An optimized CRISPRi / dCas12a gene regulation system of sphingomonas paucimobilis, a recombination engineering strain, a method and application

Abstract

The application provides an optimized CRISPRi / dCas12a gene regulation system of Sphingomonas scs, a recombinant engineering strain and method and application, and specifically belongs to the technical field of genetic engineering. The application provides an optimized CRISPRi / dCas12a gene regulation system of Sphingomonas scs, which comprises a recombinant plasmid carrying a replicon capable of stable replication in Sphingomonas scs, a PJDP promoter driving dcas12a gene transcription, dCas12a a gene, a resistance gene and a crRNA expression cassette. The optimized gene regulation system is a high-efficiency CRISPRi system suitable for S. paucimobilis Based on the optimized gene regulation system, the PHB synthesis pathway can be accurately regulated, the production of gellan gum is improved without affecting the normal growth of the strain, and gellan gum with higher transparency is directly produced.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to an optimized CRISPRi / dCas12a gene regulation system of Sphingosine Monoclonalella, recombinant engineered strains, methods, and applications. Background Technology

[0002] Gellan gum is a type of bacteria composed of Sphingosine monocytogenes (SMP). Sphingomonas paucimobilis Microbial extracellular polysaccharides produced by aerobic fermentation are widely used in food, medicine, and other fields due to their excellent gelling properties, stability, and biocompatibility. However, during the fermentation process, this strain also produces a large amount of the byproduct poly-β-hydroxybutyric acid (PHB). PHB not only competes with gellan gum for carbon sources, reducing gellan gum yield, but also severely reduces the transparency of gellan gum, increasing the difficulty and cost of subsequent purification.

[0003] Traditional methods for improving gellan gum transparency (such as alkali treatment) suffer from high energy consumption, difficulty in controlling conditions, and easy damage to the gellan gum backbone structure. While completely blocking the PHB synthesis pathway through gene knockout or mutagenesis can fundamentally eliminate PHB, it often disrupts the metabolic balance of the strain, leading to a decrease in gellan gum yield instead of an increase.

[0004] CRISPR interference (CRISPRi) technology is a novel technique for regulating gene expression at the mRNA level without cutting the DNA double strand, making it ideal for non-pattern strains with unclear genetic backgrounds. Therefore, there is an urgent need to develop a suitable method... S. paucimobilis The highly efficient CRISPRi system, which precisely regulates the PHB synthesis pathway, solves the problem of low transparency of gellan gum from the source without affecting the normal growth of the strain and high gellan gum production. Summary of the Invention

[0005] The purpose of this invention is to provide an optimized CRISPRi / dCas12a gene regulation system for *Sphingosine Monoclonalella*, a recombinant engineered strain, a method, and applications. The optimized gene regulation system described in this invention is suitable for… S. paucimobilis The highly efficient CRISPRi system, based on the gene regulation system optimized in this invention, can precisely regulate the PHB synthesis pathway, increasing gellan gum yield without affecting the normal growth of the strain, and directly producing gellan gum with higher transparency.

[0006] This invention provides an optimized CRISPRi / dCas12a gene regulatory system for *Sphingosine monocytogenes*, comprising a recombinant plasmid carrying a replicon capable of stable replication in *Sphingosine monocytogenes*, and a driver... dcas12a P of gene transcription JDPpromoter, dCas12a Gene, resistance gene, and one or more crRNA expression cassettes.

[0007] Preferably, the replicon capable of stably replicating in *Sphingosine monocytogenes* includes the pBBR1 replicon; the P... JDP The nucleotide sequence of the promoter is shown in SEQ ID NO.1; dCas12a The nucleotide sequence of the gene is shown in SEQ ID NO.2; the resistance gene includes a chloramphenicol resistance gene; the crRNA expression cassette includes a Ptac promoter, two DR fragments and an rrnB T1 terminator, and the nucleotide sequence of the crRNA expression cassette is shown in SEQ ID NO.3.

[0008] Preferably, the recombinant plasmid also carries an resistance gene; the resistance gene includes a chloramphenicol resistance gene; the nucleotide sequence of the chloramphenicol resistance gene is shown in SEQ ID NO.5.

[0009] This invention also provides a method for constructing the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* as described in the above technical solution, comprising the following steps: The recombinant plasmid of the CRISPRi / dCas12a gene regulatory system of Sphingosine monocytogenes was used to drive dcas12a The original promoter of gene expression is replaced with P JDP An optimized CRISPRi / dCas12a gene regulatory system for Sphingosine monocytogenes was obtained.

[0010] This invention also provides the application of the optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* described in the above-described technical solutions, or the optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* constructed by the construction method described in the above-described technical solutions, in any one or more of the following functions: ① Improve the efficiency of gene repression synthesis; ② Inhibit the synthesis of key genes in the poly-β-hydroxybutyrate (PHT) synthesis pathway; the key genes in the PHT synthesis pathway are... phaB Genes and / or phaC Gene; ③ Increase gellan gum production; ④ Improve the transparency of gellan gum.

[0011] This invention also provides an optimized CRISPRi / dCas12a gene regulation system for *Sphingomonas sphingosine monocytogenes* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PPH) pathway. This optimized CRISPRi / dCas12a gene regulation system for *Sphingomonas sphingosine monocytogenes*, based on the optimized CRISPRi / dCas12a gene regulation system described above, further includes a knockdown mechanism. phaB sgRNA expression and / or knockdown phaC sgRNAs expressed by genes.

[0012] Preferably, the knockdown phaB sgRNAs of gene expression include those targeting phaB gRNA or target of genes phaB Predict the gRNA in the promoter region; the knockdown phaC sgRNAs of gene expression include those targeting phaC gRNA or target of genes phaC Predict the gRNA in the promoter region.

[0013] This invention also provides a method for constructing an optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PPH) synthesis pathway, as described in the above-mentioned technical solution, comprising the following steps: Based on the optimized CRISPRi / dCas12a gene regulatory system of Sphingosine monocytogenes described in the above technical solution, a knockdown insertion was performed. phaB sgRNA expression and / or knockdown phaC The sgRNA expressed by the gene was used to obtain the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway.

[0014] This invention also provides the application of the optimized Sphingomonas CRISPRi / dCas12a gene regulation system for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate (PHB) synthesis pathway, as described in the above-described technical solutions, in improving gellan gum production and / or transparency.

[0015] The present invention also provides a recombinant engineered strain, wherein the recombinant engineered strain uses *Sphingomonas septembinos* as the host cell and introduces the optimized *Sphingomonas septembinos* CRISPRi / dCas12a gene regulatory system described in the above-mentioned technical solution or the optimized *Sphingomonas septembinos* CRISPRi / dCas12a gene regulatory system described in the above-mentioned technical solution for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway.

[0016] The present invention also provides a method for improving gellan gum yield and / or transparency, comprising the following steps: The recombinant engineered strain described in the above technical solution is inoculated into a culture medium for fermentation to produce gellan gum.

[0017] This invention provides an optimized CRISPRi / dCas12a gene regulatory system for *Sphingosine Monoclonalella*. This invention targets non-model strains. S. paucimobilis Through in-depth analysis of its transcriptome data, the highly adaptable endogenous strong promoter P was screened and utilized. JDP The original Ptac promoter was replaced, thereby significantly increasing the expression level of dCas12a protein and enhancing the system's inhibitory ability. Experimental results show that the CRISPRi system optimized in this invention has significantly improved efficiency, achieved by screening and using the endogenous strong promoter Ptac. JDP This invention increases the expression level of the key effector protein dCas12a in the CRISPRi / dCas12a system by 268% compared to the unoptimized system (using the Ptac promoter). This lays a solid foundation for achieving a stronger gene repression effect.

