is minimum (5).Human society needs renewable energy to take eventually to sustainable development For non-renewable fossil energy.On the one hand on the other hand cyanobacteria generation diesel oil can exist directly using sunlight as the energy Produce the fixed CO of process of biodiesel₂As carbon source, greenhouse effects can be alleviated.Therefore cyanobacteria generation diesel oil not only can be with Alleviating energy crisis, and environmental pollution can be alleviated.

Although cyanobacteria generation diesel oil has many big advantages, industrialization process is carried out slowly always.Cyanobacteria Bioenergy is limited in development by several factors, such as expensive bioreactor, the cost of incubation, higher richness Collection expense lacks the standard method etc. of algae culture and bio-fuel production.High production cost is algae generation bavin Primary bottleneck problem (V.L.Colin, A.Rodrfguez, H.A.Cristobal, The the role of that oil is faced synthetic biology in the design ofmicrobial cell factories for biofuel production,Journal ofBiomedicine and Biotechnology 2011(2011)1-9).So grinding at present The hot spot studied carefully is to filter out the cyanobacteria of high oil-producing, shortens the incubation time of cyanobacteria, and simplified culture condition reduces cyanobacteria generation The cost of the energy.

In recent years, with the development of synthetic biology and metabolic engineering, people utilize science of heredity, zymetology and metabolic engineering hand Section obtains many progress by the yield that transformation microbial metabolism approach improves aliphatic hydrocarbon in cyanobacteria.Tan etc. is in cyanobacteria Overexpression ACC to improve the content of intracellular acyl ACP, by cyanobacteria aliphatic hydrocarbon output increased 50% (X.Tan, L.Yao,Q.Gao,et al.Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria,Metabolic Engineering 13(2011)169- 176).Wang etc. in wild type DNC wireless by being overexpressed two copy acyl ACP reductase genes and fatty aldehyde Deformylase monooxygenase gene, make the yield of aliphatic hydrocarbon than wild type improve 8 times (W.Wang, X.Liu, X.Lu, Engineering cyanobacteria to improve photosynthetic production of alkanes, Biotechnology forBiofuels 6(2013)69)

The method for improving paraffin production in cyanobacteria at present is mainly to be realized by transformation microbial metabolism approach, this Evolvement method is although motivated, feasibility is high, but requires Evolutionary Design thinking high and can not be to being short in understanding Object is transformed, using being limited by very large, and mutation cyanobacteria bacterial strain alkane production efficiency obtained still without Method meets the requirement of modernization business application.Directed evolution technologies do not need the molecular mechanism and structure that accurately understand object to be evolved Functional relationship, but by introducing random mutation and recombination, the diversity muton being not present originally is artificially produced, and press According to specifically needing to impose selection pressure, the muton with desired character is filtered out, realizes the Simulating Evolution of molecular level, into Change more targeted and possesses better application value (W.Johannes Tyler and H.M.Zhao, Directed evolution of enzymes and biosynthetic pathways,Current Opinion in Microbiology 9(2006)261-267)。

With the sustainable development of human society, renewable energy replaces non-renewable fossil energy to be inexorable trend.It is logical Cross bioengineering and genetic engineering techniques, using qualitative evolution means obtain high yield alkyl because muton, construct high yield alkane Hydrocarbon bacterial strain.Production cost can be reduced, is had to the industrialization that realization cyanobacteria produces alkane important by improving biological production oil yield Meaning, and solve energy crisis and to provide inexhaustible new cleaning fuel for the mankind.

Summary of the invention

It is an object of the invention to overcome the deficiencies in the prior art, to solve the existing life based on wild type hydrocarbon-producing genes Produce paraffin production is lower, is difficult to meet the insufficient technical problem of industrialization demand.

The present invention is achieved by the following technical solutions: a kind of production alkane key gene muton derived from cyanobacteria, institute Gene is stated with the nucleotide sequence as shown in SEQ ID NO:1, or with as nucleotide sequence shown in SEQ ID NO:1 it is complementary Nucleotide sequence.The nucleotide sequence coded amino acid sequence is as shown in SEQ ID NO:2.

