Engineered bacillus subtillis for inserting non-natural amino acid into protein

Abstract

The invention belongs to the technical field of directed evolution modification of protein, and particularly relates to an engineered bacillus subtillis chassis cell for inserting unnatural amino acid, which is formed by uniformly replacing termination codons UAG or UGA of 57 essential genes of bacillus subtillis with UAA. On the basis, two sets of mutually orthogonal translation systems are introduced to decode the UAG and the UGA respectively, and finally site-specific insertion of the two non-natural amino acids at the preset site of the target protein is realized; a stable and reliable cell platform is provided for obtaining protein molecules carrying two different chemical functional groups and carrying out multi-site refined modification research.

Description

Technical Field

[0001] This invention belongs to the field of directed evolutionary modification technology of proteins, and relates to an engineered Bacillus subtilis strain for inserting non-natural amino acids into proteins and its applications. Background Technology

[0002] Over the past two decades, genetic codon expansion (GCE) technology has made significant progress in the insertion of unnatural amino acids (ncAAs), enabling the precise introduction of ncAAs into target proteins within cells, thereby endowing proteins with new physicochemical properties. Current GCE development focuses on two main areas: first, constructing translation tools such as aminoacyl-tRNA synthetases / inhibitory tRNAs orthogonal to the natural translation system; and second, modifying the host genome to free up "empty codons" recognized only by orthogonal translation systems, thus achieving site-specific encoding of ncAAs. Overexpression of these translation tools in unmodified chassis cells often leads to cytotoxicity and resource waste; therefore, constructing more adaptable engineered chassis is crucial for the application of GCE.

[0003] Currently, chassis modification mainly involves "simplifying" specific codons across the entire genome to free them up for encoding new amino acids. For example, in *E. coli* C321, all 321 UAG stop codons in the genome are replaced with UAA, and in synthetic yeast Sc2.0, UAG is intentionally removed from the entire genome. However, such genome-wide codon recoding often requires an extremely large amount of work, significantly limiting the widespread application of this strategy in more microbial cell factories. Summary of the Invention

[0004] The technical problem to be solved by this invention is that the large amount of work required for existing chassis modification significantly limits its application. This invention provides an engineered Bacillus subtilis chassis cell for inserting non-natural amino acids, which can provide a stable and reliable cell platform for obtaining protein molecules carrying different chemical functional groups and conducting multi-site fine modification research.

[0005] To this end, the first aspect of the present invention provides an engineered Bacillus subtilis chassis cell for inserting non-natural amino acids, which is constructed by uniformly replacing the stop codons UAG or UGA of the 57 essential genes of Bacillus subtilis with UAA.

[0006] In this invention, the 57 essential genes of Bacillus subtilis include the genes walK, rtbE, yxlC, mbl, tuaB, mrpE, mrpA, menB, menE, mrec, asps, nadD, holA, ispH, ispG, dxs, rnz, scpa, cmk, nrdE, infB, dapB, divIB, murB, ftsL, coaD, rpoA, adk, folK, folB, tilS, tmk, gyrA, rodA, pheT, dnaA, divIC, ispD, rplX, rpsG, acpS, tsaB, ppnkA, murE, murG, ftsA, plsX, trmD, uppS, coaBC, rppA, engB, rcmE, accD, mrpD, secA, and rnpA.

[0007] In some embodiments of the present invention, the sequences of the 57 essential genes of Bacillus subtilis are shown in SEQ ID NO.1-SEQ ID NO.57.

[0008] According to the present invention, the engineered Bacillus subtilis chassis cells can simultaneously insert multiple non-natural amino acids into the same protein, and the non-natural amino acids may be the same or different from each other.

[0009] Preferably, the engineered Bacillus subtilis chassis cells are capable of simultaneously inserting two non-natural amino acids into the same protein, and the two non-natural amino acids are not the same.

[0010] The present invention also provides the application of engineered Bacillus subtilis chassis cells as described in the first aspect of the present invention in protein mutation.

[0011] According to the present invention, the application includes:

[0012] Step A: Inoculate engineered Bacillus subtilis chassis cells into LB medium for culture;

[0013] Step B: After the culture reaches the logarithmic growth phase, it is transferred to S750 medium containing two non-natural amino acids for further culture to obtain a culture containing two non-natural amino acids.

