A method for producing phenylmethanol by stage fermentation of recombinant escherichia coli based on regeneration of reducing power

Abstract

The application discloses a method for producing benzyl alcohol by recombinant Escherichia coli in stages based on reduction force regeneration, and belongs to the field of metabolic engineering and industrial fermentation. The method takes the recombinant Escherichia coli for de novo synthesis of benzyl alcohol as a starting production strain, introduces an NAD+-dependent formic acid dehydrogenation (FDH) reduction force regeneration module into the strain, divides the fermentation process into a cell growth stage, an induction expression stage and a benzyl alcohol production stage, and adopts differential glucose concentration control, dissolved oxygen control and feeding strategies in different stages. In the benzyl alcohol production stage, a feeding system containing formic acid or formate is adopted to enhance intracellular NADH regeneration, promote the reduction conversion of benzaldehyde to benzyl alcohol, and inhibit the accumulation of intermediate S-MA, and the benzyl alcohol titer is as high as 18.3 g / L. The method is suitable for high-density fermentation of recombinant Escherichia coli for producing benzyl alcohol, and has the basis for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering and industrial fermentation. Specifically, it relates to a method for the staged fermentation production of benzyl alcohol by recombinant Escherichia coli based on reducing power regeneration. In particular, it relates to a method for reducing the accumulation of by-products and increasing the yield of benzyl alcohol by regenerating intracellular reducing power through formate dehydrogenase (FDH) and combining it with staged fermentation control. Background Technology

[0002] Benzyl alcohol (BALC) is an important fine chemical widely used in fragrances, solvents, and preservatives. Current industrial production methods primarily rely on chemical synthesis, mainly including the benzyl chloride hydrolysis method and the benzaldehyde hydrogenation reduction method. Compared to traditional chemical synthesis routes, the microbial fermentation method for producing benzyl alcohol offers advantages such as renewable raw materials, mild reaction conditions, and significant potential for green manufacturing.

[0003] Existing research has established a metabolic pathway for the de novo synthesis of benzyl alcohol from glucose in *E. coli*. This pathway increases the metabolic flux of phenylpyruvate (PPA) by enhancing the shikimic acid pathway. Then, using phenylpyruvate as a precursor, S-mandelic acid (S-MA) is generated under the catalysis of 4-hydroxymandelic acid synthase (HmaS). Subsequently, benzaldehyde is generated by the sequential catalysis of mandelic acid dehydrogenase (MdlB) and benzoylformate decarboxylase (MdlC), and finally reduced to benzyl alcohol by endogenous alcohol dehydrogenases (ADHs) or aldehyde reductases (AKRs).

[0004] Beijing University of Chemical Technology used Escherichia coli BW25113 as a chassis to enhance precursor supply and benzyl alcohol pathway expression through metabolic engineering, and achieved high benzyl alcohol yields by strategies such as increasing hmaS copy number. Literature reports that XBQ09 is an engineered strain obtained by integrating Plac-Aovcm17-T1 at the yjiP site on the basis of XBQ07. After introducing relevant plasmids into XBQ09, XBQ31 was obtained. XBQ31 achieved a benzyl alcohol yield of 4.66 g / L in shake flasks. In 3 L fed-batch fermentation, when OD600 reached 12, IPTG was added, and DO was maintained at 15%, ultimately achieving a benzyl alcohol titer of 10.26 g / L.

[0005] However, existing technologies still have the following shortcomings: Firstly, existing fermentation processes lack a phased control mechanism that coordinates dynamic feeding strategies with dissolved oxygen regulation strategies, leading to a mismatch between nutrient supply, oxygen supply, and cell growth cycle. This limits cell concentration and consequently affects the overall yield of benzyl alcohol.