It is invention additionally discloses a kind of recombinant vector containing the above-mentioned production alkane key gene muton derived from cyanobacteria, i.e., prominent The recombinant vector obtained after change.

Preferably, the recombinant vector derives from pEASY-1594-1711.

Invention additionally discloses a kind of plasmid pEASY-1594- to produce the building of alkane key gene derived from the wild type of cyanobacteria 1711 methods for obtaining hydrocarbon-producing genes muton for template, comprising the following steps:

(1) using the pEASY-1594-1711 plasmid of wild type as template, primer SEQ ID NO:12 and SEQ ID are used NO:13 carries out fallibility PCR to hydrocarbon key gene npun_R1711 is produced, and introduces I two restriction enzyme sites of EcoR I and Sal；

(2) by fallibility PCR product after purification pass through I double digestion of EcoR I and Sal, with also pass through double digestion processing PEASY-1594-1711 is stayed overnight in 16 DEG C of connections；

(3) connection product imports E.coliDH5 α competent cell and (is purchased from TaKaRa company, article No. through electrotransformation 9057) it, obtains containing the random mutant libraries for producing hydrocarbon key gene.

Preferably, the response procedures of the fallibility PCR are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 65 DEG C are annealed 45s, 72 DEG C of extension 1.5min after 25 circulations, then continue after extending 5min at 72 DEG C, are placed at 4 DEG C and save backup.

Preferably, in linked system, the molar ratio of Insert Fragment and carrier is 3:1, or adds in every 100ul linked system Enter 50ng carrier and 25ng segment, connection reaction condition is 16 DEG C and connects 5 hours.

Invention additionally discloses a kind of engineering bacteria, the engineering bacteria contains the production alkane key gene muton derived from cyanobacteria Recombinant vector；The gene have the nucleotide sequence as shown in SEQ ID NO:1, or with the core as shown in SEQ ID NO:1 The nucleotide sequence of nucleotide sequence complementation.

The present invention also provides a kind of application of engineering bacteria containing above-mentioned recombinant vector in biological production alkane.The work Journey bacterium is that the prokaryotic expression host Trans BL21 (DE3) containing the recombinant vector (is purchased from TRANSGEN company, article No. CD601), which can produce alkane bacterial strain directly as bacterium, and specific incubation step includes:

(1) engineering bacteria is activated prior to LB plate containing kanamycin, it is containing kanamycin is then inoculated in 5mL LB liquid medium in, be incubated overnight under 37 DEG C, 200rpm, obtain starting bacterium solution；

(2) the starting bacterium solution of step (1) is inoculated in by 1:100 with improvement M9 fluid nutrient medium containing kanamycin In, it is cultivated under 30 DEG C, 200rpm；

(3) by after culture 7 hours of step (2), the inducer IPTG of 0.5M is added, is cultivated for 40 hours；

(4) bacterium solution of step (3) is collected, that is, completes biological production alkane；

(5) by bacterium solution ultrasonication, and supernatant is collected by centrifugation, quantitative analysis can be carried out to wherein paraffin production.

The present invention has the advantage that the hydrocarbon-producing genes are derived from the wild type hydrocarbon-producing genes of cyanobacteria compared with prior art Muton, the biological production alkane total amount of the bacterial strain based on the hydrocarbon-producing genes muton improves 2.9 times compared with wild type, thus It improves biological production alkane yield, reduce biological oil-producing cost, help speed up the commercialization process of biological oil-producing.

Detailed description of the invention

Fig. 1 is the plasmid map containing production alkyl because of the recombinant vector pEASY-1594-1711 of muton；

Fig. 2 is that GC-MS analysis produces alkyl because of the yield comparison knot of muton bacterial strain and wild-type strain biological production alkane Fruit column diagram.

Specific embodiment

It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation Example.

Embodiment 1

1, test material

1) preparation of LB culture medium:

LB liquid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride；

LB solid medium: agar 15g is added in every liter of LB liquid medium；

The LB solid medium of kalamycin resistance: the heating of configured LB solid medium is completely dissolved, to temperature 55 DEG C or so the kanamycins that total amount 2 ‰ (v/v) is added are down to, are then slowly poured into culture dish, cooled and solidified is spare.