[0018] Furthermore, based on this, this application designs specific gRNAs to precisely inhibit key genes in the PHB synthesis pathway. phaB and / or phaC This blocked the metabolic pathway of carbon flow to PHB, thus simultaneously achieving the dual goals of increasing yield and purity at the source. Experimental results showed that: 1) Using the optimized system, inhibiting key genes in PHB synthesis, either alone or in combination, significantly increased the yield of gellan gum. Among these, the combined inhibition strains… S. paucimobilisThe gellan gel yield of (pBBR1-dCas12a-phaBP4-phaC4) reached 6.32 ± 0.10 g / L, which was 20.38% higher than that of the control strain (containing empty plasmid). 2) By reducing the synthesis of PHB byproducts at the source, the transparency of the gellan gel produced by this invention was greatly improved. The gellan gel produced by the combined inhibitory strain had a gel transparency that was 56.64% higher than that of the control strain, solving the problem of colloidal turbidity caused by PHB contamination in traditional methods. 3) This invention effectively inhibits the synthesis of PHB, and the content of the byproduct PHB is significantly reduced. The PHB yield of the combined inhibitory strain was only 0.238 ± 0.009 g / L, which was 31.43% lower than that of the control strain, reducing carbon source waste and simplifying the downstream purification process. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A graph showing the results of host bacteria containing candidate promoters at different growth stages, as provided by this invention. Figure 2 The graph shows the measurement results of fluorescence intensity of different promoters provided by this invention; Figure 3 The plasmid pBBR1-P provided by this invention JDP System flow diagram of -dCas12a-crRNA; Figure 4 The recombinant plasmid pBBR1-P provided by this invention JDP Figure 1; -dCas12a-crRNA sequencing alignment results; Figure 5 pBBR1-P provided for this invention JDP -dCas12a-crRNA conversion S. paucimobilis Post-colony PCR results image; Figure 6 The graph shows the expression levels of dCas12a in the dCas12a system before and after optimization, as provided by this invention. Figure 7 The diagram showing the number of transformants in the dCas12a system before and after optimization provided by this invention; Figure 8 The figure shows the genetic stability and PCR verification results of the CRISPRi system in continuous passaging provided by this invention; Figure 9 pBBR1-P provided for this invention JDP Image of colony PCR results after transformation of *S. paucimobilis* with dCas12a-PhaB-Pn; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are for electroporation with PhaB-P1. S. paucimobilis Image of colony PCR results; B, lanes 2-7, PhaB-P2 electroporation S. paucimobilis Image of colony PCR results; C, lanes 2-7, PhaB-P3 electroporation S. paucimobilis Image of colony PCR results; D, lanes 2-7, PhaB-P4 electroporation S. paucimobilis Image of colony PCR results; Lanes E 2-7, PhaB-P5 electroporation S. paucimobilis Image of PCR results for bacterial colonies in the middle; Figure 10 pBBR1-P provided for this invention JDP PCR results of *S. paucimobilis* colonies transformed with dCas12a-phaBn'; where M is the 2k Marker; lane 1 is ddH2O; and lanes A are pBBR1-P... JDP -dCas12a-phaB1' Electric transfer S. paucimobilis Image of colony PCR results; B, lanes 2-7, pBBR1-P JDP -dCas12a-phaB2' Electric transfer S. paucimobilis Image of colony PCR results; C, lanes 2-7, pBBR1-P JDP -dCas12a-phaB3' Electric transfer S. paucimobilis Image of colony PCR results; D, lanes 2-7, pBBR1-P JDP -dCas12a-phaB4' Electric transfer S. paucimobilis Image of colony PCR results; E, lanes 2-7, pBBR1-P JDP -dCas12a-phaB5' Electric transfer S. paucimobilis Image of colony PCR results; F, lanes 2-7, pBBR1-dCas12a-phaB6' electroporation S. paucimobilis Image of PCR results for bacterial colonies in the middle; Figure 11 pBBR1-P provided for this invention JDP -dCas12a-phaC-Pn conversion S. paucimobilisPost-colony PCR results; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are phaC-P1 electroporation. S. paucimobilis Image of colony PCR results; B, lanes 2-7, phaC-P2 electroporation S. paucimobilis Image of colony PCR results; C, lanes 2-7, phaC-P3 electroporation S. paucimobilis Image of colony PCR results; D, lanes 2-7, electroporation of phaC-P4 S. paucimobilis Image of colony PCR results; E, lanes 2-7, phaC-P5 electroporation S. paucimobilis Image of PCR results for bacterial colonies in the middle; Figure 12 pBBR1-P provided for this invention JDP -dCas12a-phaCn' conversion S. paucimobilis Post-colony PCR results; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are pBBR1-dCas12a-phaC1' electroporation. S. paucimobilis Image of colony PCR results; B, lanes 2-5, pBBR1-P JDP -dCas12a-phaC2' electro-transfer S. paucimobilis Image of colony PCR results; C, lanes 2-7, pBBR1-P JDP -dCas12a-phaC3' Electric converter S. paucimobilis Image of colony PCR results; D, lanes 2-7, pBBR1-P JDP -dCas12a-phaC4' electro-transfer S. paucimobilis Image of colony PCR results; E, lanes 2-7, pBBR1-P JDP -dCas12a-phaC5' Electric converter S. paucimobilis Image of colony PCR results; F, lanes 2-5, pBBR1-P JDP -dCas12a-phaC6' Electric converter S. paucimobilis Image of PCR results for bacterial colonies in the middle; Figure 13 Provided by the present invention phaB Graph showing the relative expression levels of genes; Figure 14 Provided by the present invention phaC Graph showing the relative expression levels of genes; Figure 15 The experimental strain pBBR1-P provided for this inventionJDP Figure 1.00 of the growth curve of dCas12a-phaBn' / Pn. Figure 16 The experimental strain pBBR1-P provided for this invention JDP Figure 1.00 of the growth curve of dCas12a-phaCn' / Pn. Figure 17 The graph shows the results of the gel production determination of the experimental strain provided by this invention; Figure 18 The graph shows the results of the gel production determination of the experimental strain provided by this invention; Figure 19 The graph shows the results of PHB measurement of the experimental strain provided by this invention. Figure 20 The graph shows the results of PHB measurement of the experimental strain provided by this invention. Figure 21 The image shows the results of measuring the transparency of gel formed by the experimental strain of gellan gum provided in this invention. Figure 22 The image shows the results of measuring the transparency of gel formed by the experimental strain of gellan gum provided in this invention. Figure 23 This is a PCR result of colony transformation of S. paucimobilis using pBBR1-dCas12a-phaBP4-phaC4 provided by the present invention. Figure 24 Provided by the present invention phaB and phaC Graph showing the relative expression levels of genes; Figure 25 The experimental strain pBBR1-P provided for this invention JDP Figure 1.00 of the growth curve of dCas12a-phaBP4-phaC4; Figure 26 The graph shows the results of the gel production determination of the experimental strain provided by this invention; Figure 27 The graph shows the results of PHB measurement of the experimental strain provided by this invention. Figure 28 The image shows the results of measuring the transparency of gel formed by the experimental strains provided in this invention. Detailed Implementation

[0021] This invention provides an optimized strain of Sphingosine monocytogenes (SMM) Sphingomonas paucimobilis The CRISPRi / dCas12a gene regulatory system includes a recombinant plasmid carrying a replicon and driver that can stably replicate in *Sphingosine monocytogenes*. dcas12a P of gene transcription JDP promoter, dCas12aGenes, resistance genes, and one or more crRNA expression cassettes. This invention is based on... S. paucimobilis Transcriptome data were used, with TPM values ​​as a measure of gene expression abundance, to screen for endogenous genes that were highly expressed and stable at different growth stages. Promoter sequences of these genes were predicted using bioinformatics methods and ligated to reporter genes (such as green fluorescent protein GFP). Promoter activity was assessed by measuring fluorescence intensity. Endogenous promoters with high and stable expression levels at all growth stages were selected. JDP Its sequence can be obtained from the NCBI database. In a specific embodiment, the replicon capable of stably replicating in *Sphingosine monocytogenes* includes the pBBR1 replicon, the nucleotide sequence of which can be obtained from the NCBI database, specifically the pBBR1 replicon of patent application number "202411777706.9". The P... JDP The promoter's nucleotide sequence is shown in SEQ ID NO.1: 5'-ACTCTTATCACGCACGCGCGGCTCTGGCCTTTGGCCGGCCCCGCGCCTATCTGGGCGGCT-3'; dCas12a

[0022] This invention also provides a method for constructing the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* as described in the above technical solution, comprising the following steps: The recombinant plasmid of the CRISPRi / dCas12a gene regulatory system of Sphingosine monocytogenes was used to drive dcas12a The original promoter of gene expression is replaced with P JDP An optimized CRISPRi / dCas12a gene regulatory system for Sphingosine monocytogenes was obtained.

[0023] In a specific embodiment, the recombinant plasmid of the CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* can be the recombinant plasmid in the patent application number "202411777706.9", specifically the pBBR1-dCas12a-crRNA plasmid in this patent. In order to better distinguish it from the plasmid in this application that has a promoter substitution, this embodiment of the application names this plasmid as pBBR1-Ptac-dCas12a-crRNA.

[0024] In a specific embodiment, when preparing the optimized *Sphingosine Monoclonalella* CRISPRi / dCas12a gene regulatory system of this application based on pBBR1-Ptac-dCas12a-crRNA, this invention utilizes seamless cloning technology to replace the original promoter (Ptac) driving dcas12a gene expression in the CRISPRi / dCas12a system plasmid (pBBR1-Ptac-dCas12a-crRNA) with the screened endogenous promoter P. JDP Positive transformants were screened on E. coli Top10 plates containing antibiotics (chloramphenicol), and the target recombinant strain was obtained by PCR and sequencing verification, thereby constructing the optimized plasmid pBBR1-PJDP-dCas12a-crRNA.

[0025] This invention also provides the application of the optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* described in the above-described technical solutions, or the optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* constructed by the construction method described in the above-described technical solutions, in any one or more of the following functions: ① Improve the efficiency of gene repression synthesis; ② Inhibit the synthesis of key genes in the poly-β-hydroxybutyrate (PHT) synthesis pathway; the key genes in the PHT synthesis pathway are... phaB Genes and / or phaC Gene; ③ Increase gellan gum production; ④ Improve the transparency of gellan gum.

[0026] This invention utilizes an optimized system to improve gellan gum yield and transparency by inhibiting PHB synthesis. Specifically, this invention selects key genes phaB (encoding acetyl-CoA reductase) and / or phaC (encoding PHB synthase) in the PHB synthesis pathway as inhibitory targets.

[0027] This invention also provides an optimized CRISPRi / dCas12a gene regulation system for *Sphingomonas sphingosine monocytogenes* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PPH) pathway. This optimized CRISPRi / dCas12a gene regulation system for *Sphingomonas sphingosine monocytogenes*, based on the optimized CRISPRi / dCas12a gene regulation system described above, further includes a knockdown mechanism. phaB sgRNA expression and / or knockdown phaC The sgRNA expressed by the gene. In a specific embodiment, the knockdown... phaB sgRNAs of gene expression include those targeting phaB gRNA or target of genes phaB Predict the gRNA in the promoter region; the knockdown phaC sgRNAs of gene expression include those targeting phaC gRNA or target of genes phaCThis invention predicts promoter regions using gRNAs. Based on the PAM sequence (5'-TTTV-3') recognized by the dCas12a protein, multiple sets of gRNAs are designed targeting the coding or non-coding regions of the phaB and phaC genes and their predicted promoter regions. In specific embodiments, this invention designs a total of 6 gRNAs for the phaB gene: phaB1', phaB2', phaB3', phaB4', phaB5', and phaB6', where the first three target the coding strand and the latter three target the non-coding strand; and five sites P1, P2, P3, P4, and P5 targeting the predicted phaB promoter. In specific embodiments, this invention designs a total of 6 gRNAs for the phaC gene: phaC1', phaC2', phaC3', phaC4', phaC5', and phaC6', where the first three target the coding strand and the latter three target the non-coding strand; and five sites P1, P2, P3, P4, and P5 targeting the predicted phaC promoter. In specific embodiments, the inhibitory effect is better when the gRNAs targeting the phaB gene are phaB2', phaB3', phaB5', P1, P3, P4, and P5. Specifically, the inhibitory effect is best when the gRNA targeting the phaB gene is P4, which targets its promoter region. Similarly, the inhibitory effect is better when the gRNAs targeting the phaC gene are phaC4', phaC5', phaC6', P1, P2, P4, and P5. The inhibitory effect is best when the gRNA targeting the phaC gene is phaC4', which targets its coding region. The following text will focus on the gRNA targeting the region with the best inhibitory effect. phaB P4, or for phaC The specific scheme of phaC4' will be explained.

[0028] This invention also provides a method for constructing an optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas sphingosine monocytogenes* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PPH) synthesis pathway, as described in the above-mentioned technical solution, comprising the following steps: Based on the optimized CRISPRi / dCas12a gene regulatory system of Sphingosine monocytogenes described in the above technical solution, a knockdown insertion was performed. phaB sgRNA expression and / or knockdown phaC The sgRNA expressed by the gene was used to obtain the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway.

[0029] This invention constructs both a single-gene repression system and a multi-gene combination repression system. Specifically, the construction of the single-gene repression system may include the following steps: inserting a designed gRNA (e.g., targeting...) into the gRNA... phaB P4, or for phaC The phaC4' was sent to the company for synthesis, and then annealed to form a double chain, which was then combined with the phaC4' that had undergone annealing. Bsp Optimized CRISPRi plasmid (pBBR1-P) linearized by QI restriction enzyme digestion. JDP -dCas12a-crRNA) is linked to an antibiotic-containing (e.g., chloramphenicol) compound. E. coli Positive transformants were screened on Top10 plates and verified by PCR and sequencing to obtain the target recombinant strain. Recombinant plasmids pBBR1-dCas12a-phaBP4 and pBBR1-dCas12a-phaC4' were constructed. The recombinant plasmids were then transformed into... S. paucimobilis To achieve individual inhibition phaB or phaC The engineered strain. The construction of a multi-gene combined repression system may include the following steps: taking two gRNAs with the best repressive effect (such as phaBP4 and phaC4'), tandem them using forward repeat sequences (DR) to construct a crRNA array capable of simultaneously expressing both gRNAs. This crRNA array is then cloned into the optimized CRISPRi plasmid (pBBR1-P). JDP In -dCas12a-crRNA), in antibiotics (such as chloramphenicol) E. coli Positive transformants were screened on Top10 plates and verified by PCR and sequencing to obtain the target recombinant strain. The recombinant plasmid pBBR1-dCas12a-phaBP4-phaC4 was constructed. This recombinant plasmid was then transformed into... S. paucimobilis ATCC 31461, achieving simultaneous suppression phaB and phaC Engineered strains.

