阅读说明：本技术 一种利用σ70非依赖型抗胁迫启动子实现异丁醛稳定高产的方法 (Method for realizing stable high yield of isobutyraldehyde by using sigma 70 independent stress-resistant promoter ) 是由 霍毅欣 马晓焉 于 2019-09-27 设计创作，主要内容包括：一种利用σ~(70)非依赖型抗胁迫启动子实现异丁醛稳定高产的方法,为最大化细胞工厂生产异丁醛的产量并使产率接近理论值,异丁醛合成途径必须能够持续高效地表达,以便最小化菌体生物量的增长及副产物的生成对资源的消耗。解除合成途径对σ~(70)的唯一性依赖,增强其对σ~(38)的兼容性是实现生产途径持续表达的关键。以σ~(70)非依赖型抗胁迫启动子gadA替代常规的σ~(70)型启动子驱动异丁醛的合成,实现了稳定期和酸胁迫条件下异丁醛合成途径的稳定表达。(In order to maximize the yield of isobutyraldehyde produced by a cell factory and enable the yield to be close to a theoretical value, an isobutyraldehyde synthesis pathway must be capable of being continuously and efficiently expressed so as to minimize the growth of biomass of thalli and the consumption of resources caused by the generation of byproducts, the unique dependence of the synthesis pathway on sigma 70 is removed, the key for realizing the continuous expression of the production pathway is to enhance the compatibility of the synthesis pathway on sigma 38 , and the sigma 70 independent stress-resistant promoter gadA replaces a conventional sigma 70 promoter to drive the synthesis of isobutyraldehyde, so that the stable expression of the isobutyraldehyde synthesis pathway under the conditions of a stable period and acid stress is realized.)

1. by using sigma⁷⁰Method for realizing stable high yield of isobutyraldehyde by independent stress-resistant promoter, and uses sigma to⁷⁰Replacement of conventional sigma by the independent stress-resistant promoter gadA⁷⁰The promoter drives the synthesis of isobutyraldehyde and relieves the synthesis pathway from sigma⁷⁰is uniquely dependent on, enhances its pair sigma³⁸The stable expression of the isobutyraldehyde synthetic pathway under the conditions of the stable phase and acid stress is realized.

2. Use of sigma as in claim⁷⁰The method for realizing stable high yield of isobutyraldehyde by using the independent stress-resistant promoter is characterized by comprising the following steps of:

A. According to the isobutyraldehyde synthetic route, using sigma⁷⁰Replacement of conventional sigma in isobutyraldehyde synthesis pathway by independent stress-resistant promoter⁷⁰A type promoter;

B. obtaining a corresponding gene sequence by using a PCR method, and connecting the promoter with a production way to construct a new recombinant plasmid;

C. Transferring the ligation product obtained in the step B into escherichia coli, selecting a correct single colony through colony PCR verification, and extracting to obtain a successfully ligated recombinant plasmid;

D. C, preparing competence of the strain JCL260 for fermentation, converting the plasmid obtained in the step C into the competence of the fermentation strain, and coating the competence on a flat plate for culture;

E. Transferring the single colonies obtained in the step D into a fermentation medium one by one, and culturing for 12 hours in a test tube;

F. e, transferring the seed liquid obtained in the step E, further performing fermentation culture in a fermentation culture medium, and adding oleyl alcohol with the same volume as the fermentation liquid into a fermentation bottle before culture;

The fermentation medium comprises the following components: 6.0g/l Na₂HPO₄，3.0g/l KH₂PO₄，1.0g/l NH₄Cl，0.5g/l NaCl，1mM MgSO₄，0.1mM CaCl₂10mg/l vitamin B1, 8.5% glucose, 1% yeast extract, pH adjusted to 7.0. Separately, 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin were added.

3. A method of using σ as claimed in claim 1 or 2⁷⁰A method for realizing stable high yield of isobutyraldehyde by using an independent stress-resistant promoter, which is characterized in that in the step (A), the sigma⁷⁰Independent stress-resistant promoters: the gadA promoter.

4. A method of using σ as claimed in claim 1 or 2⁷⁰The method for realizing stable high yield of isobutyraldehyde by the independent stress-resistant promoter is characterized in that the sigma constructed in the step (F)⁷⁰The fermentation period of validity of the cell factory regulated by the independent stress-resistant promoter can last until the stationary phase.

5. A method of using σ as claimed in claim 1 or 2⁷⁰independent of dependenceThe method for realizing stable high yield of isobutyraldehyde by using the stress-resistant promoter is characterized in that in the step (F), the fermentation medium comprises the following components: 6.0g/l Na₂HPO₄，3.0g/l KH₂PO₄，1.0g/l NH₄Cl，0.5g/l NaCl，1mM MgSO₄，0.1mM CaCl₂10mg/l vitamin B1, 8.5% glucose, 1% yeast extract, adjusted to pH 7.0 and added 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin.

6. A method of using σ as claimed in claim 1 or 2⁷⁰The method for realizing stable high yield of isobutyraldehyde by the independent stress-resistant promoter is characterized in that in the step (F), isovolumetric oleyl alcohol is added into fermentation liquor to extract isobutyraldehyde so as to indirectly increase the yield of isobutyraldehyde.

Technical Field

The invention relates to a method for producing isobutyraldehyde at high yield by using a stress-resistant promoter, belonging to the technical field of biological engineering.

Background art:

isobutyl aldehyde is mainly produced by hydrogenation in the industry, and isobutyl alcohol is a novel biofuel and has the advantages of high energy density, low hygroscopicity and the like; in addition, isobutyraldehyde is also used as an intermediate of drugs such as amino acids and vitamins; for synthesizing cellulose ester, essence, perfume, etc.; it can also be used for producing rubber vulcanization accelerator, antioxidant, isobutyric acid, etc.

