Phac gene based on translation rate optimization and application thereof

Abstract

The application discloses a PhaC gene based on translation rate optimization and application thereof, and belongs to the technical field of genetic engineering and protein engineering. Without changing the amino acid sequence of PhaC, the sequence of the original PhaC gene is optimized through synonymous codon substitution, a translation deceleration site is introduced in the adjacent region of the domain, and meanwhile, fast translation in the internal domain is maintained, so that the translation extension speed is matched with the protein folding kinetics (the ribosome translation rate is matched with the protein co-translational folding process), and the soluble expression level of PhaC is significantly improved. The experiment shows that the optimized sequence of the PhaC gene based on the translation rate makes the soluble expression amount of PhaC increase by 7.3 times compared with the original sequence, the protein concentration after purification is increased from 0.0461 mg / mL to 0.1575 mg / mL, about 3.4 times, and core gene resources are provided for the high-efficiency soluble expression of the PhaC protein.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering technology, specifically relating to a PhaC gene based on translation rate optimization and its applications. Background Technology

[0002] Polyhydroxyalkanoates (PHA) are a class of natural high-molecular-weight polyesters synthesized by microorganisms. They possess excellent biodegradability and biocompatibility, and have broad application prospects in fields such as bioplastics and pharmaceutical materials. PHA synthase (PhaC) is the key rate-limiting enzyme in the PHA biosynthesis pathway, and its expression level and catalytic activity directly determine the yield of PHA and the molecular weight of the polymer.

[0003] The original PhaC gene has a relatively smooth translation rate curve in E. coli, lacking the necessary translation deceleration region, resulting in insufficient folding time for the nascent peptide chain and the formation of inclusion bodies.

[0004] In practical applications, PhaC proteins are often difficult to obtain in soluble form in heterologous expression systems (such as E. coli), with the vast majority depositing as inclusion bodies in cell pellets. This problem severely hinders the study of the structure and function of PhaC and limits its potential application in PHA production. Existing optimization methods include low-temperature induction, molecular chaperone co-expression, and fusion tags, but these methods suffer from cumbersome operation, high cost, incomplete tag removal, and limited improvement on PhaC protein.

[0005] Therefore, there is an urgent need to develop a new strategy to enhance the soluble expression of PhaC at the gene level without altering its amino acid sequence. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a PhaC gene based on translation rate optimization and its application, solving the problem in the prior art of lacking a solution to improve the soluble expression level of PhaC without changing the amino acid sequence of PhaC.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a PhaC gene based on translation rate optimization, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0009] Among them, optimization refers to obtaining the PhaC gene by synonymous substitution of codons at specific positions in the original PhaC gene while keeping the amino acid sequence unchanged.

[0010] Considering that ribosomes occupy approximately 20-40 amino acids at the C-terminus of the nascent peptide chain during translation, the region upstream of the domain boundary is difficult to fold effectively before leaving the ribosome channel. Therefore, the translation deceleration site should be located approximately 20-40 codons downstream of the domain boundary. Based on this principle, this invention implements synonym substitution at the corresponding position downstream of the domain boundary.

[0011] The specific synonymous substitutions are as follows: nucleotide 589 is replaced by cgt with cga (encoding arginine); nucleotide 601 is replaced by ctg with cta (encoding leucine); nucleotide 610 is replaced by att with ata (encoding isoleucine); nucleotide 1618 is replaced by ctg with cta (encoding leucine); nucleotide 1636 is replaced by ccg with cca (encoding proline); nucleotide 1645 is replaced by gaa with gag (encoding glutamate); nucleotide 1654 is replaced by ccg with cca (encoding proline); nucleotide 1660 is replaced by acc with aca (encoding threonine); and nucleotide 1675 is replaced by cgc with cga (encoding arginine). All of these substitutions are synonymous mutations and do not change the encoded amino acids, but they significantly reduce the translation rate of the corresponding regions.

[0012] In a second aspect, the present invention provides the PhaC protein encoded by the above-mentioned translation rate-optimized PhaC gene, characterized in that the amino acid sequence is as shown in SEQ ID NO:2.

[0013] Thirdly, the present invention provides a recombinant expression vector comprising the aforementioned PhaC gene optimized for translation rate.

