A method for constructing an actinobacillus succinogenes crisperi system

Abstract

The application discloses a construction method of a succinic acid-producing actinobacillus CRISPRi system, and belongs to the technical field of biology. The CRISPRi system provided by the application takes an expression vector pLGZ92M as a skeleton vector, and main acting elements of the CRISPRi system include a cpf1 expression element and a crRNA expression element. A pckA promoter starts expression of a dcpf1 protein, a frd promoter starts expression of crRNA, and the expression is terminated by a terminator rrnB t1 terminator. The CRISPRi system provided by the application can realize inhibition of a target gene by only replacing crRNA for the target gene. The method of the application realizes gene inhibition of succinic acid-producing actinobacillus, and has the advantages of simple operation process and high transformation efficiency.

Description

Technical Field

[0001] This invention relates to a method for constructing a succinic acid-producing Actinobacillus CRISPRi system, belonging to the field of biotechnology. Background Technology

[0002] *Actinobacterium succinate* is a Gram-negative bacterium isolated from the rumen of cattle that produces high levels of succinic acid. It can naturally produce succinic acid and utilizes a broad spectrum of carbon sources, making it one of the most promising microorganisms for succinic acid production. However, due to a lack of understanding of the genetic background of *Actinobacterium succinate* and the absence of corresponding genetic manipulation tools, its genetic engineering lags behind that of *E. coli*. Currently, few genetic tools for *Actinobacterium succinate* have been reported. Kim et al. successfully constructed the vector pLGZ920 based on the shuttle plasmid pGZRS-19 of *Actinobacter pleuropneumoniae* and *Escherichia coli*, achieving the expression of exogenous genes in *Actinobacterium succinate* (US 20050164360A1). Leveraging the abundant USS sequences, the natural transformation capabilities of membrane-bound DNA absorption mechanisms, and the isocitrate dehydrogenase deficiency of *Actinobacter succinate*, this research group further established a traceless knockout method for *Actinobacter succinate* (Applied and Environmental Microbiology. Joshi, 2014, 80(10):3053.). Subsequently, the National Renewable Energy Laboratory in the United States constructed another relatively simple gene knockout method in *Actinobacter succinate* using the Cre / Frt system (Applied and Environmental Microbiology, 2017). Jiang Min et al. used suicide plasmids, transferred them into *Actinobacter succinate* through conjugation, and then constructed a gene knockout system through homologous recombination double exchange and resistance selection (CN107916247A). However, the above genetic manipulation tools are still cumbersome and have long experimental cycles. Therefore, it is necessary to establish a set of efficient and simple genetic tools to facilitate the study of the genetic background of *Actinobacter succinate* and to obtain superior strains that increase succinic acid production.

[0003] The CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Cas) gene editing system is a third-generation gene editing system developed from ZFNs and TALENs. CRISPR / Cas is an adaptive immune system widely found in bacteria and archaea. This system can cleave foreign nucleic acids under the action of RNA-guided nucleases, resisting invasion by bacteriophages and other pathogens. Utilizing this characteristic of the CRISPR / Cas system, simple and efficient gene editing tools can be constructed. Type II CRISPR / Cas systems are relatively simple; most current CRISPR tools are modified from CRISPR / Cas9 and CRISPR / Cpf1 (Cas12a) of type II. In addition to gene editing, directly inactivating key sites of Cas9 or Cpf1 proteins to obtain proteins that lack nuclease cleavage activity but still possess specific DNA recognition and binding capabilities can inhibit the expression of specific genes; this mechanism is called CRISPRi. CRISPRi has been used in an increasing number of model and non-model strains, playing a significant role in reshaping metabolic pathways, identifying new and highly efficient metabolic enzymes, and optimizing chassis cells. However, for naturally succinic acid-producing Actinobacteria, there is still a lack of CRISPR / Cas genetic manipulation tools. Summary of the Invention

[0004] The purpose of this invention is to establish a CRISPRi system method for succinic acid-producing Actinobacterium, providing an efficient genetic manipulation tool for succinic acid-producing Actinobacterium, which can effectively inhibit gene expression in succinic acid-producing Actinobacterium.