[0030] This invention also provides the application of the optimized Sphingomonas CRISPRi / dCas12a gene regulation system for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate (PHB) synthesis pathway, as described in the above-described technical solutions, in improving gellan gum production and / or transparency.

[0031] This invention also provides a recombinant engineered strain, which uses *Sphingomonas sphingosine monocytogenes* as the host cell and introduces the optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulatory system described in the above-mentioned technical solution, or the optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulatory system described in the above-mentioned technical solution that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PHB) synthesis pathway. Specifically, this invention can use electroporation to transfer the constructed optimized plasmid pBBR1-P...JDP -dCas12a-crRNA was introduced via electroporation. S. paucimobilis Positive recombinant strains were obtained from competent cells through resistance selection. In a specific embodiment, the host cell can be *Sphingosine monocytogenes*. S. paucimobilis ATCC 31461, but the present invention is not limited to this. S. paucimobilis ATCC 31461 can also be applied to other *Sphingomonas sphingosine monocytogenes* strains that produce gellan gum or other polysaccharide-producing strains with similar byproduct synthesis problems. When an optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulatory system that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PHB) synthesis pathway is introduced into the recombinant engineered strain of this invention, the gRNA in the crRNA expression cassette targets one or more key genes in the PHB synthesis pathway. These key genes include phaB (encoding acetyl-CoA reductase) and / or phaC (encoding PHB synthase). In a specific embodiment, the recombinant engineered strain may contain plasmid pBBR1-P. JDP The recombinant *Sphingosine monocytogenes* strain -dCas12a-phaBP4 expresses gRNA that targets the promoter region of the phaB gene; it may contain the plasmid pBBR1-P. JDP The recombinant *Sphingosine monocytogenes* strain -dCas12a-phaC4' expresses gRNA that targets the coding region of the phaC gene; it may also contain the plasmid pBBR1-P. JDP The recombinant sphingosine monocytogenes -dCas12a-phaBP4-phaC4 strain expresses gRNA that simultaneously targets the promoter region of the phaB gene and the coding region of the phaC gene.

[0032] The present invention also provides a method for improving gellan gum yield and / or transparency, comprising the following steps: The recombinant engineered strain described in the above technical solution is inoculated into a culture medium for fermentation to produce gellan gum. In a specific embodiment, the recombinant engineered strain can be fermented or cultured on a shaker in a culture medium containing chloramphenicol. In a specific embodiment, the concentration of chloramphenicol can be 10~25 μg / mL, specifically 15 μg / mL. In a specific embodiment, the fermentation culture is an aerobic fermentation culture, and in a specific embodiment, the fermentation culture temperature can be 28~32℃, specifically 30℃.

[0033] Regarding product extraction and analysis, after fermentation, this invention extracts gellan gum from the fermentation broth using conventional methods (alcohol precipitation, centrifugation, drying) and determines its yield. In a specific embodiment, this invention extracts PHB from the bacterial cells and determines its content using gas chromatography or ultraviolet spectrophotometry. This invention prepares a gellan gum solution (0.1% concentration) and measures its transmittance at a wavelength of 490 nm to characterize the transparency of the gellan gum.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an optimized CRISPRi / dCas12a gene regulation system, recombinant engineered strain, method, and application of *Sphingosine Monoclonalella* provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Screening and Acquisition of Highly Active Endogenous Promoters Experimental methods: 1.1 Transcriptome Analysis and Candidate Promoter Selection Bacterial culture and sampling: Sphingosine monocytogenes was cultured and sampled. S. paucimobilis ATCC 31461 was activated overnight at 30°C in YM liquid medium. The cells were then transferred to fresh YM medium at a 1% (v / v) inoculum and cultured at 30°C with shaking at 180 rpm until the mid and late logarithmic growth phases. The cells were then collected by centrifugation.

[0036] Transcriptome Sequencing and Analysis: The collected bacterial samples were sent to a biotechnology company for transcriptome sequencing (RNA-Seq). The TPM value was used as a standardized indicator of gene expression abundance to compare the expression levels of different genes at different growth stages. Endogenous genes with the highest TPM values ​​and good expression stability were screened, and their promoter sequences were predicted using promoter prediction software (such as BDGP and Softberry). In this example, the most efficient endogenous promoter screened was P. JDP .

[0037] 1.2 Fluorescence Validation of Promoter Activity To obtain usable S. paucimobilis The CRISPRi / dCas12a system was used to select the EGFP protein from the pBb(RSF1010)1k-GFPuv plasmid. The gene for this protein is generated by endogenous promoters (PphaB, PphaC, PsdhC, PsucD, Pmdh, PsucC, PacnA, PfumC, P16s, P...). JDP Transcription was initiated using PHP and synthetic promoters (PJ23119, Ptac, Pcat), with pBBR1 as the replicon. The fragments required for constructing the system and the resistance gene CMR fragment used for screening were ligated using a seamless cloning method.

[0038] 1.2.1 Obtaining GFPuv fragments Based on the principle of seamless cloning, using plasmid pBb(RSF1010)1k-GFPuv as a template, primers GFPuv-F / R were designed to amplify the GFPuv gene. The design method is the same as that in the patent with publication number CN117264989A, "A CRISPRi / dCas9 gene inhibition system of Sphingosine Monoclonalella spp. and its construction method and application".

[0039] The PCR reaction system is shown in Table 1: Table 1 PCR reaction system

[0040] The reaction procedure is as follows: ① Pre-denaturation at 98℃ for 3 min; ② Denaturation at 98℃ for 10 s, annealing at 57℃ for 15 s, extension at 72℃ for 2 min 30 s, repeat the above cycle 35 times; ③ Extension at 72℃ for 10 min. After verification by agarose gel electrophoresis, the correct bands were cut and recovered from the gel. The gel recovery procedure is described in the Gel Extraction Kit.

[0041] 1.2.2 Acquisition of the carrier Using the PBBR1-Ptac-dCas12a-crRNA plasmid (construction method is the same as that of the recombinant plasmid pBBR1-dCas12a-crRNA plasmid in patent application number "202411777706.9") as a template, PCR was performed. The PCR reaction system is shown in Table 2. Table 2 PCR reaction system

[0042] The reaction procedure is as follows: ① Pre-denaturation at 98℃ for 3 min; ② Denaturation at 98℃ for 10 s, annealing at 57℃ for 15 s, extension at 72℃ for 2 min 30 s, repeat the above cycle 35 times; ③ Extension at 72℃ for 10 min. After verification by agarose gel electrophoresis, the correct bands were cut and recovered from the gel. The gel recovery procedure is described in the Gel Extraction Kit.

[0043] 1.2.3 Promoter segments (PphaB, PphaC, PsdhC, PsucD, Pmdh, PsucC, PacnA, PfumC, P16s, P...) JDP Obtaining PHP, PJ23119, Ptac, and Pcat by S. paucimobilis Using the genome as a template, PCR was performed. The PCR reaction system is shown in Table 3. Table 3 PCR reaction system

[0044] The reaction procedure is the same as in 1.2.1. After the PCR product is verified by agarose gel electrophoresis, the correct bands are cut and recovered from the gel. The gel recovery steps are described in the Gel Extraction Kit.

[0045] 1.2.4 Seamless cloning of GFPuv fragment, promoter fragment, and PBBR1-Ptac-dCas12a-crRNA linearized vector The reaction system is shown in Table 4: Table 4 PCR reaction system

[0046] Mix gently and centrifuge for a few seconds. Incubate at 50°C for 15 minutes on a PCR instrument.

[0047] 1.2.5 Screening of positive monoclonal PBBR1-P-GFPuv plasmids (P refers to promoters PphaB, PphaC, PsdhC, PsucD, Pmdh, PsucC, PacnA, PfumC, P16s, P) JDP , PHP, PJ23119, Ptac, Pcat) 1) Thaw E. coli Top10 competent cells stored at -80℃ on ice, gently stir the bottom of the centrifuge tube to mix, and add the ligation product from 1.2.4 into the centrifuge tube; 2) Gently tap the bottom of the centrifuge tube to mix, then incubate on ice for 30 minutes; 3) Heat shock at 42℃ for 1 min 30 s; 4) Quickly remove the centrifuge tubes and incubate on ice for 2 minutes; 5) Add 700 µL of preheated LB medium to the centrifuge tube and incubate at 37°C and 160-225 rpm for 1 h. 6) After resuscitation, centrifuge at 4000 rpm for 1 min, retain about 100 µL of supernatant, rinse and mix well, and then take 50 µL and spread it on an LB agar plate containing 25 μg / mL chloramphenicol (CHL). 7) Incubate overnight at 37°C with the container upside down.

[0048] Single colonies were selected for PCR and sequencing verification to ensure that the plasmid was constructed correctly.

[0049] 1.2.6 Verification of Connection Results 1) Colony PCR: Single colonies growing on the surface of LB / CHL plates were picked and prepared into bacterial suspensions. The reaction system is shown in Table 5. Table 5 PCR reaction system

[0050] The reaction procedure is as follows: ① Pre-denaturation at 94℃ for 3 min, ② Denaturation at 94℃ for 30 s; Annealing at 58℃ for 30 s; Extension at 72℃ at 1 kb / min. Repeat the above cycle 32 times, ③ Extension at 72℃ for 10 min.

[0051] For PCR products that are verified to be correct by agarose gel electrophoresis, the corresponding bacterial suspension is incubated overnight at 37°C.

[0052] 2) Plasmid PCR: The bacterial suspension with the correct band was inoculated into LB medium containing 25 μg / mL CHL and cultured overnight at 37°C in a shaker. The next day, plasmids were extracted using the Novizan FastPure Plasmid Mini Kit and PCR reaction was performed for further verification.

[0053] 3) Sequencing verification: The extracted plasmid was sent to Genewiz Biotechnology Co., Ltd. for sequencing. The sequencing primers were a specific sequence located upstream of the insert fragment on the vector PBBR1-P-GFPuv. The sequencing results were analyzed using DNAMAN software.

[0054] 1.3 Fluorescence intensity measurement: 1.3.1 S. paucimobilis Preparation of ATCC 31461 competent cells (1) Pick S.paucimobilis Single colonies were inoculated into YM liquid medium and cultured at 30°C and 180 rpm / min for 16 h. (2) The next day, 1 mL of bacterial culture was added to an Erlenmeyer flask containing 30 mL of seed culture medium. Under the same culture conditions, the culture was cultured to the logarithmic phase, with OD600≈0.55. (3) Collect the bacterial culture into a centrifuge tube in a sterile laminar flow hood, centrifuge at 4 ℃ / 8000 rpm for 10 min, discard the supernatant, collect the cells, and repeat twice; (4) Resuspend the cells three times with pre-cooled electroporation buffer, and finally add 100L of electroporation buffer and mix well. Store competent cells at -80°C.

