阅读说明：本技术 一种利用σ54依赖型启动子实现生物燃料稳定生产的方法 (Method for realizing stable production of biofuel by using sigma 54 -dependent promoter ) 是由 霍毅欣 马晓焉 马炼杰 于 2019-09-27 设计创作，主要内容包括：本发明提供一种利用σ~(54)启动子实现将生物废料稳定转化为生物燃料的方法。本发明所提供的方法是按照包括以下步骤进行：首先确定要生产的生物燃料的合成途径,以及选择要使用的σ~(54)依赖型启动子。通过PCR基因片段,人工组装构建出σ~(54)依赖型启动子调控的特定生物燃料生产途径,与发酵菌株(ΔglnAΔgdhA)组合,构建出能高效、持续、稳定生产的细胞工厂。本发明所提供的方法可以用于不同种类的生物燃料生产。相对于传统细胞工厂,其发酵过程的有效期能延长到稳定期,不需要外源添加各种诱导物,且抗胁迫能力显著增强,所利用的氮源可以从精制氮扩展到蛋白废料,具有相当的实用价值何发展前景。(The method provided by the invention comprises the following steps of firstly determining the synthesis path of the biofuel to be produced, selecting a sigma 54 -dependent promoter to be used, manually assembling and constructing a specific biofuel production path regulated by the sigma 54 -dependent promoter through a PCR gene fragment, and combining the specific biofuel production path with a fermentation strain (delta glnA delta gdhA) to construct a cell factory capable of efficiently, continuously and stably producing.)

1. By using sigma⁵⁴Method for realizing stable production of biofuel by dependent promoter through sigma presenting negative response relation with nitrogen source supply amount⁵⁴The dependent promoter is combined with a specific fermentation strain (delta glnA delta gdhA) to realize the continuous and stable production of the biofuel by utilizing the characteristics that the promoter is not influenced by the cell cycle and is resistant to the stress environment.

2. The method of using σ as defined in⁵⁴The method for realizing stable production of the biofuel by the dependent promoter is characterized by adopting the following steps:

A. Determining sigma to be selected according to biofuel to be produced and production route thereof⁵⁴A dependent 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. Preparing competence of the strain for fermentation (Δ glnA Δ gdhA), transferring the plasmid obtained in step C into the competence of the strain for fermentation using electricity, and coating on a plate for culture;

E. Transferring the single colonies obtained in the step D into a fermentation medium one by one, and culturing for 24 hours in a test tube;

F. E, transferring the seed liquid obtained in the step E, and further performing fermentation culture in a fermentation culture medium;

The fermentation medium comprises the following components: 40g/L yeast extract, 1mM MgSO₄,0.1mM CaCl₂vitamin B1, ampicillin (100. mu.g ml)^–1) And kanamycin sulfate (50. mu.g ml)^–1)。

G. And E, transferring the seed liquid obtained in the step E, and performing fermentation culture by using waste protein subjected to alkali treatment, high-temperature treatment and protease treatment as a nitrogen source.

3. A method of using σ as claimed in claim 1 or 2⁵⁴method for stable biofuel production with a dependent promoter, characterized in that in step (A), said sigma⁵⁴The dependent promoters were: capable of promoting expression of downstream genes, any of which is expressed by sigma⁵⁴Promoters recognized and bound by RNA polymerases.

4. A method of using σ as claimed in claim 1 or 2⁵⁴The method for realizing stable production of the biofuel by the dependent promoter is characterized in that in the step (D), two ammonia assimilation genes glnA and gdhA on a genome are knocked out by the strain selected by fermentation.

5. A method of using σ as claimed in claim 1 or 2⁵⁴Method for stable production of biofuel using a dependent promoter, characterized in that in step (F), σ is constructed⁵⁴The fermentation period of validity of the cell factory regulated by the dependent promoter can be continued to a stationary phase without an additional inducer.

6. A method of using σ as claimed in claim 1 or 2⁵⁴The method for stably producing biofuel using a dependent promoter, wherein in the step (F), the fermentation medium comprises 40g/L of yeast extract and 1mM of MgSO 2₄,0.1mM CaCl₂Vitamin B1, ampicillin (100. mu.g ml)^–1) And kanamycin sulfate (50. mu.g ml)^–1)。

7. A method of using σ as claimed in claim 1 or 2⁵⁴The method for realizing stable production of the biofuel by the dependent promoter is characterized in that in the step (F), the successfully constructed cell factory can directly ferment by taking the treated waste protein as a nitrogen source.