[0005] To achieve the objectives of this invention, the following technical solution is adopted:

[0006] The first objective of this invention is to provide a CRISPRi system comprising the plasmid pCpf1-CRISPRi, the plasmid pCpf1-CRISPRi containing a gene encoding dCpf1 and a crRNA expression cassette; the gene encoding dCpf1 is located downstream of the pckA promoter, and the crRNA expression cassette is located downstream of the dCpf1 gene.

[0007] In one embodiment of the present invention, the crRNA expression cassette includes the frd promoter, the crRNA sequence, and the rrnBt1 terminator.

[0008] In one embodiment of the present invention, the nucleotide sequence of the crRNA expression cassette is shown in SEQ ID NO.1.

[0009] In one embodiment of the present invention, the dCpf1 is as follows (a) to (c):

[0010] (a) Derived from Francisella tularensis subsp. Novicida; amino acid sequence as shown in SEQ ID NO.2;

[0011] (b) Based on (a), the 917th aspartic acid is mutated to alanine;

[0012] (c) Based on (a), glutamic acid at position 1006 is mutated to alanine.

[0013] The second objective of this invention is to provide a method for constructing the CRISPRi system of succinic acid-producing Actinobacillus, the specific steps of which are as follows:

[0014] (1) Insert dCpf1 downstream of the pckA promoter of vector pLGZ92M to obtain vector pLGZ-dCpf1;

[0015] (2) A crRNA expression cassette containing the frd promoter, crRNA and terminator rrnB t1 was amplified, and its nucleotide sequence is shown in SEQ ID NO.1;

[0016] (3) The vector obtained by the inverse PCR linearization step (1) is connected with the crRNA expression cassette obtained in step (2) to form a gene repression vector, which is the CRISPRi system and named pCpf1-CRISPRi.

[0017] In one embodiment of the present invention, in step (1), the dCpf1 is as follows (a) to (c):

[0018] (a) Derived from Francisella tularensis subsp. Novicida, with the amino acid sequence shown in SEQ ID NO.2.

[0019] (b) Based on (a), the 917th aspartic acid is mutated to alanine;

[0020] (c) Based on (a), glutamic acid at position 1006 is mutated to alanine.

[0021] A third objective of this invention is to provide the application of the CRISPRi system in gene editing of succinate-producing Actinobacillus.

[0022] In one embodiment of the present invention, the application involves constructing a crRNA targeting sequence designed based on the target gene sequence into the CRISPRi system, transforming it into succinic acid-producing Actinobacillus, and obtaining a gene-suppressing strain.

[0023] In one embodiment of the present invention, the selection of crRNA target sequences is based on the following principles: 1) the positive strand of the expression sequence contains a TTN sequence, 2) the expression sequence is approximately 23 nt in length and is close to the ATG region (transcription start codon), and 3) the expression sequence is specific.

[0024] In one embodiment of the present invention, the succinate-producing Actinobacillus includes succinate-producing Actinobacillus ATCC55618, CGMCC NO.1593, CCTCC NO: M 2012036, CGMCC NO.1716, CCTCC M2011399 and their mutant strains.

[0025] The present invention also provides microbial cells containing the CRISPRi system.

[0026] The present invention also provides the application of the above-mentioned microbial cells in the preparation of products containing succinic acid.

[0027] The present invention also provides the application of the CRISPRi system in studying the biological function of specific genes in succinate-producing Actinobacteria.