[0055] 1.3.2 Verify the correct pBBR1-P JDP -GFP and control plasmids were introduced into competent cells of *Sphingosine Monoclonalis* ATCC 31461 via electroporation: (1) Take 5 μL of recombinant plasmid pBBR1-P JDP -GFP and control plasmid were added to S.paucimobilis In competent cells, mix by blowing and sucking to ensure that the plasmid and competent cells are evenly mixed.

[0056] (2) Transfer the system from the above steps to an electroshock cup, pre-cool on ice for 10 minutes, and then perform an electroshock using an electroshock device at a voltage of 2.5 kV for 4 ms (instantaneous, and then place on ice after completion).

[0057] (3) After completing the above steps, immediately add 1 mL of seed culture medium to the electroporation cup and mix well by pipetting. Transfer all the bacterial solution from the electroporation cup to a centrifuge tube and incubate at 30°C and 180 rpm for 2 hours with shaking. (4) After resuscitation, centrifuge the centrifuge tube at 5000 rpm for 2 min, discard 800 μL of supernatant, and resuspend the bacterial cells in the remaining supernatant. Spread the bacterial culture on YM solid medium plates containing 10 μg / mL CHL and incubate at 30℃ for 72 h.

[0058] The verification steps are the same as in 1.2.6 1). Since sequencing verification has already been performed during plasmid construction, sequencing verification is not performed in this step.

[0059] 1.3.3 Measurement of fluorescence intensity using an ELISA reader Positive transformants were selected and inoculated into YM liquid medium containing 15 μg / mL chloramphenicol. The culture was incubated at 30°C with shaking until the logarithmic growth phase. The GFP fluorescence intensity (excitation wavelength 488 nm, emission wavelength 507 nm) and OD of the bacterial culture were measured using a full-wavelength microplate reader. 600 Value. Expressed as fluorescence intensity / OD 600 The ratio represents promoter activity.

[0060] Experimental results: Through transcriptome data analysis, this invention uses the TPM (Transcripts Per Million) value as a standardized indicator of gene expression abundance. Its calculation, by eliminating sequencing depth and gene length biases, enables accurate comparisons of expression levels across samples. Results are as follows... Figure 1 (Figure showing the results of host bacteria expressing candidate promoters at different growth stages). S. paucimobilis Endogenous promoter P JDP TPM values ​​at different growth stages were significantly higher than those of other candidate promoters. Transcriptome analysis showed that P had the highest TPM value. JDP for S. paucimobilis The endogenous promoter with the highest startup efficiency.

[0061] like Figure 2 (The graph shows the results of fluorescence intensity measurements for different promoters), P JDP The fluorescence signal intensity driven by the promoter was significantly higher than that of other candidate promoters, confirming it as a highly active endogenous promoter.

[0062] Example 2 Construction of CRISPRi / dCas12a system plasmid pBBR1-P JDP -dCas12a-crRNA Experimental methods: Construction of plasmid pBBR1-P JDP The system procedure for -dCas12a-crRNA is as follows: Figure 3 : 2.1.1 P JDP Fragment acquisition Based on the principle of seamless cloning, primers were designed to amplify P using the genome of *Sphingomonas sphingosine monocytogenes* ATCC31461 as a template. JDP -F / R amplification P JDP The primers were added to double-distilled water to a final concentration of 100 pmol / μL, and the gRNA was annealed to form double strands. The system is shown in Table 6. Table 6 Reaction System

[0063] The annealing reaction procedure is as follows: ① 37℃ for 30 min; ② 95℃ for 5 min; then decrease to 25℃ at a rate of 5℃ / min.

[0064] 2.1.2 Acquisition of the carrier Using pBBR1-Ptac-dCas12a-crRNA plasmid as a template, PCR was performed, and the reaction system is shown in Table 7: Table 7 PCR Reaction System

[0065] The reaction procedure is as follows: ① Pre-denaturation at 94℃ for 3 min, ② Denaturation at 94℃ for 30 s; Annealing at 58℃ for 30 s; Extension at 72℃ at 1 kb / min. Repeat the above cycle 32 times, ③ Extension at 72℃ for 10 min.

[0066] After the PCR products were verified by agarose gel electrophoresis, the correct bands were cut and recovered. The gel recovery procedure is described in the GelExtraction Kit.

[0067] 2.1.3 P JDP Seamless cloning of fragments and gel-recovered pBBR1-Ptac-dCas12a-crRNA vector The reaction system is shown in Table 8: Table 8 PCR Reaction System

[0068] Mix gently and centrifuge for a few seconds. Incubate at 50°C for 15 minutes on a PCR instrument.

[0069] 2.1.4 Positive monoclonal pBBR1-P JDP Screening of -dCas12a-crRNA plasmids The experimental method is the same as in 1.2.5. 2.1.5 Verification of Connection Results 1) Colony PCR: Single colonies growing on the surface of LB / CHL plates were picked and prepared into bacterial suspensions. The reaction system is shown in Table 9. Table 9 Reaction System

[0070] The reaction procedure is described above. For PCR products verified correctly by agarose gel electrophoresis, incubate the corresponding bacterial suspension overnight at 37°C.

[0071] 2) Plasmid PCR, the specific steps are the same as above.

[0072] 3) Sequencing verification, the specific steps are the same as above.

[0073] 2.2 Construction of experimental strains S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA) 2.2.1 S. paucimobilis Preparation of ATCC 31461 competent cells The experimental method is the same as in 1.3.1.

[0074] 2.2.2 Recombinant plasmid pBBR1-P JDP -dCas12a-crRNA electroporation to S. paucimobilis The experimental method is the same as in 1.3.2.

[0075] 2.2.3 Positive strains S. paucimobilis (pBBR1-P) JDP Validation of -dCas12a-crRNA The verification steps are the same as before. Since sequencing verification has already been performed during plasmid construction, sequencing verification is not performed in this step.

[0076] 2.3 RT-qPCR determination of dcas12a expression level in the CRISPRi optimized system To construct successful experimental strains S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA) and S. paucimobilis(pBBR1-Ptac-dCas12a-crRNA) was used as the experimental group, with recombinant bacteria S. paucimobilis (pBBR1-0-dCas12a) served as a control, inoculated into YM liquid medium containing 15 μg / mL CHL and cultured at 30°C with shaking until the stationary phase, from which total bacterial RNA was extracted. The recombinant plasmid pBBR1-0-dCas12a in the control strain is characterized by containing a segment of the dcas12a gene without a promoter.

[0077] 2.3.1 Extraction of total bacterial RNA (1) Take 2 mL of bacterial culture into a 1.5 mL centrifuge tube, centrifuge at 8000 g and 4℃ for 10 min, discard the supernatant and leave the bacterial precipitate; (2) First, add 1 mL of RNAiso Plus solution to the centrifuge tube to dissolve the cell pellet, and repeatedly blow and aspirate the solution to mix it evenly until there is no obvious pellet. (3) After the centrifuge tube has been placed on ice for 5 minutes, add 200 μL of chloroform to the centrifuge tube, seal the centrifuge tube tightly, and vortex until the solution turns milky white. (4) Let stand on ice for 10 min, centrifuge at 8000g, 4C for 15 min. After centrifugation, the tube will separate into three layers: the uppermost aqueous phase contains RNA. (5) Take 400 μL of the clear upper layer solution and add it to a new centrifuge tube. Add an equal volume of 400 μL of isopropanol, gently invert and mix 10 times, and let stand on ice for 20 min. White flocculent matter can be observed. (6) Centrifuge at 8000g, 4℃ for 15min, use a pipette to remove the supernatant, leaving the white precipitate at the bottom of the centrifuge tube, which is the RNA; (7) Add 1 mL of 75% ethanol to the centrifuge tube to wash the precipitate, gently invert or tap the bottom of the tube, centrifuge at 8000 g, 4℃ for 10 min; (8) Remove the supernatant with a pipette and dry the bottom precipitate (blow air inside the cleanroom for 10 min); (9) Add 50 μL of DEPC water to dissolve the RNA, divide it into 3 portions, one portion for DNA concentration determination, another portion for reverse transcription; the remainder can be stored at -80℃.

[0078] 2.3.2 Reverse transcription reaction Reverse transcription was performed using Novizan's HiScript III RT SuperMix for qPCR (+gDNA wiper) kit.

[0079] The first step is to remove tissue DNA. The reaction system is shown in Table 10. Table 10 Reaction System

[0080] Gently blow or aspirate the mixed reaction solution, place the PCR tube in the PCR instrument, and proceed as follows: 42 ℃ for 2 min.

[0081] The second step is reverse transcription, and the reaction system is shown in Table 11: Table 11 Reaction System

[0082] Gently mix the reaction solution, place the PCR tube in the PCR instrument, and proceed according to the following procedure: ① 37℃ for 15 min ② 85℃ for 5 s. After the reaction is complete, store the reverse transcription product cDNA solution at -20℃.

[0083] 2.3.3 Real-time quantitative PCR by S. paucimobilis Using 16S rRNA as an internal reference gene, relative quantification of dCas12a was performed on recombinant bacteria containing different CRISPRi systems and their respective control groups. RT-dCas12a-F / R primers were used to target the recombinant bacteria. S.paucimobilis (pBBR1-P) JDP -dCas12a-crRNA), recombinant bacteria S.paucimobilis (pBBR1-Ptac-dCas12a-crRNA) and S.paucimobilis The dcas12a gene of (pBBR1-0-dCas12a) was relatively quantified. The control strain was used. S. paucimobilis The expression level of the dcas12a gene in (pBBR1-0-dCas12a) is defined as 1. The expression levels of the dcas12a gene in the recombinant bacteria are calculated separately. （-ΔΔCt） The value represents the relative expression level of the dcas12a gene in different CRISPRi systems. Results are expressed as mean ± standard error (mean ± SEM) of three parallel replicates. Differences between groups were analyzed using a t-test. This indicates that P < 0.05. This indicates that P < 0.01.

[0084] 2.4 Determination of the cytotoxicity of the CRISPRi / dCas12a optimized system 2.4.1 Determining the number of transformants in the CRISPRi system To compare the two systems (pBBR1-P) JDP -dCas12a-crRNA and pBBR1-Ptac-dCas12a-crRNA) in S. paucimobilisThe experiment determined the performance of the two CRISPRi systems by measuring the number of transformants. S. paucimobilis Toxicity. In experimental procedure 2.2.2, 100 μL of the revived bacterial culture was serially diluted to 10⁻⁶. -7 Each gradient of bacterial culture was spread onto YM solid medium plates containing 15 μg / mL CHL, and incubated upside down at 30°C for 72 h before counting the number of colonies.