To maximize the yield and production of isobutyraldehyde in cell factoriesThe rate is close to the theoretical value, and the isobutyraldehyde synthesis pathway must be expressed continuously and efficiently so as to minimize the increase of the biomass of the bacterial cells and the consumption of resources due to the generation of byproducts. Ideally, the thallus only grows and breeds to a certain degree, precursor substances and cofactors required by isobutyraldehyde production are synthesized in the period, when the cell number reaches the production requirement, the growth stops, and the synthesis path starts to express efficiently. If competition for resources by growth is eliminated, the pathway enzyme can convert all production resources accumulated in the early stage directly into the target compound, at which point the productivity of the cell factory peaks. However, this high-yield stage is often difficult to maintain. This is because the expression of synthetic pathways is generally dependent on housekeeping σ⁷⁰Factors, such sigma factors, which dominate transcription of essential genes, are most effective in the log phase of growth. After the cell has entered a stationary phase, stresses occur in the intracellular and extracellular environment, at which time the stress response σ³⁸A factor is generated in large quantities, and⁷⁰Compete for a limited number of RNAP core enzymes. In addition, the degree of DNA supercoiling in the stationary phase is reduced, and the change of intracellular environment such as accumulation of glutamate stress-resistant substances is more beneficial to sigma³⁸Binding to core enzymes. Thus, transcription of pathway genes is often coupled with growth, and once cell growth is arrested, expression of synthetic pathways is decreased and intracellular resources begin to be largely allocated to survival maintenance activities such as stress resistance. Thus, the synthetic pathway pair σ is eliminated⁷⁰Is uniquely dependent on, enhances its pair sigma³⁸The compatibility of (A) is the key to achieving sustained expression of the production pathway.

At σ⁷⁰Replacement of conventional sigma by the independent stress-resistant promoter gadA⁷⁰The promoter drives the synthesis of isobutyraldehyde, and realizes the stable expression of the synthesis pathway of isobutyraldehyde under the conditions of a stable phase and acid stress. The mechanism research finds that the gadA promoter is not influenced by the change of the intracellular sigma factor during the growth period of the thallus and can be changed by the sigma factor⁷⁰and sigma³⁸The RNAPs are transcribed respectively, show the resistance to stress conditions such as low pH, glutamate accumulation, DNA supercoiled relaxation and the like, and can exert transcriptional activity under almost the whole cell growth cycle and stress conditions.

Disclosure of Invention

The invention aims to provide a method for realizing stable high yield of isobutyraldehyde by utilizing sigma⁷⁰replacement of conventional sigma by the independent stress-resistant promoter gadA⁷⁰The promoter drives synthesis of isobutyraldehyde, stable expression of isobutyraldehyde synthesis pathway under the conditions of stable phase and acid stress is realized, the final isobutyraldehyde yield of the strain is finally determined through a shake flask fermentation experiment, and a new method is provided for realizing stable and high isobutyraldehyde yield.

According to the technical scheme provided by the invention, the method utilizes sigma⁷⁰The method for realizing stable high yield of isobutyraldehyde by using independent stress-resistant promoter adopts

The method comprises the following steps:

1. Amplification of gadA promoter P including the entire upstream noncoding region (-1 to-183) with the genome of E.coli MG1655 as a template_gadA183

2. The isobutyraldehyde synthetic pathway consists of two operons, alsS-ilvC/D and kivd, and PgadA183 was inserted upstream of the RBS of the alsS-ilvC/D operon by Gibson assembly on two plasmids, pSA69 and pSA65, respectively, replacing the original promoter PLlacO1, resulting in plasmid pMX 1.

3. the PLlacO1 on the pSA65 plasmid was replaced with PgadA183 in a manner similar to that described in 2 and 3, resulting in plasmid pMX 2.

4. the JCL260 strain is used as a chassis host for producing isobutyraldehyde, pMX1 and pMX2 are transferred into JCL260 competent cells, and the cells are spread on a plate for culture.

5. And (4) transferring the single colony obtained by the conversion in the step (4) to a small amount of culture medium for culture to obtain a seed solution.

6. Inoculating the seed liquid into a fermentation culture medium for fermentation verification.

The oleyl alcohol can be used for efficiently extracting isobutyraldehyde from a water phase, the oleyl alcohol with the same volume as that of fermentation liquid is added into a fermentation bottle before culture, and equal volumes of the oleyl alcohol and the water phase are respectively sampled when the yield is monitored.

The formula of the M9 culture medium used for isobutyraldehyde fermentation is as follows: 6.0g/l Na₂HPO₄，3.0g/l KH₂PO₄，1.0g/l NH₄Cl，0.5g/l NaCl， 1mM MgSO₄，0.1mM CaCl₂10mg/l vitamin B1, 8.5% glucose, 1% yeast extract, pH adjusted to 7.0. Separately, 100. mu.g/ml ampicillin and 50. mu.g/ml kanamycin were added.

Drawings

FIG. 1: the gadA promoter is an independent stress-resistant promoter in a growth period, and can drive the high-efficiency expression of a biofuel synthesis way under various physiological conditions;

FIG. 2: glucose addition, initial pH, broth volume, yield of isobutyraldehyde production driven by gadA promoter by oleyl alcohol extraction (a), glucose consumption (b), OD₆₀₀(c) And the influence of the yield (d).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials, reagents, etc. used in the following examples are commercially available without specific reference.

The following examples are further illustrative of the present invention and are not to be construed as limiting the spirit of the present invention.