[0006] Secondly, the reduction of benzaldehyde depends on the level of intracellular reducing power. At the same time, there is a side reaction of PPA to PLA in the system, which competes with the synthesis of benzyl alcohol in terms of metabolic flux and reduction equivalent distribution. In order to maintain a high "NADH / NAD+" ratio and keep the dissolved oxygen level low, the enzyme activity of MdlB will be inhibited, which will lead to the accumulation of intermediate S-MA. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the staged fermentation production of benzyl alcohol by recombinant Escherichia coli based on reducing power regeneration, so as to solve the problem of insufficient coordination between cell growth, heterologous protein expression, reducing power supply and benzyl alcohol synthesis in the prior art, and further solve the problem of S-MA accumulation during fermentation, thereby increasing the yield of benzyl alcohol.

[0008] The objective of this invention can be achieved through the following technical solutions: A method for producing benzyl alcohol by staged fermentation of recombinant Escherichia coli based on reducing power regeneration is as follows: (1) Selecting the starting production strain Recombinant Escherichia coli containing a benzyl alcohol biosynthesis metabolic module was selected as the starting production strain.

[0009] Preferably, the benzyl alcohol biosynthetic metabolic module includes an exogenous enzymatic reaction module for converting aromatic precursors into benzaldehyde and / or reducing benzaldehyde to benzyl alcohol and / or an endogenous metabolic module for enhancing expression.

[0010] Preferably, the starting strain is an engineered strain constructed using Escherichia coli K-12 derivative or B-line derivative as the host.

[0011] More preferably, the starting strain is an engineered strain constructed based on Escherichia coli BW25113.

[0012] More preferably, the starting strain is XBQ09 or a benzyl alcohol production strain derived from XBQ09.

[0013] (2) Constructing a restorative force regeneration module A reducing power regeneration module was introduced into the starting production strain to enhance the intracellular NADH regeneration capacity during benzyl alcohol production.

[0014] Preferably, the reducing power regeneration module includes an NAD+ (oxidized coenzyme I)-dependent formate dehydrogenase (FDH) expression module.

[0015] More preferably, the FDH is obtained by optimizing the expression of a coding gene from a heterologous microorganism through adaptation to a host, and is introduced into the starting production strain by plasmid expression or chromosome integration.

[0016] More preferably, the FDH is derived from yeast, bacteria, or fungi. Even more preferably, the FDH is derived from Candida boidinii.

[0017] More preferably, formic acid or formate is added to the fermentation system during the benzyl alcohol production period to catalyze the oxidation of formic acid by the FDH and promote the regeneration of intracellular NADH (reduced coenzyme I), thereby improving the conversion efficiency of benzaldehyde to benzyl alcohol.

[0018] (3) Staged fermentation control The recombinant *E. coli* was subjected to staged fermentation culture, including at least a cell growth stage, an induction expression stage, and a benzyl alcohol production stage. Different glucose concentration control, dissolved oxygen control, and feeding strategies were employed at each stage to meet the metabolic requirements of cell amplification, heterologous protein expression, and target product accumulation, respectively.

[0019] Preferably, dissolved oxygen is controlled at above 60% during the cell growth stage, at 30-60% during the induction expression stage, and at 20-40% during the benzyl alcohol production stage.

[0020] More preferably, the glucose concentration is controlled at above 5 g / L during the cell growth stage, at 0-2 g / L during the induction expression stage, and at 1-5 g / L during the benzyl alcohol production stage.

[0021] Preferably, when the bacterial cells grow to an OD600 of 10-15, an inducer is added to the fermentation system to initiate the expression of the benzyl alcohol biosynthesis metabolism module and the reducing power regeneration module.

[0022] More preferably, the inducer is IPTG with a final concentration of 1 mM.

[0023] (4) Production period reduction force coupled feeding control During the benzyl alcohol production stage, a feeding system containing formic acid or formate is used for dynamic feeding, which works in conjunction with the reducing power regeneration module to enhance intracellular NADH regeneration, promote the reduction conversion of benzaldehyde to benzyl alcohol, and inhibit the accumulation of intermediate S-MA.

[0024] Preferably, the benzyl alcohol production stage is switched in 6-10 h after induction, and the concentration of formic acid or formate in the fermentation broth is controlled to be no higher than 2 g / L.

