A prenyltransferase mutant and its use

Abstract

The present invention belongs to the field of bioengineering technology and specifically relates to an isoprenyltransferase mutant and its use. To address the prior art problem of microbial inability to synthesize polyisoprene, the present invention utilizes semi-rational enzyme design. Without changing the amino acid sequence of the enzyme's catalytic domain, the present invention obtains a substrate-specific isoprenyltransferase mutant by truncating the N-terminal hydrophobic amino acid, among other methods. The amino acid sequence of the mutant is shown in SEQ ID NO: 5. The gene encoding the mutant is recombined into an expression vector to obtain a recombinant expression vector, which is then transformed into the host cell Escherichia coli to ultimately produce an engineered bacterium capable of producing polyisoprene, thereby achieving heterologous synthesis of polyisoprene.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to an isoprenyl transferase mutant and a use thereof. Background Art

[0002] Natural rubber is a secondary metabolite mainly synthesized in higher plants. Its basic skeleton structure is cis-1,4-polyisoprene ((C5H8) n ) has a wide range of applications due to its unique physical and chemical properties, such as elasticity, wear and impact resistance, effective heat dissipation, and ductility at low temperatures. Natural rubber is primarily sourced from rubber trees. The development of petroleum-based synthetic rubber is unsustainable and increasingly impacted by pressure to conserve energy and reduce emissions. Therefore, diversifying natural rubber raw materials is urgent.

[0003] The use of synthetic biology to construct a natural rubber cell factory can solve the problem of insufficient rubber raw materials. The natural rubber biosynthesis pathway involves three main stages: the first stage, the production of the initial C5 unit isoprenyl pyrophosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP); the second stage, the production of polyisoprene precursor modules, the continuous condensation of IPP and DMAPP as a substrate to produce (E)-oligoisoprene (C 10-20 ), geranyl pyrophosphate (GPP) (C 10 ), farnesyl pyrophosphate (FPP) (C 15 ) and geranylgeranyl pyrophosphate (GGPP) (C 20 ); the third stage involves the elongation and modification of polyisoprene chains, characterized by the sequential condensation of IPP with the substrate (E)-oligoisoprene to form linear (Z,E)-mixed polyisoprene. While E. coli possesses the complete metabolic pathways for the first two stages, it lacks the key enzyme in the third stage, isoprenyltransferase (cPT).

[0004] Currently, cPT, a key enzyme in natural rubber synthesis, has been isolated from terrestrial plants and microorganisms. However, due to factors such as intracellular expression of the enzyme as inclusion bodies and a lack of cofactors, natural rubber synthesis in heterologous cells has not been successful. This suggests that these terrestrial cPTs involved in natural rubber biosynthesis are incompatible with microorganisms such as Escherichia coli. The ocean's variable environment, characterized by large pressure and temperature fluctuations, oligotrophic factors, and frequent tidal activity, contributes to the diversity of marine life. This diversity also holds the potential for diverse intracellular metabolic processes and enzymes that catalyze biochemical reactions. The unique enzymes of these extreme marine organisms are crucial mediators in catalyzing the biosynthesis of high-end chemical materials.

[0005] Isoprenylation is a key enzyme in the polyisoprene biosynthesis pathway. It contains an isopentenyltransferase domain and can continuously link oligoisoprenes such as GPP, FPP, and GGPP to IPP in a head-to-tail fashion to produce long-chain polyisoprenes. Escherichia coli does not contain cPT, which efficiently catalyzes the synthesis of long-chain polyisoprenes. Summary of the Invention

[0006] In order to solve the technical problem in the prior art that microorganisms cannot ferment and synthesize polyisoprene, the present invention uses semi-rational enzyme design to obtain an isoprenyltransferase mutant that is highly compatible with the chassis cell Escherichia coli and can be expressed soluble, and applies it to the construction of genetically engineered bacteria for producing polyisoprene, providing a material basis for the fermentation synthesis of polyisoprene.

[0007] The present invention first provides a technical solution: an isoprenyltransferase mutant, the amino acid sequence of which is shown in SEQ ID NO: 5 in the sequence listing.

[0008] The present invention further provides a gene encoding an isoprenyltransferase mutant, which is obtained by truncating the N-terminal sequence of the isoprenyltransferase gene, and the nucleotide sequence thereof is shown in SEQ ID NO: 4 in the sequence listing.

[0009] The present invention further provides a recombinant expression vector comprising the gene encoding the isoprenyl transferase mutant.