Technical Field

The invention relates to the utilization of sigma⁵⁴A method for stably converting biological waste into biofuel by a dependent promoter belongs to the technical field of biological engineering.

Background

Biofuel is a new fuel which is obtained by converting biomass and can replace non-renewable resources such as petroleum, and is an important direction for developing and utilizing renewable energy sources. Has considerable diversity in raw materials and industries, and also has considerable convenience in storage and transportation. The most outstanding advantage is its environmental protection nature, and all matters of biofuel can directly get into the biomass circulation of earth in the use, really realizes zero release.

taking isobutanol as an example of one important organic synthetic raw material. An ideal isobutanol microbial cell factory should have a relatively high feedstock conversion efficiency while allowing for sustained expression of the relevant functional genes. However, in actual production, the yield and productivity tend to be far below the theoretical level, mainly due to the following: first, sigma used by traditional cell factories due to the robustness of cellular evolutionary development over the long term⁷⁰The activity of the dependent promoter is remarkably reduced after the fermentation enters a stable period due to sigma factor competition, the expression quantity of downstream enzyme genes is directly influenced, and the cell factory almost stops running. Secondly, various wastes are generated in the process of producing the target product in the factory, and the accumulation of the wastes can not only break the balance of the culture environment, but also generate toxic action on the cells and influence the improvement of the yield.

The invention is based on sigma which presents a negative response relation with the nitrogen source supply⁵⁴Based on the dependent promoter, the promoter can show stronger transcription activity under the condition of insufficient nitrogen source (in the later fermentation period), and can resist the stress environment in the fermentation condition. The method solves various problems faced by the traditional cell factory and realizes the stable production of the biofuel such as isobutanol and the like.

Final draft in actual production process, sigma⁵⁴The dependent promoters glnAp2, argTp appear to compare to the classical. sigma⁷⁰The obvious advantages of the dependent promoter rrnB and the like, and simultaneously has the sigma with the strongest activity for producing isobutanol at present⁷⁰Promoter P_LlacO₁Yield remained equal even under stress conditions with yield relative to P_LlacO₁Higher as shown in figure 1.

disclosure of Invention

The invention aims to provide a method for utilizing sigma⁵⁴the dependent promoter realizes a method for stably converting waste protein into biofuel such as isobutanol and the like. Using sigma capable of responding to the availability of a nitrogen source in the environment⁵⁴The promoter controls the expression of the pathway of production of the product of interest. By using the stress-resistant condition of the promoter and the characteristic that the expression activity and the nitrogen source supply amount present a negative response relation and combining with the fermentation strain for knocking out ammonia assimilation genes glnA and gdhA, the cell factory which can get rid of the influence of the production period and can continuously work to the fermentation stable period is successfully constructed. Successfully realizes the stable production of the biofuel such as isobutanol and the like.

According to the technical scheme provided by the invention, the method utilizes sigma⁵⁴The method for realizing stable production of biofuel such as isobutanol by using the dependent promoter comprises the following steps:

1. determining sigma to use⁵⁴Dependent on the promoter, the production pathway of the biofuel to be used is determined.

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

3. And (4) transferring the ligation product obtained in the step two into escherichia coli, selecting a correct single colony through colony PCR verification, and extracting to obtain the successfully ligated recombinant plasmid.

4. The competence of the strain used for fermentation (Δ glnA Δ gdhA) was prepared, and the plasmid obtained in step 3 was transferred into the strain competence using electricity, spread on a plate, and cultured.

5. Transferring the single colonies obtained by the transformation in the step 4 into a fermentation medium one by one, and culturing in a test tube for 24 hours.

6. And (5) transferring the seed liquid obtained in the step (5), and further performing fermentation culture in a fermentation culture medium.

The fermentation medium comprises the following components: 40g/L yeast extract, 1mM MgSO₄,0.1mM CaCl₂,10^–4% vitamin B1, ampicillin (100. mu.g ml)^–1) And kanamycin sulfate (50. mu.g ml)^–1)。

7. And (5) transferring the seed liquid obtained in the step (5), and performing fermentation culture by using waste protein subjected to alkali treatment, high-temperature treatment and protease treatment as a nitrogen source.

Drawings

FIG. 1 comparison of the yield of different promoters under ideal fermentation conditions and stress conditions (200mM NaCl)

FIG. 2glnAP₂Driven isobutanol production system

FIG. 3 shows glnAP₂Comparison of the yield of isobutanol plant fermentation for promoters

Figure 4argTp driven isobutanol production system

FIG. 5 is a graph comparing the fermentation yield of isobutanol cell factory with argTp as promoter

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.