[0014] Step C involves separating and purifying the culture containing two non-natural amino acids to obtain a purple protein containing non-natural amino acids.

[0015] In some embodiments of the present invention, the concentration of each non-natural amino acid in the S750 culture medium containing two non-natural amino acids is 1 mM.

[0016] In some embodiments of the present invention, the culture temperature is 30°C and the culture time is 24 hours.

[0017] According to the present invention, the S750 culture medium also contains IPTG.

[0018] In some preferred embodiments of the present invention, the concentration of IPTG in the S750 culture medium is 1 mM.

[0019] This invention utilizes codon expansion technology to introduce non-natural amino acids into proteins, providing a promising approach to enhancing protein structure and function. Specifically, this invention relates to an engineered Bacillus subtilis chassis cell capable of simultaneously inserting two non-natural amino acids into the same protein. Specifically, it involves uniformly replacing the stop codons (UAG or UGA) of 57 essential genes in Bacillus subtilis with UAA to construct the chassis strain Bs57. Based on this, two orthogonal translation systems are introduced to decode UAG and UGA respectively, ultimately achieving site-specific insertion of the two non-natural amino acids at predetermined sites in the target protein. This provides a stable and reliable cellular platform for obtaining protein molecules carrying two different chemical functional groups and conducting multi-site refined modification studies. Attached Figure Description

[0020] The present invention will now be described in further detail with reference to the accompanying drawings:

[0021] Figure 1 This is a schematic diagram of a multiplex gene editing system; where A is the plasmid pSpRY-m3 carrying three guide RNA cassettes, and the nSpRY fusion protein, after being activated by IPTG, targets different DNA sites under the guidance of the guide RNA; B is the CRISPR / Cas9 system carrying two guide RNA cassettes and a DNA repair template.

[0022] Figure 2 The flowchart for constructing strain Bs57 is shown below; the left figure shows the synonymous replacement of the stop codons UAG and UAA in 22 essential genes; the right figure shows the synonymous replacement of the stop codons UGA and UAA in 35 essential genes.

[0023] Figure 3 The results of the growth analysis of strain Bs57 are shown.

[0024] Figure 4 The diagram shows the process of inserting two non-natural amino acids into a protein. The left diagram shows the design of insertion sites for non-natural amino acids using the SIFT tool. The middle diagram shows the expression of the target protein by plasmid pHT01 and the use of orthogonal pairs of non-natural amino acids integrated into the genome for the recognition and insertion of the two non-natural amino acids. The right diagram shows the transcription and translation process of the non-natural amino acid-inserted protein.

[0025] Figure 5 This is a secondary mass spectrometry analysis of a protein containing two non-natural amino acids. Detailed Implementation

[0026] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0027] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0028] I. Terminology

[0029] The terms "original" and "primary" are both relative to the "mutation" provided in this invention.

[0030] In this invention, the terms "protein" and "protein protein" can be used interchangeably.

[0031] II. Implementation Plan

[0032] As mentioned earlier, current chassis modification primarily involves "simplifying" specific codons across the entire genome to free them up for encoding new amino acids. Examples include replacing all 321 UAG stop codons with UAA in *E. coli* C321 and intentionally removing UAG from the entire genome in synthetic yeast Sc2.0. However, such genome-wide codon recoding often requires an extremely large amount of work, significantly limiting the widespread application of this strategy in more microbial cell factories.

[0033] To address the aforementioned issues, based on our previous work replacing the UAG codons of 22 essential genes with UAA stop codons in the Bs22 chassis cell, we performed synonymous substitutions on the UGA stop codons in all essential genes, totaling 35. This resulted in the construction of an engineered Bacillus subtilis chassis cell, Bs57, capable of simultaneously inserting two non-natural amino acids into the same protein. The process includes the following steps:

[0034] (1) Bacillus subtilis has 257 essential genes, of which 35 terminate with the UGA codon, 23 with the UAG codon, and 199 with the UAA codon. To shorten the construction time of chassis cells, we used a multiplex genome editing system developed in our laboratory, such as... Figure 1 As shown.

[0035] (A) The plasmid pSpRY-m3 carries three guide RNA boxes. After the nSpRY fusion protein is activated by IPTG, it targets different DNA sites under the guidance of the guide RNA.

[0036] (B) The CRISPR / Cas9 system carries two guide RNA boxes and a DNA repair template.

[0037] The following are the guide RNA sequences used for gene editing (Table 1).