[0014] Based on the above technical solution, the backbone of the expression vector is a pET series vector.

[0015] Based on the above technical solution, the backbone of the expression vector is pET-28a(+).

[0016] Fourthly, the present invention provides an engineered bacterium comprising the above-described recombinant expression vector.

[0017] Based on the above technical solution, the host bacterium of the engineered bacteria is Escherichia coli.

[0018] Among them, Escherichia coli was identified as Escherichia coli.

[0019] Based on the above technical solution, the host bacterium of the engineered bacteria is E. coli BL21(DE3).

[0020] Fifthly, the present invention provides the application of the above-mentioned translation rate-optimized PhaC gene, the above-mentioned recombinant expression vector, or the above-mentioned engineered bacteria in PhaC protein expression.

[0021] In a sixth aspect, the present invention provides the application of the above-described translation rate-optimized PhaC gene, the above-described recombinant expression vector, or the above-described engineered bacteria in the purification of PhaC protein.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention optimizes the original PhaC gene sequence through synonymous codon substitution without altering the PhaC amino acid sequence. Translation deceleration sites are introduced into the domain contiguous regions while maintaining rapid translation within the domains. This matches the translation elongation rate with protein folding kinetics (the ribosomal translation rate matches the protein co-translational folding process), thereby significantly improving the soluble expression level of PhaC. Experiments show that the optimized PhaC gene sequence increases the soluble expression level of PhaC by 7.3 times compared to the original sequence, and the purified protein concentration increases from 0.0461 mg / mL to 0.1575 mg / mL, approximately 3.4 times. This provides a core gene resource for the efficient soluble expression of PhaC protein.

[0023] 2. The optimization of this invention only involves synonymous codon substitution and does not change the amino acid sequence of the PhaC protein, thus fully preserving the enzyme's natural catalytic activity and substrate specificity.

[0024] 3. The optimized sequence of the present invention can be directly used for the efficient expression and purification of PhaC protein without the need for complex inclusion body refolding or tag excision steps, which simplifies the process and reduces production costs. Attached Figure Description

[0025] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0026] Figure 1 The following is a comparison of the translation rate curves of the original PhaC gene and the PhaC gene based on translation rate optimization in Example 1 of the present invention: A is the translation rate curve of the original sequence, and B is the translation rate curve of the PhaC gene based on translation rate optimization. Figure 2 This is an SDS-PAGE analysis result of the soluble expression levels of the original PhaC gene sequence and the PhaC gene sequence optimized based on translation rate in Example 3 of the present invention. Detailed Implementation

[0027] In recent years, the relationship between protein translation rate and co-translational folding has been gradually revealed. The speed at which ribosomes move on mRNA is not constant; translation is slower in certain regions, a phenomenon known as "translational pauses." These pause sites are closely related to the distribution of protein domains and can coordinate the synthesis and folding of nascent polypeptide chains.

[0028] Specifically, the translation speed needs to be moderately slowed down in the connecting regions between domains to allow folding time for the upstream domains; while the translation speed within the domains needs to be maintained at a relatively fast rate to ensure the integrity and continuity of the polypeptide chain and avoid problems such as ribosome stagnation and degradation of the new chain caused by slow translation.

[0029] Therefore, efficient expression of exogenous proteins depends on the precise regulation of translation rate: slowing down at the domain boundary and maintaining high speed inside the domain, so that translation elongation and protein folding are synchronized.

[0030] Analysis of the structural features of PhaC revealed that the PhaC protein has multiple domains, and the correct folding of the domain boundary regions is crucial for maintaining the overall conformation and catalytic activity of the enzyme.

[0031] Based on the above understanding, this invention applies this strategy to the gene optimization of PhaC protein. Without changing the amino acid sequence, a deceleration site is introduced at an appropriate position through synonymous codon substitution, resulting in an optimized PhaC gene sequence with significantly enhanced soluble expression.

[0032] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0033] Materials used in the examples: The host bacterium *Escherichia coli* BL21(DE3) was purchased from Novagen; the expression vector pET-28a(+) was from BGI Genomics; the plasmid extraction kit and SDS-PAGE were purchased from New England Biolabs; the Ni-NTA affinity chromatography column used for protein purification was purchased from GE; gene synthesis was performed by BGI Genomics; and all other reagents were domestically produced analytical grade.

