A method of biosynthesizing N-hydroxypiperidine acid

Abstract

The application discloses a kind of lysine as substrate biosynthesis N-hydroxypiperidine acid technology, belong to metabolic engineering technical field.The application uses escherichia coli BL21 (DE3) as starting strain, first constructs recombinant plasmid pET-28a-pipA-pntAB and engineering bacteria, to realize with lysine as substrate conversion generation L-piperidine acid intermediate.On this basis, construct recombinant plasmid PACYCDuet-SNaC and it is transformed into engineering bacteria, to realize L-piperidine acid is further converted into N-hydroxypiperidine acid.Then, in engineering strain, the concentration of coenzyme NADPH is improved by overexpressing glucose dehydrogenase coding gene gdh.Lysine degradation gene cadA is knocked out in host bacteria.Finally, by means of directed evolution, the activity of L-PA hydroxylase encoded by SNaC gene is improved.The experimental results of the above strategy show that the engineering strain can convert 40g / L L-lysine into 32.5g / L L-PA and 187.6mg / L NHP in a 5L fermenter.

Description

[Technical Field]

[0001] This invention belongs to the field of metabolic engineering technology. It refers to the technology of constructing an engineered strain of Escherichia coli that synthesizes N-hydroxypiperidine acid using L-lysine by combining metabolic engineering and directed evolution, and optimizing its fermentation system to synthesize N-hydroxypiperidine acid. [Background Technology]

[0002] N-hydroxypiperidine acid, as a rigid cyclic non-protein amino acid, plays an important role in plant immune defense. It is a crucial signal transduction molecule for systemic acquired resistance in plants and plays a key role in agriculture. The synthesis of N-hydroxypiperidine acid mainly relies on chemical and biocatalytic methods, but both methods have significant technical bottlenecks.

[0003] Traditional chemical methods use piperidinone derivatives as raw materials, reducing the carbonyl group with metal hydrides (such as sodium borohydride) to generate the target product. However, this method requires the use of large amounts of flammable and explosive metal reducing agents, and post-treatment necessitates the neutralization of strongly alkaline wastewater, posing safety hazards and incurring high environmental costs. Furthermore, chemical methods struggle to achieve stereoselectivity control, requiring additional chiral resolution steps. While recent studies have attempted to simplify the process through novel reactions such as free radical coupling, these still rely on precious metal catalysts, and the optical purity of the products remains insufficient.

[0004] Biocatalysis has become a research hotspot due to its high stereoselectivity and environmental friendliness. However, current enzyme catalysis systems face challenges such as low catalytic efficiency and low product yield. Furthermore, research on the biosynthesis of N-hydroxypiperidinic acid is still in its early stages, with existing studies primarily focusing on designing biosynthetic pathways using plant-derived enzymes. These studies are heavily reliant on coenzymes, and difficulties in coenzyme regeneration and insufficient supply during the reaction process lead to low product yields. [Summary of the Invention]

[0005] [Purpose of the Invention]

[0006] This invention addresses key issues in existing biosynthesis processes of N-hydroxypiperidine acid, including low enzyme catalytic efficiency, insufficient regeneration of the coenzyme NADPH, severe substrate degradation, and uncoordinated product synthesis pathways. By integrating genome mining, directed evolution of enzyme molecules, metabolic engineering, and fermentation process optimization technologies, a stable and scalable method for the biosynthesis of N-hydroxypiperidine acid using L-lysine as a substrate has been developed. Specific objectives include:

[0007] (1) Constructing engineered strains for the biosynthesis of intermediate product L-piperidine acid

[0008] First, the lysine cyclic deaminase (LCD) encoding gene *pipA* was amplified from *Streptomyces pristinaespiralis* ATCC 25486, and the NAD enhancement gene *pntAB* was amplified from *Escherichia coli*. These two genes were then ligated into the plasmid vector *pET-28a* to construct the recombinant plasmid *pET-28a-pipA-pntAB*, which was then transformed into the host bacterium BL21(DE3). The constructed engineered strain can transform the substrate lysine to produce L-piperidine acid, laying the foundation for the synthesis of N-hydroxypiperidine acid.

[0009] (2) Screening and optimization of highly active L-piperidinic acid hydroxylase

[0010] By screening novel L-piperidinate hydroxylase (L-PA hydroxylase) genes from Streptomyces through genome mining, and improving enzyme activity through error-prone PCR directed evolution technology, the technical bottleneck of low catalytic efficiency of traditional hydroxylases can be solved.