[0038] Table 1 Guide RNA sequences used for gene editing

[0039]

[0040] (2) First, synonym substitution editing was performed simultaneously on three genes using the PLCBE toolbox. After five rounds of sequential editing, all stop codons UGA in the 15 essential genes were replaced with UAA. Based on this, we used the Cas9 system to perform synonym substitution on two genes per round, for a total of 10 rounds. In summary, we performed a total of 23 rounds of editing on Bacillus subtilis to obtain strain Bs57. The construction flowchart of this strain is shown below. Figure 2 As shown.

[0041] The following are the gene editing methods used for each required gene stop codon replacement (Table 2).

[0042] Table 2. Codon Substitution Methods for 35 Essential Genes

[0043]

[0044] (3) Growth curve analysis showed that synonymous substitution of essential gene codons did not have a negative impact on strain growth. Figure 3 ).

[0045] (4) To confirm whether non-natural amino acids could be inserted into proteins, we selected superfolded green fluorescent protein (sfGFP) and purple protein (plasmid purchased from Addgene) as target proteins. First, we used the SIFT algorithm to predict the insertion site of ncAA (http: / / sift-dna.org). This algorithm uses sequence homology to predict the consequences of all potential amino acid substitutions at each position in the protein sequence. Therefore, we used the SIFT algorithm to predict UAG and UGA substitution sites to reduce the impairment of protein function. Subsequently, we integrated the *Methanococcus jansii* tyrosine tRNA synthetase variant and the *Methanococcus martensii* pyrrolidone variant into the genome, and the target protein was overexpressed in the pHT01 plasmid to purify the protein containing the two non-natural amino acids. Figure 4 ).

[0046] (5) Finally, we successfully expressed a green fluorescent protein containing 4-azidophenylalanine and norbornene-type lysine derivatives. Figure 5 The modified green fluorescent protein exhibited an average fluorescence intensity approximately 10.1% that of the wild-type green fluorescent protein. Similarly, we expressed a purple protein containing the UAG and UGA codons. Secondary mass spectrometry analysis (Bruker AutoFlex Max, Bruker) confirmed the successful incorporation of the two non-natural amino acids into the protein. These results demonstrate the successful construction of a protein containing two site-specific incorporations of non-natural amino acids in Bs57.

[0047] (B) Secondary mass spectrometry analysis of purple protein with two non-natural amino acids inserted (Bruker AutoFlexMax, Bruker).

[0048] As can be seen from the above, the engineered Bacillus subtilis chassis cell constructed by the method of the present invention, which can simultaneously insert two non-natural amino acids into the same protein, is constructed by uniformly replacing the stop codons UAG or UGA of the 57 essential genes of Bacillus subtilis with UAA. In this invention, this chassis cell is named engineered Bacillus subtilis chassis cell Bs57.

[0049] In this invention, the 57 essential genes of Bacillus subtilis include the genes walK, rtbE, yxlC, mbl, tuaB, mrpE, mrpA, menB, menE, mrec, asps, nadD, holA, ispH, ispG, dxs, rnz, scpa, cmk, nrdE, infB, dapB, divIB, murB, ftsL, coaD, rpoA, adk, folK, folB, tilS, tmk, gyrA, rodA, pheT, dnaA, divIC, ispD, rplX, rpsG, acpS, tsaB, ppnkA, murE, murG, ftsA, plsX, trmD, uppS, coaBC, rppA, engB, rcmE, accD, mrpD, secA, and rnpA.