[0028] Beneficial effects:

[0029] The CRISPRi system provided by this invention uses the expression vector pLGZ92M as its backbone vector. Its main active elements include a Cpf1 expression element and a crRNA expression cassette. The pckA promoter initiates the expression of the dCpf1 gene, and the frd promoter initiates the expression of the crRNA expression cassette. This invention uses the ackA gene of *Actobacter succinate-producing* as a target gene for gene editing. Results show that the CRISPRi system provided by this invention can successfully edit genes in *Actobacter succinate-producing*. Furthermore, the CRISPRi system only requires replacing the crRNA targeting the target gene to achieve inhibition of the target gene; it is simple to operate and easy to implement. Attached Figure Description

[0030] Figure 1 CRISPRi plasmid diagram. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0032] The culture media involved in the following examples:

[0033] LB liquid medium: glucose 10g / L, yeast extract 5g / L, NaCl 10g / L.

[0034] LB solid medium: glucose 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 16-18g / L.

[0035] Tryptone soybean broth (TSB) liquid culture medium: TSB 30 g / L.

[0036] Tryptone soybean broth (TSB) solid medium: TSB 30 g / L, agar 16–18 g / L

[0037] Table 1. Primers involved in the following examples

[0038]

[0039]

[0040] Example 1: Construction of the CRISPRi System

[0041] 1) Carrier selection

[0042] The pULcrRNA and pDZLcas12a vectors were donated by Professor Wang Jin's research group at Shanghai Normal University and have been published in the literature Zhou Y, Liu X, Wu, et al. CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei[J]. Frontiers in Bioengineering and Biotechnology, 2020, 8.

[0043] 2) Construction of pLGZ-Cpf1 vector

[0044] Using pDZLcas12a as a template, the Cpf1 fragment (amino acid sequence shown in SEQ ID NO.2) was amplified using primers 12a-plgzhomo-S / 12a-plgzhomo-A and recovered by gel electrophoresis. The recovered fragment was then ligated with the vector pLGZ92M, which had been double-digested with SacI and XbaI, using a one-step cloning kit. The ligation was then heat-shocked into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. After single colonies emerged, colonies were picked, inoculated into LB liquid medium, and incubated overnight at 37°C. The plasmid was extracted, verified by PCR, and sent to Genewiz for sequencing. The correctly sequenced plasmid was identified as the vector pLGZ-Cpf1.

[0045] The pLGZ92M plasmid has been published in: Zhang Qun. Preliminary study on the response of Actinobacillus succinate to succinic acid stress [D]. Jiangnan University.

[0046] 3) Construct a backbone vector containing dCpf1

[0047] Using pLGZ-Cpf1 as a template, linearized fragments mutated from aspartic acid at position 917 to alanine and from glutamic acid at position 1006 to alanine were amplified using primers d12a-d917-S / d12a-d917-A and d12a-e1006-S / d12a-e1006-A, respectively, and recovered. The recovered fragments were then heat-shocked and transformed into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. After single colonies grew, colonies were picked, inoculated into LB liquid medium, and incubated overnight at 37°C. Plasmids were extracted and sent to Genewiz for sequencing. The correctly sequenced fragments were identified as the target vectors pLGZ-dCpf1-917 and pLGZ-dCpf1-1006.

[0048] 4) Construct an expression cassette that provides the frd promoter, crRNA, and terminator rrnB t1.

[0049] Using the genome of *Actinomyces succinate-producing* as a template, the *frd* promoter fragment was amplified using primers *frd-12ahomo-S* / *frd-crRNAhomo-A*, and recovered after gel electrophoresis. Using the *pULcrRNA* plasmid as a template, the *crRNA* expression cassette was amplified using primers *crRNA-frdhomo-S* / *crRNA-frdhomo-A*, and recovered after gel electrophoresis. The recovered fragments were then amplified using fusion PCR with primers *frd-12ahomo-S* / *crRNA-frdhomo-A* to form the *frd-crRNA-rrnB T1* expression cassette, the nucleotide sequence of which is shown in SEQ ID NO. 1.