[0010] The present invention further provides a recombinant host cell comprising the recombinant expression vector.

[0011] The present invention further provides an engineered bacterium for producing polyisoprene, comprising the gene encoding the isoprenyltransferase mutant. The engineered bacterium is produced by introducing the recombinant expression vector into a host cell for induction transformation.

[0012] The present invention further provides the use of any one of the isoprenyltransferase mutant, the gene, the recombinant expression vector, the recombinant host cell, and the engineered bacteria in producing polyisoprene. It also provides the use of any one of the isoprenyltransferase mutant, the gene, the recombinant expression vector, and the recombinant host cell in constructing an engineered bacteria for producing polyisoprene.

[0013] The present invention further provides a method for producing polyisoprene, which utilizes the engineered bacteria to carry out shake flask fermentation to prepare the polyisoprene product.

[0014] The present invention has the beneficial effects of increasing the soluble expression of PucPT through N-terminal truncation without altering catalytic activity through semi-rational protein design, resulting in a superior isoprenyltransferase mutant with improved compatibility with chassis cells. Finally, through plasmid recombination, transformation induction, and shake flask fermentation, an engineered polyisoprene-producing bacterium was successfully constructed. This engineered bacterium enables large-scale production of polyisoprene in a green and pollution-free manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Comparison of the substrate binding pockets of PucPT and ΔN-ter-PucPT in Example 1 of the present invention; the gray sphere represents the cofactor Mg 2+ , the stick structure represents the substrate FPP, the blue represents the PucPT substrate binding pocket, and the green represents the △N-ter-PucPT substrate binding pocket;

[0016] Figure 2 This is a comparison of the mass spectrometry product identification results in Example 3 of the present invention; wherein A is the mass spectrometry identification result of the control strain pET-15b-BL21(DE3), and B is the mass spectrometry identification result of the polyisoprene engineered strain ΔN-ter-PucPT-pET-15b-BL21(DE3). DETAILED DESCRIPTION

[0017] The present invention will be described in further detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0018] Example 1: Optimization of the isoprenyltransferase gene

[0019] The amino acid sequence of prenyltransferase is shown in SEQ ID NO: 1, which is derived from the intertidal plant Porphyra umbilicalis (Gen-Bank: OSX73815.1). The present invention optimizes the N-terminus of the coding sequence to adapt it to the chassis cell Escherichia coli by truncating the sequence shown in SEQ ID NO: 2 from the N-terminus of SEQ ID NO: 1 to obtain the final sequence SEQ ID NO: 4.

[0020] Using AlphaFold 3-I-TASSER-Protein-Sol triple prediction analysis, the catalytic pocket of the expressed △N-ter-PucPT protease was consistent with the wild type, and the N-terminal hydrophobic amino acid was reduced (as shown in the sequence list SEQ ID NO: 3). The truncated amino acids are shown in the sequence list SEQ ID NO: 5 and Figure 1 shown.

[0021] Example 2: Construction of recombinant expression vectors and strains of polyisoprene engineering bacteria

[0022] (1) Construction of recombinant expression vector

[0023] The isoprenyl transferase gene ΔN-ter-PucPT with the sequence shown in SEQ ID NO: 4 was synthesized into the pET-15b plasmid vector to successfully obtain the ΔN-ter-PucPT-pET-15b recombinant expression vector.

[0024] (2) Construction of engineered bacteria for producing polyisoprene

[0025] The constructed isoprenyltransferase recombinant expression plasmid △N-ter-PucPT-pET-15b was transformed into Escherichia coli BL21 (DE3) competent cells, and transformants were screened using LB solid plates; the bacterial plates were placed in a 37°C incubator for 12 hours, and normally growing positive transformants were selected and inoculated into glass test tubes containing LB liquid culture medium; the test tubes were incubated on a shaker at 37°C and 150 rpm for 6 hours, 1.5 mL of the bacterial solution was transferred to 150 mL of LB liquid culture medium, and the culture was continued at 37°C and 150 rpm for 6 hours to obtain seed culture medium; 150 mL of the seed solution was inoculated into a shake flask containing 15 L of culture medium, and the culture was continued at 37°C and 150 rpm until the OD 600 =0.6, then transferred to 16 ° C, 120 rpm and continued to shake the flask fermentation for 1 hour; then added 0.05 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for induction, and continued to shake the flask fermentation at 16 ° C, 120 rpm for 14 hours to obtain.