[0050] The present invention consists of a 57-foot-long sequence consisting of a sequence of SEQ IDNO.1-SEQ ID NO.57 is based on NCBI ID: walK(NCBI ID:937793), rtbE(NCBI ID:937530),yxlC(NCBI ID:937793),mbl(NCBI ID:936916),tuaB(NCBI ID: 936770), mrpE(NCBI ID: 2914191), mrpA(NCBI ID: 938828), menB(NCBI ID: 937195), menE(NCBI ID: 937132), mrec(NCBI ID: 937498, asps(NCBI ID: 937549), nadD(NCBI ID: 937818), holA(NCBI ID: 937833), ispH(NCBI ID: 936770), ispG(NCBI ID: 937984), dxs(NCBI ID: 938609), rnz(NCBI ID: 938694), scpa(NCBI ID: 938950), cmk(NCBI ID: 938982), nrdE(NCBI ID: 940091), infB(NCBI). ID: 939630), dapB(NCBI ID: 939024), divIB(NCBI ID: 939701), murB(NCBI ID: 939804), ftsL (NCBI ID: 939860), coaD(NCBI). ID: 936889, rpoA(NCBI ID: 938921), adk(NCBI ID: 938508), folK(NCBI ID: 936904), folB(NCBI ID: 937976), tilS(NCBI ID: 936922), tmk(NCBI ID: 937018, gyrA (NCBI ID: 940002), rodA (NCBI ID: 937294), pheT (NCBI ID: 937445), dnaA (NCBI ID: 939978), divIC (NCBI). ID: 936786, ispD(NCBI ID: 936855), rplX (NCBI ID: 936799), rpsG (NCBI ID: 935997), acpS (NCBI ID: 938194), tsaB(NCBI). ID: 939874), ppnkA(NCBI ID: 936414), murE(NCBIID: 937567), murG (NCBI ID: 935909), ftsA (NCBI ID: 936145), plsX (NCBI ID: 938066), trmD (NCBI ID: 936650), uppS (NCBI ID: 939640), coaBC (NCBI ID: 938487), rppA (NCBI ID: 937961), engB (NCBI ID: 938009), rcmE (NCBI ID: 935934), accD (NCBI ID: 936186), mrpD (NCBI ID: 938858), secA (NCBI ID: 936711), rnpA (NCBI ID: 937930).

[0051] According to the present invention, the engineered Bacillus subtilis chassis cells can simultaneously insert multiple non-natural amino acids into the same protein, and the non-natural amino acids may be the same or different from each other.

[0052] Preferably, the engineered Bacillus subtilis chassis cells are capable of simultaneously inserting two non-natural amino acids into the same protein, and the two non-natural amino acids are not the same.

[0053] This invention also provides the application of engineered Bacillus subtilis chassis cells as described above in protein mutation, comprising:

[0054] Step A: Inoculate engineered Bacillus subtilis chassis cells into LB medium for culture;

[0055] Step B: After the culture reaches the logarithmic growth phase, it is transferred to S750 medium containing two non-natural amino acids and IPTG and cultured at 30°C for 24 hours to obtain a culture containing two non-natural amino acids.

[0056] Step C involves separating and purifying the culture containing two non-natural amino acids to obtain a purple protein containing the two non-natural amino acids.

[0057] In step A above, the concentration of each non-natural amino acid in the S750 culture medium containing two non-natural amino acids is 1 mM, and the concentration of IPTG is 1 mM.

[0058] The non-natural amino acids mentioned in this invention include, but are not limited to, 4-azidophenylalanine, norbornene-type lysine, coumarin, and 5-hydroxytryptophan.

[0059] Specifically, the non-natural amino acids mentioned in this invention include, but are not limited to, 4-azidophenylalanine with click chemistry groups, norbornene-type lysine, coumarin with fluorescent probe properties, and 5-hydroxytryptophan with medical efficacy.

[0060] In some preferred embodiments of the present invention, a protein purification nickel column is used to separate and purify a culture containing two non-natural amino acids.

[0061] This invention utilizes codon expansion technology to introduce non-natural amino acids into proteins, providing a promising approach to enhancing protein structure and function. Specifically, this invention relates to an engineered Bacillus subtilis chassis cell capable of simultaneously inserting two non-natural amino acids into the same protein. This is achieved by uniformly replacing the stop codons (UAG or UGA) of 57 essential genes in Bacillus subtilis with UAA, resulting in the successful chassis strain Bs57. Furthermore, two orthogonal translation systems are introduced to decode UAG and UGA respectively, ultimately achieving site-specific insertion of the two non-natural amino acids at predetermined sites in the target protein. This provides a stable and reliable cellular platform for obtaining protein molecules carrying two different chemical functional groups and conducting multi-site refined modification studies.