2) it improves M9 buffer: weighing the Na of 6g₂HPO₄, 3g KH₂PO₄, 0.5g Nacl dissolution and constant volume it is ultrapure in 1L In water, autoclave sterilization obtains A liquid.Remaining ingredient individually high temperature and pressure or filtration sterilization are weighed, and is added in A liquid Following components: the NH of 2g/L₄The MgSO of Cl, 0.25g/L₄ⅹ7H₂O, the CaCl of 11mg/L₂, 27mg/L FeCl₃ⅹ6H₂O、 The ZnCl X 4H of 2mg/L₂O, the Na of 2mg/L₂MoO₄ⅹ2H₂O, the CuSO of 1.9mg/L₄ⅹ5H₂O, the H of 0.5mg/L₃BO₃、1mg/L Thiamine, 200mM Bis-Tris (PH 7.25) and 0.1% (v/v) Triton-X100, be mixedly configured into 1L jointly Improvement M9 buffer.

The improvement M9 buffer liq culture medium of kalamycin resistance: it is added in configured improvement M9 fluid nutrient medium The kanamycins of total amount 2 ‰ (v/v).

2, derived from the acquisition of the production hydrocarbon key gene of cyanobacteria:

1) wild type produces the acquisition of hydrocarbon key gene:

Using plasmid pAL112 as template, PCR amplification is done with primer described in SEQ ID NO:3 and SEQ ID NO:4, is obtained Wild type produces hydrocarbon key gene, the opinion that the preparation method of the plasmid pAL112 was delivered referring to Xuefeng Lu et al. in 2013 Text (Aiqiu, Liu., et al., Fatty alcohol production in engineered E.coli Marinobacter fatty acyl-CoA reductases,Appl Microbiol Biotechnol,97(2013) 7061-7071.)。

SEQ ID NO:3:P1:AACCGCTCGAGTGCCATGTCCGGTTTTCAAC；

SEQ ID NO:4:P2:AACCGCTCGAGCGCAAAAAGGCCATCCGTCAGGATG.

2) wild type produces the building of hydrocarbon key gene recombinant vector

Bacterium living beings produce alkane and need while including two crucial hydrocarbon-producing genes, for convenience of fallibility PCR and mutant is passed through Library database technology studies hydrocarbon-producing genes, need to add respectively at two genes, that is, both ends orf1594 and npun_R1711 Restriction enzyme site, and they are regulated and controled respectively using two ptrc promoters, construct plasmid pEASY- as shown in Figure 1 1594-1711。

(1) using plasmid pAL112 as template, using primer SEQ ID NO:3 and SEQ ID NO:4 amplification containing there are two produce The segment of hydrocarbon key gene is connected into carrier T pEASY-T5 after PCR product is then added " A ", identifies through sequencing, obtains plasmid pEASY-1594-1711-RC；

(2) using plasmid pEASY-1594-1711-RC as template, both ends has been used to have the primer of II restriction enzyme site of Bgl SEQ ID NO:5 and SEQ ID NO:6 amplified fragments, then by PCR product from connecting, acquisition introduces restriction enzyme site Bgl's II Plasmid pEASY-1594-1711-RC-Bgl2；

(3) using plasmid pEASY-1594-1711-RC-Bgl2 as template, used both ends with I restriction enzyme site of EcoR Primer SEQ ID NO:7 and SEQ ID NO:8 amplified fragments, then by PCR product from connecting, acquisition introduces restriction enzyme site EcoR I plasmid pEASY-1594-1711-RC-Bgl2-EcoR1；

(4) using plasmid pEASY-1594-1711-RC-Bgl2-EcoR1 as template, respectively with primer SEQ ID NO:9 and SEQ ID NO:10 expands ptrc promoter, and introduces I restriction enzyme site of Cla；With primer SEQ ID NO:5 and SEQ ID NO:11 Amplification vector, and I restriction enzyme site of Cla is introduced, by obtaining matter as shown in Figure 1 by both ends PCR product double digestion and after connecting Grain pEASY-1594-1711.