[0026] The LB solid medium used for bacterial culture was prepared by weighing the required nutrients or salt components according to Table 1.

[0027] Table 1 LB solid culture medium formula

[0028]

[0029]

[0030] Dissolve the above components in ddH2O, adjust the pH to 7.4, and dilute to 1 L with ddH2O. Finally, sterilize under high pressure steam for 20 min. When the culture medium temperature drops to 50°C, add ampicillin antibiotics at a final concentration of 100 μg / mL. Pour the culture medium into plates at a rate of 10 mL per plate in a clean bench. After solidification, invert and place in a 4°C refrigerator for subsequent use.

[0031] Preparation of LB liquid medium for bacterial culture: excluding agar powder, the remaining components of the liquid medium are the same as those of the solid medium. After sterilization, wait until the temperature drops to room temperature, add ampicillin to a final concentration of 100 μg / mL, and place in a 4°C refrigerator for subsequent use.

[0032] The present invention transforms the N-terminus of PucPT, not only truncating the N-terminal hydrophobic amino acids to enhance the solubility of the protease, but also leaving its substrate binding pocket unchanged, thus not affecting the catalytic properties of the enzyme.

[0033] Example 3: Evaluation of the engineered strain ΔN-ter-PucPT-pET-15b-BL21 (DE3) constructed by the present invention: 1 L of fermented bacterial solution was centrifuged at 8000 rpm for 10 min to remove the supernatant, and 10 mL of distilled water was added to resuspend the bacterial solution, and the solution was disrupted by ultrasonication; 8 mL of methanol, 8 mL of ammonia water, and 30 mL of pentane were added and mixed and shaken, and the mixture was centrifuged at 5000 rpm for 10 min. The supernatant was taken and rotary evaporated, and the product yield was 10 μg / L; after evaporation, the product was dissolved in 2 mL of methanol, and the polyisoprene product was identified by HPLC-MS. Compared with the control group ( Figure 2 Middle A), experimental group ( Figure 2 In Figure B), a large polyisoprene fragment product appeared at a high molecular weight of m / z 1348. Based on the calculation of relative molecular weight, under the bombardment of positive ions, the CO single bond connecting the pyrophosphate group to the adjacent carbon atom is weak and preferentially dissociates. The CC single bond at the end of the free carbon chain is unstable, leaving a more stable C=C double bond at the end of the carbon chain after dissociation. Based on the molecular weight, it can be inferred that the product reaches C 100 ( Figure 2 ). Combined Figure 1 and Figure 2 The results showed that by truncating the N-terminal hydrophobic amino acid, recombining the isoprene transferase mutant gene △N-ter-PucPT into the expression vector pET-15b, and then transforming it into the chassis cell BL21 (DE3), the construction and application of polyisoprene engineering bacteria can be achieved.

[0034] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A prenyltransferase mutant, characterized in that: Its amino acid sequence is shown in SEQ ID NO: 5 in the sequence listing.

2. A gene encoding the isoprenyltransferase mutant according to claim 1, characterized in that: The isoprenyl transferase gene is obtained by truncating the N-terminal sequence, and its nucleotide sequence is shown in SEQ ID NO: 4 in the sequence listing.

3. A recombinant expression vector, characterized in that: Comprising the gene according to claim 2.

4. A recombinant host cell, characterized in that: Comprising the recombinant expression vector according to claim 3.

5. An engineered bacterium for producing polyisoprene, characterized in that: Comprising the gene encoding the isoprenyl transferase mutant according to claim 2.

6. The engineered bacteria according to claim 5, characterized in that: The recombinant expression vector according to claim 3 is introduced into a host cell to induce transformation.

7. Use of any one of the isoprenyltransferase mutant of claim 1, the gene encoding the isoprenyltransferase mutant of claim 2, the recombinant expression vector of claim 3, and the recombinant host cell of claim 4 in constructing an engineered bacterium for producing polyisoprene.

8. Use of any one of the isoprenyltransferase mutant according to claim 1, the gene encoding the isoprenyltransferase mutant according to claim 2, the recombinant expression vector according to claim 3, the recombinant host cell according to claim 4, and the engineered bacteria according to claim 5 or 6 in producing polyisoprene.

9. A method for producing polyisoprene, characterized in that: The polyisoprene product is prepared by shake flask fermentation using the engineered bacteria according to claim 5 or 6.

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