[0062] III. Examples

[0063] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0064] Example 1:

[0065] (1) Construction of strains and plasmids

[0066] The recombinant plasmids were constructed using *Escherichia coli* strain JM109 (ATCC 53323, USC), and *Bacillus subtilis* (ATCC 23857, USC) cultured at 30°C in LB or LB agar. If necessary, antibiotics such as ampicillin (100 μg / mL), chloramphenicol (5 μg / mL), or kanamycin (50 μg / mL) were added to the culture medium. The construction of plasmids pCas and pSpRY laid the foundation for genome editing research. Primers contained a 20 bp specific spacer region of the target sequence as an overlap region. The resulting PCR products were directly transformed into *E. coli* using a single PCR method. Fragments self-ligated to form circular structures, thus constructing plasmids containing guide RNA expression chimeras. The specific donor DNA, containing two homologous arms of similar length, was amplified from the wild-type *Bacillus subtilis* genome by PCR. The donor DNA was integrated into a plasmid containing a single guide RNA via Gibson assembly.

[0067] (2) Transformation by Bacillus subtilis

[0068] The day before, the bacterial strain was streaked onto LB agar plates to promote the transformation of genomic DNA or linearized DNA. Colonies no older than one day were inoculated into large glass tubes containing 5 mL of GM I medium and incubated overnight at 30°C. The next day, 1 mL of the bacterial culture was added to 9 mL of GM I medium containing 100 μL of glucose. The mixture was placed on a shaker and incubated at 37°C for 3 hours. Subsequently, 2 mL of the bacterial culture was added to 18 mL of GMII medium and incubated at 37°C with shaking for 1.5 hours, followed by centrifugation at 5000 rpm for 5 minutes to collect the cells. DNA (1-2 μg) was added to the cells, and the cells were incubated at 30°C with shaking for another 2 hours. The cells were then plated onto LB agar plates containing appropriate antibiotics and incubated overnight at 30°C to screen for positive transformants.

[0069] (3) Use the PLCBE toolbox and CRISPR-Cas9 system to perform essential gene selection and genome modification.

[0070] The replacement of the essential gene's stop codon UGA with UAA was achieved using the PLCBE toolkit in conjunction with the CRISPR-Cas9 system. Guide RNA facilitates UGI targeting of DNA, and UGI catalyzes the conversion of cytosine to uracil. Simultaneously, nSpRY cleaves the non-target strand to initiate DNA repair. During repair, uracil is converted to thymine, while guanine on the complementary strand is converted to adenine. The *Streptococcus pyogenes* Cas9, encoded by the guide RNA, induces a double-strand break in the target sequence. The plasmid introduces the replacement homologous sequence. The repair template, derived from genomic DNA, was obtained through PCR amplification, and the UAA stop codon was introduced using rapid conversion PCR technology.

[0071] (4) Plasmid removal and iterative editing

[0072] To remove the temperature-sensitive plasmids pCas and pBAC, colonies were transferred to antibiotic-free LB agar plates and incubated at 42°C. Individual colonies were then streaked onto LB agar plates at 42°C to isolate them. Finally, plasmid loss was assessed by transferring individual colonies to LB agar plates containing kanamycin. The resulting modified strains can then be used as initial strains for the next round of genome editing. Individual colonies without plasmids were selected and cultured on LB agar with or without the appropriate antibiotics to verify plasmid carrier status.

[0073] (5) Selection of UGA integration site and cloning of target protein expression constructs

[0074] Positive transformants were validated by single-colony PCR. Protein residue tolerance was predicted using the SIFT algorithm to screen for UAG integration sites. This algorithm can predict amino acid substitutions with minimal impact on protein function based on sequence homology (http: / / sift.jcvi.org). We selected tolerance residues closest to the translation start codon (UTG) to facilitate genome editing. The plasmids used to express sfGFP- and purple proteins were derived from plasmid pHT01 and had a C-terminal His6 tag added. The introduction of the stop codon was achieved via rapid substitution PCR.

[0075] (6) Expressing proteins containing non-natural amino acids

[0076] Fresh colonies (engineered Bacillus subtilis chassis cells used for inserting non-natural amino acids) were selected from LB agar plates and added to 5 mL of LB liquid medium. Once the culture reached the logarithmic growth phase, it was transferred to S7 medium containing 1 mM of non-natural amino acids (two distinct non-natural amino acids: 4-azidophenylalanine and a norbornene-type lysine derivative) and IPTG. 50In the culture medium, incubate at 30°C for 24 hours.