The PCR primer and its restriction enzyme site of table 1:GFP and AID

3) directed evolution obtains hydrocarbon-producing genes muton

Using pEASY-1594-1711 plasmid as template, using primer pair SEQ ID NO:12 and SEQ ID NO:13 to production Hydrocarbon key gene npun_R1711 carries out fallibility PCR, and introduces I two restriction enzyme sites of EcoR I and Sal.By fallibility after purification PCR product passes through I double digestion of EcoR I and Sal, connect with the pEASY-1594-1711 by the processing of same double digestion in 16 DEG C Overnight.Wherein in linked system, the molar ratio of Insert Fragment and carrier is 3:1, or 50ng is added in every 100ul linked system Carrier and 25ng segment, connection reaction condition are 16 DEG C and connect 5 hours；Connection product imports E.coliDH5 α through electrotransformation Competent cell (is purchased from TaKaRa company, article No. 9057), obtains random mutant libraries.

The primer is as shown in table 2 below:

Table 2: primer sequence and its restriction enzyme site

The reaction system of the fallibility PCR, is shown in Table 3

Table 3

The response procedures of the fallibility PCR are as follows: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 65 DEG C of annealing 45s, 72 DEG C are prolonged It stretches 1.5min, after 25 circulations, then continues after extending 5min at 72 DEG C, be placed at 4 DEG C and save backup.

The passage dilution taken turns by 10, which is evolved, screens, and contains hydrocarbon-producing genes muton after finally being evolved, by its turn Entering expressive host Trans BL21 (DE3) (being purchased from TRANSGEN company, article No. CD601) is the biological production alkane work after evolving Journey bacterium.

3, the hydrocarbon-producing genes muton biological production alkane experiment after evolving

1) by the engineering bacteria containing muton plasmid after the engineering bacteria containing hydrocarbon-producing genes wild plasmid and evolution point It is not inoculated in the LB solid medium tablets containing kalamycin resistance, 37 DEG C of overnight incubations；

2) picking single colonie is inoculated in 5mL LB liquid medium containing kanamycin, and at 37 DEG C, 200rpm was descended Night culture obtains starting bacterium solution；

3) the starting bacterium solution of step (2) is inoculated in by 1:100 volume ratio and is trained with improvement M9 liquid containing kanamycin It supports in base (100mL system is in 250mL conical flask), is cultivated under 30 DEG C, 200rpm；

4) after step 3) is cultivated 7 hours, the inducer IPTG of 0.5M is added, is cultivated for 40 hours；

5) bacterium solution is collected, that is, completes biological production alkane；

4, the hydrocarbon-producing genes muton biological production alkane experiment after GC-MS detection is evolved

1) bacterium solution containing alkane is subjected to ultrasonication 30 minutes (power 30%, 10s in Ultrasonic Cell Disruptor；5s off)；

2) 5000g is centrifuged 10 minutes, collects supernatant；

3) culture medium for taking 2mL to contain alkane is added 2mL and contains 7 μ g/mL icosane hydrocarbon as interior target ethyl acetate Solution is uniformly mixed；

4) it is centrifuged 10 minutes in 5000g, collects upper solution, carry out GC-MS analysis；

5) the alkane mixed sample for being dissolved in ethyl acetate that each component concentration is 7 μ g/mL is configured simultaneously, carries out GC-MS Analysis；

As a result as shown in Fig. 2, wt is the engineering bacteria containing wild type hydrocarbon-producing genes, and M28 is to be mutated containing hydrocarbon-producing genes The engineering bacteria of son.As can be seen that the paraffin production of the production hydrocarbon key gene muton after evolving has 2.9 times compared with wild type in figure Promotion, illustrate evolve after hydrocarbon-producing genes muton improve biological production alkane yield.It is mutated with the hydrocarbon-producing genes after evolving Daughter bacteria strain carries out biological production alkane, is expected to reduce biological oil-producing cost, helps speed up the commercialization process of biological oil-producing.