[0050] 5) Constructing the pCpf1-CRISPRi system carrier

[0051] Using the vector from step 3) as a template, the linearized pLGZ-dCpf1 vector was amplified by reverse PCR using primers Plgz-12a-30-S / Plgz-12a-30-A. This vector was then ligated to the frd-crRNA-rrnB T1 expression cassette via one-step cloning. The vector was then heat-shock transformed into *E. coli* JM109 and plated on LB agar plates containing 100 μg / mL ampicillin, incubated overnight at 37°C. After single colonies emerged, colonies were picked, inoculated into LB liquid medium, and incubated overnight at 37°C. Plasmids were extracted and sent to Genewiz for sequencing. Correctly sequenced plasmids were identified as the CRISPRi system. Figure 1 They were named pCpf1-CRISPRi-917 and pCpf1-CRISPRi-1006.

[0052] Example 2: Inhibition of succinate-producing Actinobacter gene by the CRISPRi system

[0053] The example uses the ackA gene of succinic acid-producing Actinobacillus acetate kinase as a target gene to further illustrate the technical solution of the present invention.

[0054] Using plasmids pCpf1-CRISPRi-917 and pCpf1-CRISPRi-1006, containing mutation sites of D917A or E1006A, respectively, linearized crRNA fragments containing the target gene were amplified using primers ackacrrna2-S / ackacrrna2-A. After gel electrophoresis and recovery, the fragments were heat-shocked and transformed into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Once single colonies emerged, they were picked, inoculated into LB liquid medium, and incubated overnight at 37°C. Plasmids were then extracted and sent to Genewiz for sequencing.

[0055] Using *Actinomyces succinate* F3-II-F as a blank control, the correctly sequenced plasmid was electroporated into *Actinomyces succinate* F3-II-F, plated on TSB plates containing 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain single colonies, namely D917-F3-II-F with a mutation at the D917 site and E1006-F3-II-F with a mutation at the E1006 site.

[0056] Since acetic acid production in succinic acid-producing Actinobacillus is mainly catalyzed by the ackA gene, fermentation was conducted after single colonies grew to verify the acetic acid yield and determine the inhibitory effect of CRISPRi. The shake-flask fermentation conditions are described in (Selection of high-yield succinic acid fermentation strains [J]. Industrial Microbiology, Liu Yupeng, Zhu Leilei, Zheng Pu, Ni Ye, & Sun Zhihao. 2007, 37(2), 6). By detecting the acetic acid concentration in the bacterial culture, it was found that the CRISPRi targeting the ackA gene, with the D917 mutation site, inhibited the target gene by 0-35%; the E1006 mutation site inhibited the target gene by 73%-100% (Table 2).

[0057] Table 2

[0058]

[0059] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A CRISPRi system, characterized in that, The CRISPRi system comprises the plasmid pCpf1-CRISPRi, which uses the expression vector pLGZ92M as its backbone vector and contains a gene encoding dCpf1 and a crRNA expression cassette. The gene encoding dCpf1 is located downstream of the pckA promoter, and the crRNA expression cassette is located downstream of the gene encoding dCpf1. The crRNA expression cassette contains the frd promoter, the crRNA sequence, and the rrnB t1 terminator. The nucleotide sequence of the crRNA expression cassette is shown in SEQ ID NO.1; the dCpf1 is based on the amino acid sequence shown in SEQ ID NO.2, with the glutamic acid at position 1006 mutated to alanine.

2. The application of the CRISPRi system of claim 1 in gene editing of succinate-producing Actinobacillus.

3. The application according to claim 2, characterized in that, The application involves constructing a crRNA sequence designed based on the target gene sequence into the CRISPRi system of claim 1, transforming it into succinate-producing Actinobacillus, and obtaining a gene-suppressing strain.

4. Microbial cells containing the CRISPRi system of claim 1, characterized in that, The microbial cells are succinic acid-producing Actinobacillus.

5. The use of the microbial cells according to claim 4 in the preparation of products containing succinic acid.

Images