[0077] (7) Protein purification and mass spectrometry analysis

[0078] A purple protein containing non-natural amino acids was purified from cells using a GE Healthcare His Spin Trap column. The cell pellet was resuspended in His lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl) and sonicated. The resulting lysate was centrifuged (20 min, 16000 g, 4 °C) for clarification and then loaded onto the His Spin Trap column. The column was washed four times, and the protein was eluted with 3 column volumes (CV) of His elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM imidazole). The purified purple protein containing two non-natural amino acids was then concentrated using ultrafiltration. The purity and structure of the obtained protein were analyzed by SDS-PAGE. The purified protein was digested with trypsin and analyzed by secondary mass spectrometry. The results showed that 4-azidophenylalanine and norbornene-type lysine derivatives could be successfully inserted into the purple protein.

[0079] The culture media used in this study are listed in Table 3:

[0080] Table 3 Culture medium formulation

[0081]

[0082]

[0083] Comparative experiments showed that unengineered chassis cells failed to simultaneously insert two non-natural amino acids into the same protein. However, the strain Bs57 constructed in this invention successfully produced a protein containing two non-natural amino acid insertions for the first time, achieving a breakthrough from "0" to "1". Furthermore, non-natural amino acids, as a class of amino acids with diverse structures and functions, can endow proteins with new functions, enhance their stability or specificity, and thus promote progress in biotechnology, pharmaceutical research and development, and materials science. The 4-azidophenylalanine and norbornene-type lysine used in this invention each carry orthogonal bio-orthogonal click chemistry reaction groups, which do not interfere with each other in the same system, enabling simultaneous, efficient, and highly specific covalent coupling of two different functional molecules. Based on this dual orthogonal click chemistry system, different types of functional molecules, such as small molecule drugs, fluorescent probes, and nanomaterials, can be precisely linked to target proteins or cell surfaces, significantly expanding the freedom and complexity of functional modification. Therefore, this invention provides a solid and universal technical foundation for multimodal intracellular imaging, precise drug delivery, and the construction of multifunctional biomaterials.

[0084] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. An engineered Bacillus subtilis chassis cell for inserting non-natural amino acids, which is constructed by uniformly replacing the stop codons UAG or UGA of 57 essential genes of Bacillus subtilis with UAA.

2. The engineered Bacillus subtilis chassis cells according to claim 1, characterized in that, The 57 essential genes of Bacillus subtilis include walK, rtbE, yxlC, mbl, tuaB, mrpE, mrpA, menB, menE, mrec, asps, nadD, holA, ispH, ispG, dxs, rnz, scpa, cmk, nrdE, infB, dapB, divIB, murB, ftsL, coaD, rpoA, adk, folK, folB, tilS, tmk, gyrA, rodA, pheT, dnaA, divIC, ispD, rplX, rpsG, acpS, tsaB, ppnkA, murE, murG, ftsA, plsX, trmD, uppS, coaBC, rppA, engB, rcmE, accD, mrpD, secA, and rnpA.

3. The engineered Bacillus subtilis chassis cells according to claim 2, characterized in that, The sequences of the 57 essential genes of the Bacillus subtilis are shown in SEQ ID NO.1-SEQ ID NO.

57.

4. The engineered Bacillus subtilis chassis cells according to claims 1-3, characterized in that, The engineered Bacillus subtilis chassis cells are capable of simultaneously inserting multiple non-natural amino acids into the same protein, with each non-natural amino acid being either the same or different from the others.

5. The engineered Bacillus subtilis chassis cells according to claim 4, characterized in that, The engineered Bacillus subtilis chassis cells are capable of simultaneously inserting two non-natural amino acids into the same protein, and the two non-natural amino acids are not the same.

6. The application of engineered Bacillus subtilis chassis cells as described in any one of claims 1-5 in protein mutation.

7. The application according to claim 6, comprising: Step A: Inoculate engineered Bacillus subtilis chassis cells into LB medium for culture; Step B: After the culture reaches the logarithmic growth phase, it is transferred to S750 medium containing two non-natural amino acids for further culture to obtain a culture containing two non-natural amino acids. Step C involves separating and purifying the culture containing two non-natural amino acids to obtain a purple protein containing non-natural amino acids.

8. The application according to claim 7, characterized in that, The S750 culture medium containing two non-natural amino acids has a concentration of 1 mM for each non-natural amino acid.

9. The application according to claim 7, characterized in that, The culture temperature is 30°C, and the culture time is 24 hours.

10. The application according to any one of claims 7-9, characterized in that, The S750 culture medium also contains IPTG; preferably, the concentration of IPTG in the S750 culture medium is 1 mM.

Images