[0085] 2.4.2 Determination of bacterial growth curve Successfully constructed recombinant bacteria S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA), S. paucimobilis (pBBR1-Ptac-dCas12a-crRNA) was used in the experimental group and S. paucimobilis (pBBR1MCS-2) control group: Growth curves were measured by inoculating the above strains into YM liquid medium containing 15 μg / mL CHL and culturing for 36 h. The bacterial solutions were then inoculated into 50 mL Erlenmeyer flasks containing 20 mL of medium, shaken, and cultured. The bacterial solutions were sampled every 2 h, and the OD value was measured using a microplate reader. 600 The absorbance values ​​were calculated and plotted as growth curves.

[0086] 2.5 Determining the genetic stability of the CRISPRi / dCas12a optimized system It can be detected by the strain *S. paucimobilis* (pBBR1-P) containing the CRISPRi system. JDP The genetic stability of CRISPRi was assessed by continuous passage experiments of dCas12a-crRNA. The experimental method was the same as that described in (Shin J, Baek JY, Chung DR, et al. Instability of the IncFII-Type Plasmid Carrying blaNDM-5 in a Klebsiellapneumoniae Isolate [J]. Journal of microbiology and biotechnology, 2017, 27(9): 1711-1715).

[0087] Experimental results (1) CRISPRi / dCas12a system plasmid pBBR1-P JDP Construction of dCas12a-crRNA pBBR1-P JDP-dCas12a-crRNA ligation product transformed to E. coli The top 10 competent cells were picked the following day and colonies grown on LB / CHL plates were PCR-verified, followed by shaking and plasmid extraction. Three plasmids with correct bands were selected, and unidirectional sequencing was performed using dCas12a-V-R1 and dCas12a-V-R2 as sequencing primers. The sequencing results were compared using DNAMAN software. Figure 4 (recombinant plasmid pBBR1-P) JDP The sequencing alignment results of the dCas12a-crRNA are shown in the figure. The recombinant plasmid pBBR1-P JDP -dCas12a-crRNA plasmid was successfully constructed.

[0088] (2) Experimental strains S. paucimobilis (pBBR1-P) JDP Construction of -dCas12a-crRNA The validated colony PCR products were verified by 1% agarose gel electrophoresis. The results are as follows: Figure 5 (pBBR1-P) JDP -dCas12a-crRNA conversion S. paucimobilis Post-colony PCR results; where M is the 2k Marker; lane 1 is ddH2O; lanes A (2-7) are pBBR1-P JDP -dCas12a-crRNA electroporation S. paucimobilis As shown in the colony PCR (in microscopy), the correct band amplified by the extracted plasmid should be between 750 and 1000 bp. The recombinant strain was verified. S. paucimobilis (pBBR1-P) JDP The dCas12a-crRNA was successfully constructed.

[0089] (3) Determination of dcas12a expression level in CRISPRi optimization system Recombinant bacteria were detected by RT-qPCR. S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA) and recombinant bacteria S. paucimobilis The relative expression level of the dCas12a gene in (pBBR1-Ptac-dCas12a-crRNA). Both used 16S rRNA as an internal reference gene. S. paucimobilis (pBBR1-0-dcas12a) is the control strain. The pBBR1-0-dcas12a plasmid does not contain the dcas12a promoter. The relative expression level of dcas12a in the control strain is defined as 1, and 2 is calculated based on the obtained Ct value. -ΔΔCtThe relative expression levels of the dcas12a gene were obtained. Results are expressed as mean ± standard error (mean ± SEM) of three parallel replicates. Differences between groups were analyzed using a t-test. This indicates that P < 0.05. This indicates that P < 0.01. The results are as follows: Figure 6 (The graph shows the expression levels of dCas12a in the dCas12a system before and after optimization.) The optimized dCas12a system showed an expression level approximately 82.76 times that of the control group, while the unoptimized system showed an expression level approximately 22.49 times that of the control group. This means the optimized dCas12a system showed a 268% increase in expression, indicating that the new promoter significantly outperforms the old promoter in driving dCas12a expression in *Sphingosine monocytogenes*, thus enhancing the expression of dCas12a in... S. paucimobilis The level of expression.

[0090] (4) Determination of cytotoxicity of the CRISPRi / dCas12a optimized system To analyze and compare the effects of the two CRISPRi systems on S. paucimobilis Toxicity, experiments were conducted using strains S. paucimobilis (pBBR1MCS-2) served as the control group, and the number of transformants in the two dCas12a systems was determined under the same electroconversion conditions.

[0091] The results are as follows Figure 7 (The results of the number of transformants in the dCas12a system before and after optimization are shown in the figure.) The number of transformants that can grow on the plate of the dCas12a system plasmid after transformation optimization and the cytotoxicity are not significantly different from those of the dCas12a system plasmid before transformation optimization, indicating that the two have similar toxicity to Sphingosine monocytogenes.

[0092] (5) Determination of the genetic stability of the CRISPRi / dCas12a optimized system The experiment was conducted using strains S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA) and strain S. paucimobilis The genetic stability of the CRISPRi system was assessed through successive passage experiments using (pBBR1-Ptac-dCas12a-crRNA), and the results are as follows: [[ID= (Figure showing the genetic stability and PCR verification results of the CRISPRi system in continuous subculture) shows that it can be stably subcultured for 50 generations under antibiotic pressure of 15 µg / mL CHL.

[0093] Example 3 Suppressing alone using the CRISPRi / dCas12a system ​ , ​ Genes to reduce PHB synthesis Experimental methods: 3.1 Constructing Targets ​ The CRISPRi / dCas12a gene system 3.1.1 Design and Synthesis of gRNA For the CRISPRi / dCas12a system, the PAM sequence recognized by the dCas12a protein is 3'-TTTV-5'. Therefore, the target site of the gRNA should be immediately downstream of this sequence and should not have any special secondary structures. Sequence specificity was also determined using NCBI. Based on this, six gRNAs were designed targeting the phaB gene: phaB1', phaB2', phaB3', phaB4', phaB5', and phaB6'. The first three target the coding strand, and the latter three target the non-coding strand. Primers phaBn'-F / R (n=1, 2, 3, 4, 5, 6) were synthesized and purified using PAGE. In addition, five sites targeting the predicted phaB promoter, P1, P2, P3, P4, and P5, were designed. Primers Pn-F / R were also synthesized and purified using PAGE (n=1, 2, 3, 4, 5).

[0094] 3.1.2 gRNA annealing to form sticky ends Primers were added to double-distilled water to a final concentration of 100 pmol / μL, and gRNA was annealed to form double strands. The system is shown in Table 12. Table 12 Reaction System

[0095] The annealing reaction procedure is as follows: ① 37℃ for 30 min; ② 95℃ for 5 min; then decrease to 25℃ at a rate of 5℃ / min.

[0096] 3.1.3 Vector pBBR1-P JDP -dCas12a-crRNA linearization The CRISPRi / dCas12a system vector was used with restriction endonuclease ​ Q is linearized. The reaction system is shown in Table 13: Table 13 Reaction System

[0097] The reaction procedure was as follows: incubation at 50℃ for 5 h, followed by gel extraction and recovery of the correct bands after verification by 1% agarose gel electrophoresis. Gel recovery was performed using the OMEGA Gel Extraction Kit; please refer to the kit's instruction manual for specific procedures.

[0098] 3.1.4 Ligation of gRNA annealing product with linearized vector The linearized dCa12a vector was ligated with the gRNA annealing product using T4 DNA Ligase. The ligation system is shown in Table 14. Table 14 Connection System

[0099] The reaction procedure is as follows: 16°C, overnight. The reaction product can be stored at -20°C.

[0100] 3.1.5 pBBR1-P JDP -dCas12a-phaBn' / Pn linkage product chemically converted to ​ Top 10 The experimental method is the same as in 1.2.5.

[0101] 3.1.6 Verification of Connection Results (1) Colony PCR: Single colonies growing on the surface of LB / CHL plates were picked and prepared into bacterial suspensions. The PCR verification reaction system is shown in Table 15. Table 15 Reaction System

[0102] The reaction procedure was the same as in Example 2. After the PCR products were verified by agarose gel electrophoresis, they were incubated overnight at 37°C.

[0103] (2) Plasmid PCR, the reaction system is the same as (1).

[0104] (3) Sequencing verification, the experimental steps are the same as above.

[0105] 3.2 Constructing Targets ​ The CRISPRi / dCas12a gene system 3.2.1 Design and Synthesis of gRNA For the CRISPRi / dCas12a system, the PAM sequence recognized by the dCas12a protein is 5'-TTTV-3'. Therefore, the target site of the gRNA should be immediately downstream of this sequence and should not have any special secondary structures. Sequence specificity should be determined using NCBI. Based on this, targeting... ​Six gRNAs were designed for the gene: phaC1', phaC2', phaC3', phaC4', phaC5', and phaC6'. The first three target the coding strand, and the latter three target the non-coding strand. Primers phaCn-F / R (n=1, 2, 3, 4, 5, 6) were synthesized and purified by PAGE. In addition, five sites targeting the predicted phaC promoter (P1, P2, P3, P4, P5) were designed; primers Pn-F / R were also synthesized and purified by PAGE (n=1, 2, 3, 4, 5).

[0106] 3.2.2 gRNA annealing to form sticky ends Primers were added to double-distilled water to a final concentration of 100 pmol / μL, and gRNA was annealed to form double strands. The system is shown in Table 16. Table 16 Reaction System

[0107] The annealing reaction procedure is as follows: ① 37℃ for 30 min; ② 95℃ for 5 min; then decrease to 25℃ at a rate of 5℃ / min.

[0108] 3.2.3 Vector pBBR1-P JDP -dCas12a-crRNA linearization The CRISPRi / dCas12a system vector was used with restriction endonuclease ​ Q is linearized. The reaction system is shown in Table 17: Table 17 Reaction System

[0109] The reaction procedure was as follows: incubation at 50℃ for 5 h, followed by gel extraction and recovery of the correct bands after verification by 1% agarose gel electrophoresis. Gel recovery was performed using the OMEGA Gel Extraction Kit; please refer to the kit's instruction manual for specific procedures.

[0110] 3.2.4 Ligation of gRNA annealing product with linearized vector The linearized dCa12a vector was ligated with the gRNA annealing product using T4 DNA Ligase. The ligation system is shown in Table 18. Table 18 Reaction System

[0111] The reaction procedure is as follows: overnight at 16°C. The reaction products can be stored at -20°C.

[0112] 3.2.5 pBBR1-P JDP-dCas12a-phaCn' / Pn linkage product chemically converted to ​ Top 10 The experimental method is the same as in 1.2.5.

[0113] 3.2.6 Verification of Connection Results (1) Colony PCR: Single colonies growing on the surface of LB / CHL plates were picked and prepared into bacterial suspensions. The PCR verification reaction system is shown in Table 19: Table 19 Reaction System

[0114] The reaction procedure was the same as in Example 2. After the PCR products were verified by agarose gel electrophoresis, they were incubated overnight at 37°C.

[0115] (2) Plasmid PCR, the reaction system is the same as (1).

[0116] (3) Sequencing verification, the experimental steps are the same as above.

[0117] 3.3 Construction of experimental strains ​ (pBBR1-P) JDP -dCas12a-phaBn' / Pn) 3.3.1 ​ Preparation of ATCC 31461 competent cells The experimental method is the same as in 1.3.1.