It should be noted that, in this document, such as first and second or the like relational terms are used merely to one if it exists A entity or operation with another entity or operate distinguish, without necessarily requiring or implying these entities or operation it Between there are any actual relationship or orders.Moreover, the terms "include", "comprise" or its any other variant are intended to Cover non-exclusive inclusion, so that the process, method, article or equipment for including a series of elements not only includes those Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or setting Standby intrinsic element.In the absence of more restrictions, the element limited by sentence "including a ...", it is not excluded that There is also other identical elements in the process, method, article or apparatus that includes the element.

The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.

SEQUENCE LISTING

<110>Hefei Institutes of Physical Science, Chinese Academy of Sciences

<120>a kind of derived from the production alkane key gene muton of cyanobacteria and its application

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Claims

1. a kind of alkane-producing key gene mutant derived from cyanobacteria, is characterized in that, described gene has nucleotide sequence as shown in SEQ ID NO:1, or with nucleoside as shown in SEQ ID NO:1 The nucleotide sequence complementary to the acid sequence.

2 . The cyanobacteria-derived key gene mutant for alkane production according to claim 1 , wherein the amino acid sequence encoded by the nucleotide sequence is shown in SEQ ID NO: 2. 3 .

3. A recombinant vector containing the cyanobacteria-derived alkane-producing key gene mutant as claimed in claim 1.

4. The recombinant vector containing the cyanobacteria-derived alkane-producing key gene mutant according to claim 3, wherein the recombinant vector is pEASY-1594-1711.

5. a kind of with the plasmid pEASY-1594-1711 that the wild-type alkane-producing key gene that originates from cyanobacteria is constructed is the method for template to obtain hydrocarbon-producing gene mutant, it is characterized in that, comprise the following steps:

(1) Using the wild-type pEASY-1594-1711 plasmid as a template, using primers to carry out error-prone PCR on the key gene npun_R1711 for hydrocarbon production, and introducing two restriction sites, EcoR I and Sal I;

(2) The purified error-prone PCR product was double digested with EcoR I and Sal I, and then ligated with pEASY-1594-1711 that had also undergone double digestion at 16°C overnight;

(3) The ligation product was electroporated into E.coli DH5α competent cells to obtain a random mutant library containing key genes for hydrocarbon production.

6. the plasmid pEASY-1594-1711 that the wild-type alkane-producing key gene that is derived from cyanobacteria is constructed according to claim 5 is the method for template obtaining hydrocarbon-producing gene mutant, it is characterized in that, the reaction of described error-prone PCR The program was: pre-denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing at 65 °C for 45 s, extension at 72 °C for 1.5 min, after 25 cycles, and then extended at 72 °C for 5 min, and stored at 4 °C for later use.

7. the plasmid pEASY-1594-1711 that the wild-type alkane-producing key gene derived from cyanobacteria is constructed according to claim 5 is the method for obtaining the hydrocarbon-producing gene mutant as a template, it is characterized in that, in the connection system, insert fragment and The molar ratio of the vector was 3:1, or 50 ng of vector and 25 ng of fragments were added to each 100 ul of the ligation system, and the ligation reaction conditions were 16°C for 5 hours.

8. An engineering bacterium, characterized in that, the engineering bacterium contains a recombinant vector derived from a cyanobacterial key gene mutant for alkane production; the gene has the nucleotide sequence shown in SEQ ID NO: 1, or is the same as SEQ ID NO: 1. A nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:1.

9. a kind of application using the engineering bacteria as claimed in claim 8 in biological production of alkane.

10. the application of engineering bacteria according to claim 9 in biological production of alkane, it is characterized in that:

Include the following steps:

(1) The engineered bacteria are activated prior to the LB plate containing kanamycin, then inoculated into 5 mL of LB liquid medium containing kanamycin, and cultured overnight at 37° C. and 200 rpm to obtain a starting bacterial liquid;

(2) The initial bacterial solution was inoculated into M9 liquid medium containing kanamycin at a volume ratio of 1:100, and cultured at 30°C and 200rpm;

(3) After culturing for 7 hours, add 0.5M inducer IPTG, and continue culturing for 40 hours;

(4) bacterial liquid collection, namely complete biological alkane production.