[0034] Example 1 This embodiment involves PhaC protein structure analysis and translation rate optimization design.

[0035] 1. Structural characteristics of PhaC protein.

[0036] The PhaC protein consists of 559 amino acids (SEQ ID NO:2). Structure prediction using AlphaFold revealed that PhaC possesses multiple domains, and the connecting regions between these domains play a crucial role in the correct folding of the protein.

[0037] 2. Analysis of the original gene translation rate.

[0038] The original PhaC gene is 1680 bp in length (SEQ ID NO:3). Translation rate distribution in *E. coli* was simulated using RiboTempo software. The results showed that the translation rate of the original sequence fluctuated little across its entire length, and no obvious deceleration regions were formed at the domain boundaries. Figure 1 A). This continuous high-speed translation pattern is not conducive to the co-translational folding of multi-domain proteins, which is an important reason for the formation of inclusion bodies in PhaC.

[0039] 3. Design and introduction of translation deceleration sites.

[0040] To introduce translation deceleration at appropriate locations, this invention, based on the ribosome coverage length pattern (ribosomes occupy approximately 20-40 amino acids at the C-terminus of the nascent peptide chain), places the deceleration site approximately 30 codons downstream of the domain boundary. Specifically, nine synonymous substitutions are implemented: position 589 cgt→cga (Arg), position 601 ctg→cta (Leu), position 610 att→ata (Ile), position 1618 ctg→cta (Leu), position 1636 ccg→cca (Pro), position 1645 gaa→gag (Glu), position 1654 ccg→cca (Pro), position 1660 acc→aca (Thr), and position 1675 cgc→cga (Arg). These substitutions do not alter the amino acid sequence but significantly reduce the translation rate in the corresponding regions. The optimized translation rate curve shows a distinct deceleration peak at the corresponding positions. Figure 1 B).

[0041] 4. Optimize gene sequences.

[0042] The optimized nucleotide sequence of the PhaC gene is shown in SEQ ID NO:1, and the encoded amino acid sequence is shown in SEQ ID NO:2.

[0043] Example 2 This embodiment describes the construction of the expression vector.

[0044] 1. Construction of the original PhaC gene expression vector.

[0045] The original PhaC gene (SEQ ID NO:3) was synthesized by BGI Genomics. The synthesized product was digested with NcoI and XhoI, ligated into the pET-28a(+) vector, transformed into E. coli DH5α, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Positive clones were screened, and sequencing confirmed their correctness, yielding the recombinant plasmid pET-28a(+)-PhaC.

[0046] 2. Construction of an optimized PhaC gene expression vector.

[0047] The optimized PhaC gene (SEQ ID NO:1) was synthesized by BGI Genomics. It was cloned into the pET-28a(+) vector using the same method described above to obtain the recombinant plasmid pET-28a(+)-PhaC-opt.

[0048] Example 3 This embodiment performs PhaC protein expression and solubility analysis.

[0049] 1. Obtaining the expression strain.

[0050] pET-28a(+)-PhaC and pET-28a(+)-PhaC-opt were transformed into E. coli BL21(DE3), plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked to obtain BL21-PhaC (control) and BL21-PhaC-opt (optimized) strains.

[0051] 2. Induced expression.

[0052] A single colony was picked and inoculated into 5 mL of LB medium containing kanamycin, and cultured overnight at 37°C and 200 rpm with shaking. The next day, the colony was transferred to 100 mL of fresh LB medium (containing kanamycin) at a 1:100 ratio, and cultured at 37°C and 200 rpm until OD600≈0.6. IPTG was then added to a final concentration of 0.1 mM, and the culture was induced at 30°C for 4 h.

[0053] 3. Sample processing and SDS-PAGE detection.

[0054] Bacterial cells were collected, washed with PBS, resuspended, and sonicated (300 W, 3 s sonication, 5 s intervals, total 15 min). The cells were then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant and precipitate were collected separately. The precipitate was resuspended in an equal volume of PBS. All samples were subjected to SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue.

[0055] The results are as follows Figure 2As shown: the original PhaC band was very weak in the supernatant and mainly existed in the precipitate, indicating that it existed in the form of inclusion bodies; while the optimized PhaC-opt showed a clear target band (about 62.4 kDa) in the supernatant, indicating that its soluble expression was greatly improved.