[0118] 3.3.2 Recombinant plasmid pBBR1-P JDP -dCas12a-phaBn' / Pn transfer to ​ The experimental method is the same as in 1.3.2.

[0119] 3.3.3 Validation of positive strains in experimental strains The verification steps are the same as described above. Since sequencing verification has already been performed during plasmid construction, no further sequencing verification is required in this step.

[0120] 3.4 Construction of experimental strains ​ (pBBR1-P) JDP -dCas12a-phaCn' / Pn) 3.4.1 ​ Preparation of ATCC 31461 competent cells The experimental method is the same as in 1.3.1.

[0121] 3.4.2 Recombinant plasmid pBBR1-P JDP -dCas12a-phaCn' / Pn transfer to ​ The experimental method is the same as in 1.3.2.

[0122] 3.4.3 Validation of positive strains in experimental strains The verification steps are the same as described above. Since sequencing verification has already been performed during plasmid construction, no further sequencing verification is required in this step.

[0123] 3.5 RT-qPCR was used to determine the expression level of the target gene. To successfully construct the CRISPRi / dCas12a strain ​ (pBBR1-P) JDP -dCas12a-phaBn' / phaCn') and S. ​ (pBBR1-P) JDP -dCas12a-phaBPn / phaCPn) was the experimental group, with S. ​ (pBBR1-P) JDP The bacterial culture (-dCas12a-crRNA) was used as a control and inoculated into YM liquid medium containing 15 μg / mL CHL. The bacterial culture was cultured at 30°C with shaking until the stationary phase, and then used to extract total bacterial RNA.

[0124] 3.5.1 Extraction of total bacterial RNA The extraction method is the same as in 2.3.1.

[0125] 3.5.2 Reverse transcription reaction The experimental method is the same as in 2.3.2.

[0126] 3.5.3 Real-time quantitative PCR The reverse transcription product cDNA was used as a template for quantitative real-time PCR of the test samples, and the reaction was performed using Green qPCRMasterMix in a CFX Opus 96 Real-Time PCR System. ​ The 16S rRNA of ATCC31461 was used as an internal reference gene for recombinant bacteria. ​ (pBBR1-P) JDP -dCas12a-phaBn' / phaCn'), and ​ (pBBR1-P) JDP -dCas12a-phaBPn / phaCPn) and control group S. ​ (pBBR1-P) JDP The phaB and phaC genes in dCas12a-crRNA were relatively quantified. The reaction procedure and analytical method were the same as in 2.3.3.

[0127] 3.6 Determining the growth curve of the strain during shake-flask fermentation. Successfully constructed CRISPRi strain ​ (pBBR1-P) JDP -dCas12a-phaBn' / phaCn') and ​ (pBBR1-P) JDP The experimental group consisted of empty vector strains (-dCas12a-phaBPn / phaCPn). S. ​ (pBBR1-P) JDP The growth curve was measured using dCas12a-crRNA as the control group. For specific methods, please refer to 2.4.2.

[0128] 3.7 Determination of Gellan Gum Production (1) The successfully constructed CRISPRi strain ​ (pBBR1-P) JDP -dCas12a-phaBn' / phaCn'), ​ (pBBR1-P) JDP -dCas12a-phaBPn / phaCPn) and empty control strain S. ​ (pBBR1-P) JDP -dCas12a-crRNA) was inoculated into YM medium containing 10 μL / mL CHL and cultured at 30°C with shaking for 24 h to obtain the seed culture. 10 ml of the seed culture was transferred to a 500 mL Erlenmeyer flask containing 90 mL of fermentation medium and fermented at 30°C with shaking for 72 h.

[0129] (2) Take 10 g of fermentation broth from different fermentation times (48 h, 72 h) into centrifuge bottles, add 90 mL of H2O, heat the centrifuge bottles in boiling water for 15 min, centrifuge at 8000 rpm for 45 min, pour the supernatant into 300 mL of isopropanol, and let stand overnight at 4℃. Collect the gellan gum polysaccharide by centrifugation, dry it at 80℃ using an MA35 infrared moisture analyzer, and determine its weight.

[0130] 3.8 Determination of PHB content Weigh 10 g of fermentation broth into a centrifuge tube, centrifuge to collect the cell bodies, add 40% sodium hypochlorite solution (v / v), place in a shaker at 30℃, and react for 30 min. After the reaction is complete, centrifuge at 10000 rpm / min for 20 min and collect the solid precipitate. Resuspend the precipitate in distilled water, centrifuge at 10000 rpm / min for 20 min, and finally collect the precipitate. Dry at 40℃ to obtain the crude PHB extract, and weigh it.

[0131] 3.9 Determination of the transparency of gellan gum (1) Weigh 0.1g of gellan gum sample and put it into a beaker. Add a measured amount of 85℃ deionized water to the beaker.

[0132] (2) Keep warm on a magnetic stirrer and stir until the sample is completely dissolved.

[0133] (3) Remove and cool to 60°C. While still hot, pour the above sample solution into the cuvette to 2 / 3 of its height. Allow it to cool naturally to room temperature and store in a refrigerator at 4°C for 24 hours.

[0134] (4) Read and record the transmittance at a wavelength of 490 nm. Each sample was measured in triplicate, and the experimental results are the average of the three experiments ± standard deviation. The higher the transmittance, the better the transparency.

[0135] Experimental results (1) Individual target ​ Construction of CRISPRi strain To obtain strains containing the target phaB and its predicted promoter CRISPRi / dCas12a system, plasmid pBBR1-P was used. JDP -dCas12a-phaBPn / n' (n=1-5, n'=1-6) are electrotransformed to respectively ​ In competent cells, single colonies were picked from plates, revived, and plasmids were extracted. Using the extracted plasmids as templates and phaBPn / n'-F as the upper primer and crRNA-VR as the lower primer, PCR was performed to verify and screen for positive clones. ​ , ​ As shown: At least one sample of each transformation product amplified the target band of the correct size. Recombinant plasmid pBBR1-P JDP -dCas12a-phaBPn / n' have all been successfully converted to ​ Strains containing the target phaB and its CRISPRi / dCas12a system have been successfully constructed.

[0136] ​ pBBR1-P JDP Image of colony PCR results after transformation of *S. paucimobilis* with dCas12a-PhaB-Pn; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are for electroporation with PhaB-P1. ​ Image of colony PCR results; B, lanes 2-7, PhaB-P2 electroporation ​ Image of colony PCR results; C, lanes 2-7, PhaB-P3 electroporation ​ Image of colony PCR results; D, lanes 2-7, PhaB-P4 electroporation​ Image of colony PCR results; Lanes E 2-7, PhaB-P5 electroporation ​ Image of PCR results for bacterial colonies in the middle.

[0137] ​ pBBR1-P JDP PCR results of *S. paucimobilis* colonies transformed with dCas12a-phaBn'; where M is the 2k Marker; lane 1 is ddH2O; and lanes A are pBBR1-P... JDP -dCas12a-phaB1' Electric transfer S. ​ Image of colony PCR results; B, lanes 2-7, pBBR1-P JDP -dCas12a-phaB2' Electric transfer S. ​ Image of colony PCR results; C, lanes 2-7, pBBR1-P JDP -dCas12a-phaB3' Electric transfer S. ​ Image of colony PCR results; D, lanes 2-7, pBBR1-P JDP -dCas12a-phaB4' Electric transfer S. ​ Image of colony PCR results; E, lanes 2-7, pBBR1-P JDP -dCas12a-phaB5' Electric transfer S. ​ Image of colony PCR results; F, lanes 2-7, pBBR1-dCas12a-phaB6' electroporation ​ Image of PCR results for bacterial colonies in the middle.

[0138] (2) Individual target ​ Construction of CRISPRi strain To obtain target ​ Strains of the CRISPRi / dCas12a system, which predicts the promoter, will use plasmid pBBR1-P JDP -dCas12a-phaCPn / n' (n=1-5, n'=1-6) are electrotransformed to respectively ​ In competent cells, single colonies were picked from plates, revived, and plasmids were extracted. Using the extracted plasmids as templates and phaCPn / n'-F as the upper primer and crRNA-VR as the lower primer, PCR was performed to verify and screen for positive clones. ​ , ​ As shown: At least one sample of each transformation product amplified the target band of the correct size. Recombinant plasmid pBBR1-P JDP-dCas12a-phaCPn / n' have all been successfully converted to ​ Contains target ​ The strain of the CRISPRi / dCas12a system, which includes the predicted promoter, has been successfully constructed.

[0139] ​ pBBR1-P JDP -dCas12a-phaC-Pn conversion ​ Post-colony PCR results; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are phaC-P1 electroporation. ​ Image of colony PCR results; B, lanes 2-7, phaC-P2 electroporation S. paucimobilis Image of colony PCR results; C, lanes 2-7, phaC-P3 electroporation S. paucimobilis Image of colony PCR results; D, lanes 2-7, electroporation of phaC-P4 S. paucimobilis Image of colony PCR results; E, lanes 2-7, phaC-P5 electroporation S. paucimobilis Image of PCR results for bacterial colonies in the middle.

[0140] Figure 12 pBBR1-P JDP -dCas12a-phaCn' conversion S. paucimobilis Post-colony PCR results; where M is the 2k Marker; lane 1 is ddH2O; and lanes 2-7 are pBBR1-dCas12a-phaC1' electroporation. S. paucimobilis Image of colony PCR results; B, lanes 2-5, pBBR1-P JDP -dCas12a-phaC2' electro-transfer S. paucimobilis Image of colony PCR results; C, lanes 2-7, pBBR1-P JDP -dCas12a-phaC3' Electric converter S. paucimobilis Image of colony PCR results; D, lanes 2-7, pBBR1-P JDP -dCas12a-phaC4' electro-transfer S. paucimobilis Image of colony PCR results; E, lanes 2-7, pBBR1-P JDP -dCas12a-phaC5' Electric converter S. paucimobilis Image of colony PCR results; F, lanes 2-5, pBBR1-P JDP -dCas12a-phaC6' Electric converter S. paucimobilis Image of PCR results for bacterial colonies in the middle.

[0141] (2) phaB, phaC Determination of gene expression levels To test the CRISPRi / dCas12a system's performance phaB The transcriptional repression effect of genes was investigated using RT-qPCR. The experimental strains were... S. paucimobilis (pBBR1-P) JDP -dCas12a-phaBn') and S. paucimobilis (pBBR1-P) JDP Total RNA was extracted and measured after the culture of dCas12a-phaBPn (dCas12a-phaBPn) reached the stationary phase. phaB The expression level of the control strain phaB The relative expression level of a gene is defined as 1. The results are as follows: Figure 13 ( phaB Relative gene expression levels (Figure) P<0.05 As shown in the figure (P<0.01): like Figure 13 As shown, in the target phaB and its predicted promoter P JDP Among the CRISPRi strains, phaB Gene expression levels ranged from 12.19% to 77.70%, with gRNA P4 targeting the promoter region showing the best performance in inhibition, achieving a knockdown efficiency of 77.70%.