[0011] (3) Constructing engineered strains with efficient coenzyme regeneration and substrate metabolism synergy

[0012] A multi-plasmid co-expression system was constructed in Escherichia coli BL21(DE3) to integrate the exogenous glucose dehydrogenase gene (gdh) from Bacillus subtilis, thereby enhancing NADPH supply capacity. The host bacterium's L-lysine degradation gene (cadA) was knocked out to block substrate bypass metabolism, reduce substrate loss, and further increase NHP production. By fusing the SnaC mutant with a coenzyme regeneration module, a high-efficiency engineered strain ΔcadA BL21(DE3) was constructed to further enhance NHP production.

[0013] (4) Establish a high-yield fermentation process system

[0014] Integrating the methods for increasing the yield of N-hydroxypiperidine acid biosynthesis in (2), we developed a fermentation strategy based on the exogenous addition of coenzyme precursor (L-aspartic acid) and dynamic feeding, combined with pH control and nutrient feeding strategies, to systematically increase the yield of N-hydroxypiperidine acid.

[0015] [Technical Solution of the Invention]

[0016] This invention constructs a biosynthetic pathway for N-hydroxypiperidine acid (NHP) using lysine as a substrate in *Escherichia coli* BL21(DE3). The yield was further improved through metabolic engineering, directed evolution, and fermentation optimization. Based on the constructed engineered N-hydroxypiperidine acid biosynthetic strain, on the one hand, enzyme activity was enhanced using error-prone PCR directed evolution technology; on the other hand, the exogenous glucose dehydrogenase gene (gdh) from *Bacillus subtilis* was integrated to strengthen NADPH supply; the host strain's L-lysine degradation gene (cadA) was knocked out to block substrate bypass metabolism, reduce substrate loss, and further increase NHP yield. Finally, by fusing the SnaC mutant with a coenzyme regeneration module, a high-efficiency engineered strain ΔcadA BL21(DE3) was constructed and fermented. In a 5L fermenter, the NHP yield reached 187.6 mg / L. This invention can provide a reference for the subsequent industrial production of N-hydroxypiperidine acid. [Attached Image Description]

[0017] Figure 1 This invention provides a comparison of NHP production yield in fermenters using metabolically engineered bacteria.

[0018] Figure 2 The changes in L-lysine and L-piperidine acid concentrations during fermentation of engineered bacteria after integrating and optimizing conditions according to this invention [Detailed Implementation Method]

[0019] Example 1: Fermentation of engineered bacteria containing the glucose dehydrogenase gene gdh overexpression

[0020] (1) First, the lysine cyclic deaminase LCD encoding gene pipA was amplified from *Streptomyces simulans* and the cofactor NAD enhancement gene pntAB was amplified from *Escherichia coli*. The two were then ligated into the plasmid vector pET-28a to construct the recombinant plasmid pET-28a-pipA-pntAB. After transformation into the host bacterium BL21(DE3), the intermediate product L-piperidine acid could be synthesized using L-lysine as a substrate. Subsequently, the amplified target gene SNaC was ligated into the plasmid vector PACYCDuet-1 to construct the recombinant plasmid PACYCDuet-SNaC. After verification, the linearized plasmid PACYCDuet-SNaC, digested with a single enzyme, was ligated with the amplified NADPH-enhancing gene gdh. BL21(DE3) containing both plasmids pET-28a-pipA-pntAB and PACYCDuet-SNaC-gdh was then fermented in a fermenter. 40 mg / L L-aspartic acid was added to the fermenter, and glucose and L-lysine were added in a fed-batch manner. Initially, 20 g / L L-lysine and 20 g / L glucose were added, respectively. After 36 hours of fermentation, 20 g / L L-lysine and 20 g / L glucose were added. Fermentation broth samples were collected every 24 hours, with a fermentation time of 72 hours. An initial control group consisting of BL21(DE3) containing both pET-28a-pipA-pntAB and PACYCDuet-SNaC was maintained under the same conditions. When the engineered E. coli BL21(DE3) strain containing pET-28a-pipA-pntAB and PACYCDUet-1-SNaC fermented in a fermenter for 72 hours, samples were taken and the results were analyzed. The results showed that the basic strain accumulated 75.2 mg / L NHP during the fermentation process. Based on this, the gdh gene was introduced into the PACYCDUet-1 vector to increase NADPH, and 175.3 mg / L NHP was synthesized in 72 hours.