[0056] 4. Comparison of soluble expression levels.

[0057] ImageJ was used to perform grayscale analysis on the target bands in SDS-PAGE gels. The relative soluble expression level of PhaC-opt was calculated using the original soluble PhaC grayscale value as a baseline (set to 1). The results showed that the optimized soluble expression level was 7.3 times higher than the original.

[0058] Example 4 This example demonstrates the purification and concentration determination of PhaC protein.

[0059] 1. Protein purification.

[0060] BL21-PhaC-opt was cultured to 1 L using the method described above. After induction of expression, the bacterial cells were collected, resuspended in PBS buffer, sonicated, and the supernatant was collected by centrifugation. The supernatant was filtered through a 0.45 μm filter and loaded onto a Ni-NTA affinity chromatography column, eluted sequentially with buffers containing 20 mM, 40 mM, 200 mM, and 500 mM imidazole, and the 500 mM imidazole elution peak was collected. The original PhaC was purified using the same procedure.

[0061] 2. Protein concentration determination.

[0062] The concentration of purified protein was determined using the BCA method. The results showed that the concentration of the original PhaC after purification was 0.0461 mg / mL, while the concentration of the optimized PhaC-opt after purification was 0.1575 mg / mL, which was 3.4 times that of the original sequence.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0064] Sequence List: PhaC gene sequence based on translation rate optimization SEQ ID NO:1 Amino acid sequence of PhaC protein SEQ ID NO:2 MSNKNNEDLQRQASDNTLNLNPVIGIRGKDLLSSARMVLLQAIKQPFHSAKHVAHFGLELKNVLLGQSGLQPEADDRRFNDPAWSQNPLYKRYLQTYLAWRKELHSWIDESNLSSQDASRGHFVINLMTEAMAPTNSMANPAAVKRFFETGGKSLLDGLSHLAKDMVNNGGMPSQVNMDAFEVGQNLATTEGAVVFRNDVLELIQYKPITESVYERPLLVVPPQINKFYVFDLSPEKSLARFCLRSNLQTFIVSWRNPTKAQREWGLSTYIEALKEAIDVILKITGAKDLNILGACSGGITTVALLGHYQAIGETKVNAFTQMVSVLDFNLDSQVALFADEQTLEAAKRRSYQAGVLEGKDMAKVFAWMRPNDLIWNYWVNNYLLGNEPPAFDILYWNNDTTRLPAAFHGELVEMFKTNALTRPNALEVCGTPIDLKQVTSDFFCLAGTTDHITPWEACYRSALLLGGKCEFVLSNSGHIQSILNPPGNPKARFSTGSEMPKDPKAWLENATKHADSWWLHWQQWIGERSGKTKKASFTLGNKAFPAGEASPGTYVHER Original sequence of PhaC gene SEQ ID NO:3

Claims

1. A PhaC gene based on translation rate optimization, characterized in that, The nucleotide sequence is shown in SEQ ID NO:

1.

2. The PhaC protein encoded by the PhaC gene as described in claim 1, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that, It includes the PhaC gene based on translation rate optimization as described in claim 1.

4. The recombinant expression vector according to claim 3, characterized in that, The backbone of the expression vector is a pET series vector.

5. The recombinant expression vector according to claim 3, characterized in that, The backbone of the expression vector is pET-28a(+).

6. An engineered bacterium, characterized in that, It includes the recombinant expression vector as described in any one of claims 3 to 5.

7. The engineered bacteria according to claim 6, characterized in that, The host bacterium of the engineered bacteria is Escherichia coli.

8. The engineered bacteria according to claim 6, characterized in that, The host bacterium of the engineered bacteria is E. coli BL21(DE3).

9. The application of the PhaC gene based on translation rate optimization as described in claim 1, the recombinant expression vector as described in any one of claims 3 to 5, or the engineered bacteria as described in any one of claims 6 to 8 in the expression of PhaC protein.

10. The use of the PhaC gene based on translation rate optimization as described in claim 1, the recombinant expression vector as described in any one of claims 3 to 5, or the engineered bacteria as described in any one of claims 6 to 8 in the purification of PhaC protein.

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