[0142] To test the CRISPRi / dCas12a system's performance phaC The transcriptional repression effect of genes was investigated using RT-qPCR. The experimental strains were... S. paucimobilis (pBBR1-P) JDP -dCas12a-phaCn') and S. paucimobilis (pBBR1-P) JDP Total RNA was extracted and measured after the culture of dCas12a-phaCPn (dCas12a-phaCPn) reached the stationary phase. phaC The expression level of the control strain phaC The relative expression level of a gene is defined as 1. The results are as follows: Figure 14 ( phaC Relative gene expression levels (Figure) P<0.05 As shown in the figure (P<0.01): like Figure 14 As shown, in the target phaC and its predicted promoter P JDP Among the CRISPRi strains, phaC Gene expression levels ranged from 0.22% to 90.80%, with gRNA phaC4' targeting the gene coding region showing the best performance, achieving a knockdown efficiency of up to 90.80%.

[0143] (3) Growth curve of strain and determination of residual sugar This experiment analyzed the shake-flask fermentation process of all experimental strains, including the determination of three aspects: shake-flask fermentation growth curves, residual sugar content, and gum production. The strains were used as the basis for the analysis. S. paucimobilis (pBBR1-Ptac-dCas12a-crRNA) was used as a control to measure the experimental strains. S. paucimobilis (pBBR1-P) JDP -dCas12a-phaB / phaCn') and S. paucimobilis (pBBR1-P) JDP The growth of (-dCas12a-phaB / phaCPn) in the fermentation broth. The growth curve is shown below. Figure 15 (Experimental strain pBBR1-P) JDP (Results of growth curve determination of -dCas12a-phaBn' / Pn) S. paucimobilis (pBBR1-P) JDP -dCas12a-phaBn') and S. paucimobilis (pBBR1-P) JDP The growth rate of -dCas12a-phaBPn in the fermentation broth and its relationship with OD 600 The values ​​were not significantly different from those of the control bacteria. The growth curve is shown below. Figure 16 (Experimental strain pBBR1-P) JDP (Results of growth curve determination of -dCas12a-phaCn' / Pn) S. paucimobilis (pBBR1-P) JDP -dCas12a-phaCn') and S. paucimobilis (pBBR1-P) JDP The growth rate of -dCas12a-phaCPn in the fermentation broth and its relationship with OD 600 The values ​​were not significantly different from those of the control bacteria.

[0144] (4) Determination of gellan gum production Figure 17 The graph shows the results of the gel production measurement of the experimental strain. P<0.05 (P<0.01 #, P<0.05 ##, P<0.01), such as Figure 17 As shown, the fermentation broths of the control strain and the CRISPRi strain were weighed and gelled, with phaBP4 and phaBP5 sites as the target sites. S. paucimobilis The strain showed a significant increase in gelatin production at 48 h. The same characteristic was observed in the gelatin production at 72 h for these experimental strains. phaB The relative expression level was also lower. Compared with the control,S. paucimobilis (pBBR1-P) JDP The gellan gum yield of S. paucimobilis (pBBR1-P) was 6.06 ± 0.10 g / L, showing the most significant increase, compared with the control S. paucimobilis (pBBR1-P). JDP Compared to (-dCas12a-crRNA)(phaB0'), it increased by approximately 15.73%.

[0145] Figure 18 The graph shows the results of the gel production measurement of the experimental strain. P<0.05 (P<0.01 #, P<0.05 ##, P<0.01), such as Figure 18 As shown, the fermentation broths of the control strain and the CRISPRi strain were weighed and gelled, with phaC4' and phaCP5 sites as the target sites. S. paucimobilis The strain showed a significant increase in gelatin production at 48 h. The same characteristic was observed in the gelatin production at 72 h for these experimental strains. phaC The relative expression level was also lower. Compared with the control, S. paucimobilis (pBBR1-P) JDP The gellan gum yield of the (-dCas12a-phaC4') group was 5.80 ± 0.10 g / L, showing the most significant increase compared to the control. S. paucimobilis (pBBR1-P) JDP Compared to (-dCas12a-crRNA)(phaC0'), it increased by approximately 12.45%.

[0146] (5) Determination of PHB content Weigh out the crude PHB extracts from the fermentation broths of the control strain and the CRISPRi strain, and target the phaB3', phaB4', phaB5', phaB6', P1, P2, P3, P4, and P5 sites. S. paucimobilis The PHB production of these strains was significantly reduced at 48 h compared to the control strain. The same pattern of PHB changes was observed at 72 h, and the relative expression levels of phaB in these experimental strains were also lower. Compared to the control, S. paucimobilis (pBBR1-P) JDP The PHB production of dCas12a-phaBP4 was 0.291 ± 0.008 g / L, showing the most significant decrease compared to the control S. paucimobilis (pBBR1-P). JDP Compared to (-dCas12a-crRNA)(phaB0'), it was reduced by approximately 16.90%. The results are as follows... Figure 19 (Figure showing the results of PHB measurement for experimental strains) P<0.05 The values ​​are shown in the table (P<0.01, P<0.05, P<0.01).

[0147] Weigh out the crude PHB extracts from the fermentation broths of the control strain and the CRISPRi strain, and target the phaC3', phaC4', phaC5', phaC6', P1, P2, P3, P4, and P5 sites. S. paucimobilis The strain exhibited a significantly reduced PHB production at 48 h compared to the control strain. The same characteristic change in PHB levels was observed at 72 h in these experimental strains. phaC The relative expression level was also lower. Compared with the control, S. paucimobilis (pBBR1-P) JDP The PHB production of the α-dCas12a-phaC4' group was 0.261 ± 0.010 g / L, showing the most significant decrease compared to the control. S. paucimobilis (pBBR1-P) JDP Compared to (-dCas12a-crRNA)(phaC0'), it was reduced by approximately 24.10%. The results are as follows... Figure 20 (Figure showing the results of PHB measurement for experimental strains) P<0.05 The values ​​are shown in the table (P<0.01, P<0.05, P<0.01).

[0148] (6) Determination of the transparency of gelling gum Weigh the fermentation broths of the control strain and the CRISPRi strain, and compare their gel transparency when gelled. S. paucimobilis (pBBR1-P) JDP The transparency of (-dCas12a-phaBP4) was most significantly increased, compared with the control. S. paucimobilis (pBBR1-P) JDP Compared to -dCas12a-crRNA, it increased by approximately 51.07%. The results are as follows... Figure 21 (Results of transparency measurement of gel formed by experimental strains in gelling gum) P<0.05 As shown in the figure, P<0.01).

[0149] Weigh the fermentation broths of the control strain and the CRISPRi strain, and measure their gel clarity as shown in the figure. Figure 22 (Results of transparency measurement of gel formed by experimental strains in gelling gum) P<0.05 (P<0.01) Compared with the control, S. paucimobilis (pBBR1-P) JDP The transparency of (-dCas12a-phaC4') increased most significantly, compared with the control. S. paucimobilis (pBBR1-P) JDP Compared to -dCas12a-crRNA, it increased by approximately 44.21%.

[0150] Example 4 Combination suppression using the CRISPRi / dCas12a system phaB and phaC Genes to reduce PHB synthesis Experiments have revealed the target phaB gRNA phaBP4 in the promoter region and its target phaC The gRNA phaC4 in the coding region showed the most significant inhibitory effect, reducing PHB production by 16.9% and 24.1%, respectively (p<0.001). However, single-target inhibition strategies still have limitations: metabolic redundancy in the PHB synthesis pathway may lead to the continued accumulation of some substrates through alternative pathways, and the perturbation of the strain's metabolic network by single-gene inhibition may trigger adaptive feedback (such as dynamic imbalance of the acetyl-CoA pool), limiting further improvement in PHB blocking efficiency. To inhibit the PHB synthesis pathway and optimize metabolic regulation, this embodiment constructs a tandem CRISPRi / dCas12a dual-target combined inhibition system based on the above-mentioned optimal gRNAs (phaBP4 and phaC4). This strategy simultaneously inhibits... phaB and phaC Genes can synergistically reduce PHB synthesis at both the substrate supply and product assembly levels.

[0151] Experimental methods: 4.1 Construction of recombinant plasmid pBBR1-P JDP -dCas12a-phaBP4-phaC4 4.1.1 Design and Synthesis of gRNA For the CRISPRi / dCas12a system, dCas12a can autoprocess precursor crRNA by recognizing a short, 19-base-pair specific repetitive sequence (DR) and release crRNA from the precursor transcript. Utilizing this property, dCas12a has been developed for multiplex genome editing via a single construct in which crRNAs targeting different targets are co-assembled, separated by repetitive sequences (DRs), forming a crRNA array. Accordingly, the two gRNAs phaBP4 and phaC4, which showed the best inhibitory effect in Example 3, were tandemly synthesized using primers phaBP4-phaC4'-F / R, and purified by PAGE.

[0152] 4.1.2 gRNA annealing to form sticky ends The experimental procedure is the same as in 3.1.2, except that the required primers are replaced.

[0153] 4.1.3 Vector pBBR1-P JDP -dCas12a-crRNA linearization The experimental procedure is the same as in 3.1.3.

[0154] 4.1.4 Ligation of gRNA annealing product with linearized vector The experimental procedure is the same as in 3.1.4, except that the gRNA annealing product is replaced.

[0155] 4.1.5 Chemical conversion of the linking product to E. coli Top 10 The experimental method is the same as in 1.2.5.

[0156] 4.1.6 Verification of Connection Results (1) Colony PCR: Single colonies growing on the surface of LB / CHL plates were picked and prepared into bacterial suspensions. The PCR verification reaction system is shown in Table 20: Table 20 Reaction System

[0157] The reaction procedure was the same as in Example 2. After the PCR products were verified by agarose gel electrophoresis, they were incubated overnight at 37°C.

[0158] (2) Plasmid PCR, the reaction system is the same as (1).

[0159] (3) Sequencing verification, the experimental steps are the same as above.

[0160] 4.2 Construction of experimental strain S . paucimobilis (pBBR1-P) JDP -dCas12a- phaBP4- phaC4) 4.2.1 S. paucimobilis Preparation of ATCC 31461 competent cells The experimental method is the same as in 1.3.1.

[0161] 4.2.2 Recombinant plasmid pBBR1-dCas12a-phaBP4-phaC4 was electroporated to S. paucimobilis The experimental method is the same as in 1.3.2.

[0162] 4.2.3 Validation of positive strains in experimental strains The verification steps are the same as described above. Since sequencing verification has already been performed during plasmid construction, no further sequencing verification is required in this step.

[0163] 4.3 RT-qPCR assay phaB , phaC expression level To successfully construct the CRISPRi / dCas12a strain S. paucimobilis (pBBR1-P) JDP -dCas12a-phaBP4-phaC4) was the experimental group, with S. paucimobilis (pBBR1-P) JDP The bacterial culture (-dCas12a-crRNA) was used as a control and inoculated into YM liquid medium containing 15 μg / mL CHL. The bacterial culture was cultured at 30°C with shaking until the stationary phase, and then used to extract total bacterial RNA.

[0164] 4.3.1 Extraction of total bacterial RNA The extraction method is the same as in 2.3.1.