[0021] Example 2: Fermentation of an engineered strain with the lysine degradation gene cadA knocked out.

[0022] The lysine degradation gene cadA in BL21(DE3) was knocked out. Based on the constructed plasmid PACYCDuet-SNaC-gdh, the host bacterium was further knocked out to construct a recombinant strain ΔcadA BL21(DE3) containing both PACYCDuet-1-SNaC-gdh and pET-28a-pipA-pntAB. Fermentation was also carried out, with 40 mg / L L-aspartic acid added to the fermenter. Glucose and L-lysine were added in a fed-batch manner. Initially, 20 g / L L-lysine and 20 g / L glucose were added, respectively. After 36 h of fermentation, 20 g / L L-lysine and 20 g / L glucose were added again. Fermentation broth samples were collected every 24 h, with a fermentation time of 72 h. After knocking out the lysine degradation gene cadA, the NHP accumulation was 178.2 mg / L.

[0023] Example 3: Fermentation of engineered bacteria containing the mutant L-PA hydroxylase gene

[0024] Due to the low activity of L-PA hydroxylase, error-prone PCR was used for directed evolution. The selected positive mutant gene sequence underwent a mutation at position 38 (G to A) and position 83 (T to A). The amino acid sequence also showed mutations at positions 13 (glycine to aspartic acid) and 28 (valine to aspartic acid). Enzyme activity analysis revealed that the enzyme activity encoded by the mutant SNaC was 0.162 U / mg, representing a 19.1% increase compared to the wild type. The constructed plasmid PACYCDuet-1-SNaC-gdh was back-amplified and ligated with the mutant SNaC. The recombinant plasmid PACYCDuet-Mutant SNaC-gdh and pET-28a-pipA-pntAB were simultaneously transformed into the ΔcadA BL21(DE3) strain for fermentation. 40 mg / L L-aspartic acid was added to the fermenter, and glucose and L-lysine were added in a fed-batch manner. Initially, 20 g / L L-lysine and 20 g / L glucose were added, respectively. After 36 hours of fermentation, 20 g / L L-lysine and 20 g / L glucose were added again. Fermentation broth samples were collected every 24 hours, and the fermentation time was 72 hours. Fermentation of ΔcadA BL21(DE3) containing pET-28a-pipA-pntAB and PA CYCDUet-1-Mutant SNaC-gdh in a fermenter resulted in an NHP accumulation of 187.6 mg / L after 72 h. Analysis of the control group (E. coli BL21(DE3) without the recombinant plasmid) revealed no detectable NHP product under otherwise identical conditions. Compared to the initial engineered strain, NHP production increased by 1.49-fold after recombinant expression of the gdh gene, knockout of the lysine degradation gene cadA, and positive mutation of the SNaC gene.

[0025] The above results indicate that the present invention integrates genes related to the biosynthesis of L-piperidine acid and N-hydroxypiperidine acid in Escherichia coli to further construct and optimize the N-hydroxypiperidine acid biosynthesis system, thereby constructing a method for synthesizing N-hydroxypiperidine acid using lysine as a substrate and the intermediate product L-piperidine acid.

Claims

1. A method for the biosynthesis of N-hydroxypiperidine acid (NHP) based on metabolically engineered strains, characterized in that: (a) Constructing an engineered strain capable of converting L-lysine to L-piperidinic acid: The lysine cyclic deaminase encoding gene pipA and the cofactor NAD enhancement gene pntAB were co-cloned into an expression vector and transformed into the host strain; (b) The L-piperidinic acid hydroxylase gene SNaC and the exogenous glucose dehydrogenase gene gdh were introduced to construct a multi-plasmid co-expression system; the L-lysine degradation gene cadA in the host strain was knocked out to block substrate bypass metabolism; (c) Achieving efficient NHP synthesis through a dynamic fed-batch fermentation process and by regulating the carbon source and substrate concentrations.

2. The method according to claim 1, characterized in that: The host bacterium is Escherichia coli.

3. The method according to claim 1, characterized in that: The nucleotide sequence of the mutant SNaC described in step (b) is shown in Seq ID NO.

1.

4. The method according to claim 1, characterized in that: The exogenous glucose dehydrogenase gene gdh mentioned in step (b) is derived from Bacillus subtilis.

5. The method according to claim 1, characterized in that: The dynamic feeding strategy described in step (c) includes: initially adding 10-30 g / L L-lysine and 20-80 g / L glucose, fermenting for 36-72 h, and fermentation pH of 6.8-7.2.

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