[0165] 4.3.2 Reverse transcription reaction The experimental method is the same as in 2.3.2.

[0166] 4.3.3 Real-time quantitative PCR The reverse transcription product cDNA was used as a template for quantitative real-time PCR of the test samples, and the reaction was performed using Green qPCRMasterMix in a CFX Opus 96 Real-Time PCR System. S. paucimobilis The 16S rRNA of ATCC31461 was used as an internal reference gene for recombinant bacteria. S. paucimobilis (pBBR1-P) JDP -dCas12a-phaBP4-phaC4) and control group S. paucimobilis (pBBR1-P) JDP -dCas12a-crRNA) phaB and phaC Genes were quantified relatively. The reaction procedure and analytical methods were the same as in 2.3.3.

[0167] 4.4 Determining the growth curve of the strain during shake-flask fermentation The successfully constructed CRISPRi strain S. paucimobilis (pBBR1-P) JDP -dCas12a-phaBP4-phaC4) is the experimental strain, and the empty vector strain is... S. paucimobilis (pBBR1-P) JDPThe growth curve of the control strain (-dCas12a-crRNA) was determined using the same experimental method as 2.4.2.

[0168] 4.5 Determination of gellan gum production The experimental method is the same as in 3.7, except that the required strain is replaced with the CRISPRi strain. S. paucimobilis (pBBR1-dP) JDP Cas12a-phaBP4-phaC4) is sufficient.

[0169] 4.6 Determination of PHB content The experimental method is the same as in 3.8, except that the required strain is replaced with the CRISPRi strain. S. paucimobilis (pBBRP) JDP 1-dCas12a-phaBP4-phaC4) is sufficient.

[0170] 4.7 Determining the transparency of gellan gum The experimental method is the same as in 3.9, except that the required strain is replaced with the CRISPRi strain. S. paucimobilis (pBBR1-P) JDP The command is -dCas12a-phaBP4-phaC4.

[0171] Experimental results (1) Combined targets phaB and phaC Construction of CRISPRi strain To obtain strains using the combined target CRISPRi / dCas12a system, plasmid pBBR1-P was used. JDP -dCas12a-phaBP4-phaC4 electroconversion to S. paucimobilis In competent cells, single colonies were picked from plates, revived, and plasmids were extracted. Using the extracted plasmids as templates and phaBP4-F as the upper primer and crRNA-VR as the lower primer, PCR was performed to screen for positive clones. Figure 23 (The image shows the colony PCR results after transformation of *S. paucimobilis* with pBBR1-dCas12a-phaBP4-phaC4; M, 2k Marker; lane 1, ddH2O; lanes 2-7, phaBP4-phaC4 electroporation results of *S. paucimobilis* colonies.) As shown, at least one sample of each transformation product amplified the target band of the correct size. Recombinant plasmid pBBR1-P JDP -dCas12a-phaBP4-phaC4 have all been successfully converted to S. paucimobilis A strain containing the combined target CRISPRi / dCas12a system has been successfully constructed.

[0172] (2) phaB , phaC Determination of gene expression levels To test the effect of the phaBP4-phaC4 combined inhibitory system on phaB and phaC To investigate the transcriptional repression effect of genes, this experiment utilized RT-qPCR. The experimental strains... S. paucimobilis (pBBR1-P) JDP Total RNA was extracted and measured after the culture of (-dCas12a-phaBP4-phaC4) reached the stationary phase. phaB and phaC The expression level of the control strain phaB and phaC The relative expression level of each gene was defined as 1. The results are as follows: Figure 24 ( phaB and phaC The results of the relative expression levels of genes are shown in the figure below: (3) Growth curve and residual sugar determination This experiment used bacterial strains. S. paucimobilis (pBBR1-P) JDP (-dCas12a-crRNA) was used as a control to measure the experimental strains. S. paucimobilis (pBBR1-P) JDP The growth of (-dCas12a-phaBP4-phaC4) in the fermentation broth. The growth curve is shown below. Figure 25 (Experimental strain pBBR1-P) JDP (Figure showing the growth curve determination results of -dCas12a-phaBP4-phaC4) S. paucimobilis (pBBR1-P) JDP The growth rate of (-dCas12a-phaBP4-phaC4) in the fermentation broth and its relationship with OD 600 The values ​​were not significantly different from those of the control bacteria.

[0173] (5) Determination of gellan gum production Weigh out the fermentation broth of the control strain and the phaBP4-phaC4 combined inhibition system strain to form a cryogel, targeting the phaBP4-phaC4 site. S. paucimobilis The strain showed a significant increase in cryolite production at 48 h. The same pattern of change in gel production was observed at 72 h. Compared to the control, S. paucimobilis (pBBR1-P) JDP The gellan gum production of (-dCas12a-phaBP4-phaC4) was 6.32 ± 0.10 g / L, showing the most significant increase, increasing by approximately 20.38%; compared with the highest gellan gum production measured when inhibited alone. S. paucimobilis (pBBR1-P) JDPCompared to -dCas12a-phaBP4), it increased by approximately 4.84%. The results are as follows... Figure 26 (Results of gum production measurement of experimental strains) P<0.05 The values ​​are shown in the table (P<0.01, P<0.05, P<0.01).

[0174] (5) Determination of PHB content Weigh out the fermentation broth of the control strain and the phaBP4-phaC4 combined inhibition system strain to form a cryogel, targeting the phaBP4-phaC4 site. S. paucimobilis The strain showed a significant decrease in PHB production at 48 h compared to the control strain. The same characteristic pattern was observed in PHB production at 72 h. Compared to the control, S. paucimobilis (pBBR1-P) JDP The PHB production of (-dCas12a-phaBP4-phaC4) was 0.238 ± 0.009 g / L, with the most significant increase, followed by a decrease of approximately 31.43%; compared with the lowest PHB production measured when inhibited alone. S. paucimobilis (pBBR1-P) JDP Compared to (-dCas12a-phaC4'), it decreased by approximately 8.81%. The results are as follows... Figure 27 (Figure showing the results of PHB measurement for experimental strains) P<0.05 As shown in the figure, P<0.01).

[0175] (6) Determination of the transparency of gellan gum Weigh the fermentation broths of the control strain and the CRISPRi strain, and compare their gel transparency when gelled. S. paucimobilis (pBBR1-P) JDP The transparency of (-dCas12a-phaBP4-phaC4) increased most significantly, by approximately 56.64%; compared with the highest transparency measured when inhibited alone. S. paucimobilis (pBBR1-P) JDP Compared to -dCas12a-phaBP4), it increased by approximately 5.43%. The results are as follows... Figure 28 (Results of transparency measurement of gel formed by experimental strains in gelling gum) P<0.05 As shown in the figure, P<0.01).

[0176] The primers used in the experiment are shown in Table 21, the reagents required for the experiment are shown in Table 22, and the plasmid information required for the experiment is shown in Table 23.

[0177] Table 21 Primer Sequences

[0178] Table 22 Reagents required for the experiment

[0179] Table 23 Plasmids required for the experiment

[0180] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An optimized CRISPRi / dCas12a gene regulatory system for *Sphingosine monocytogenes*, characterized in that, This includes a recombinant plasmid carrying a replicon and a driver that can stably replicate in *Sphingosine monocytogenes*. dcas12a P of gene transcription JDP promoter, dCas12a Gene, resistance gene, and one or more crRNA expression cassettes.

2. The optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* according to claim 1, characterized in that, The replicons that can stably replicate in *Sphingosine monocytogenes* include the pBBR1 replicon; the P... JDP The nucleotide sequence of the promoter is shown in SEQ ID NO.1; dCas12a The nucleotide sequence of the gene is shown in SEQ ID NO.2; The resistance gene includes a chloramphenicol resistance gene; the crRNA expression cassette includes a Ptac promoter, two DR fragments and an rrnB T1 terminator, and the nucleotide sequence of the crRNA expression cassette is shown in SEQ ID NO.

3.

3. A method for constructing the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* as described in claim 1 or 2, comprising the following steps: The recombinant plasmid of the CRISPRi / dCas12a gene regulatory system of Sphingosine monocytogenes was used to drive... dcas12a The original promoter of gene expression is replaced with P JDP An optimized CRISPRi / dCas12a gene regulatory system for Sphingosine monocytogenes was obtained.

4. The application of the optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulatory system according to claim 1 or 2, or the optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulatory system constructed by the construction method according to claim 3, in any one or more of the following functions: ① Improve the efficiency of gene repression synthesis; ② Inhibit the synthesis of key genes in the poly-β-hydroxybutyrate (PHT) synthesis pathway; the key genes in the PHT synthesis pathway are... phaB Genes and / or phaC Gene; ③ Increase gellan gum production; ④ Improve the transparency of gellan gum.

5. An optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas pyrenoidosa* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PHT) synthesis pathway, characterized in that, The optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulation system for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate (PHHB) synthesis pathway, based on the optimized *Sphingomonas sphingosine monocytogenes* CRISPRi / dCas12a gene regulation system described in claim 1 or 2, further includes knockdown... phaB sgRNA expression and / or knockdown phaC sgRNAs expressed by genes.

6. The optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas pyrenoidosa* for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway according to claim 5, characterized in that, The knockdown phaB sgRNAs of gene expression include those targeting phaB gRNA or target of genes phaB Predict the gRNA in the promoter region; the knockdown phaC sgRNAs of gene expression include those targeting phaC gRNA or target of genes phaC Predict the gRNA in the promoter region.

7. A method for constructing the optimized CRISPRi / dCas12a gene regulatory system of *Sphingomonas oligokinase* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate (PHT) synthesis pathway as described in claim 5 or 6, comprising the following steps: Based on the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* as described in claim 1 or 2, a knockdown was inserted. phaB sgRNA expression and / or knockdown phaC The sgRNA expressed by the gene was used to obtain the optimized CRISPRi / dCas12a gene regulatory system of *Sphingosine Monoclonalella* that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway.

8. The application of the optimized Sphingomonas CRISPRi / dCas12a gene regulation system for inhibiting the synthesis of key genes in the poly-β-hydroxybutyrate (PPH) synthesis pathway as described in claim 5 or 6, or the optimized Sphingomonas CRISPRi / dCas12a gene regulation system for inhibiting the synthesis of key genes in the PPH synthesis pathway constructed by the method described in claim 7, in improving gellan gum yield and / or transparency.

9. A recombinant engineered bacterial strain, characterized in that, The recombinant engineered strain uses *Sphingomonas oligosaccharidosis* as the host cell and introduces the optimized *Sphingomonas oligosaccharidosis* CRISPRi / dCas12a gene regulatory system as described in claim 1 or 2, or the optimized *Sphingomonas oligosaccharidosis* CRISPRi / dCas12a gene regulatory system as described in claim 5 or 6 that inhibits the synthesis of key genes in the poly-β-hydroxybutyrate synthesis pathway.

10. A method for increasing gellan gum yield and / or transparency, characterized in that, Includes the following steps: The recombinant engineered strain described in claim 9 is inoculated into a culture medium for fermentation to produce gellan gum.

Images