[0025] The beneficial effects of this invention are: (1) By dividing the fermentation process into a cell growth stage, an induction expression stage and a benzyl alcohol production stage, and adopting differentiated carbon and nitrogen source feeding and dissolved oxygen control strategies at different stages, this invention can better match the different metabolic needs of cell amplification, exogenous enzyme expression and target product accumulation, thereby improving the stage adaptability of the fermentation process.

[0026] (2) The present invention introduces a reducing power regeneration module in the benzyl alcohol production stage, and preferably combines it with formic acid or formate feed, which is beneficial to enhance intracellular NADH regeneration and promote the reduction conversion of benzaldehyde to benzyl alcohol.

[0027] (3) By combining the regeneration of reducing power during the production period with staged fermentation control, this invention helps to suppress the accumulation of intermediate S-MA and increase the yield of benzyl alcohol.

[0028] (4) This invention improves the process based on the fermentation of the disclosed benzyl alcohol producing bacteria, and has a clear engineering implementation basis and potential for scale-up application. Attached Figure Description

[0029] The invention will now be further described with reference to the accompanying drawings.

[0030] Figure 1 It is the plasmid pCS-pheA in Example 1 fbr -aroG fbr -FDH diagram. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] SEQ ID NO:1: Codon-optimized FDH sequence (5'-3'): Artificial sequence: Example 1: Construction of production strain and reducing power regeneration module This embodiment uses a recombinant *Escherichia coli* strain capable of producing benzyl alcohol from glucose fermentation as the starting production strain. The starting production strain is *XBQ09*, a benzyl alcohol production strain constructed using *Escherichia coli* BW25113 as a chassis. In the disclosed system, *XBQ09* is used in conjunction with plasmids pET-Aovcm17-Aovcm17-mdlB-mdlC-Aovcm17 and pCS-pheA. fbr -aroG fbr Co-transformation resulted in the production strain XBQ31. To enhance intracellular reducing power supply during benzyl alcohol production, an NAD+-dependent formate dehydrogenase (FDH) reducing power regeneration module was introduced into the starting production strain. The FDH was derived from *Candida boidinii*, with accession number AJ011046.2, an original open reading frame length of 1095 bp, encoding 364 amino acid residues. The FDH encoding gene was codon-optimized according to *E. coli* host expression preferences, and the optimized nucleotide sequence is shown in SEQ ID NO:1 above.

[0033] With plasmid pCS-pheA fbr -aroG fbr As a vector, the plasmid contains the pLlacO1 promoter, p15A replicon, and kanamycin resistance marker, and carries pheA. fbr and aroG fbr Expression module. The codon-optimized FDH expression cassette was inserted into plasmid pCS-pheA using the Gibson assembly method. fbr -aroG fbr In the process, the recombinant plasmid pCS-pheA was obtained. fbr -aroG fbr -FDH (The spectrum of this plasmid is as follows) Figure 1 (As shown). The FDH expression cassette includes a lac promoter, a lac operator, an RBS, an FDH coding sequence, and an rrnB T1 terminator, wherein the rrnB T1 terminator is used to terminate FDH expression transcription. Then, the recombinant plasmid pCS-pheA fbr -aroG fbrFDH and plasmid pET-Aovcm17-Aovcm17-mdlB-mdlC-Aovcm17 (containing an ampicillin resistance marker) were co-transformed into the starting production strain XBQ09. Positive transformants were screened on LB solid medium containing kanamycin and ampicillin, and the correct construction of the target strain was verified by colony PCR and sequencing. A benzyl alcohol production engineered strain containing a reducing power regeneration module was obtained and named HYSW-BA01. In subsequent fermentation, the engineered strain provides substrate for the FDH-catalyzed reaction by supplementing formic acid or formate, thereby promoting intracellular NADH regeneration, enhancing the reduction conversion of benzaldehyde to benzyl alcohol, and laying the foundation for reducing the accumulation of intermediate S-MA and increasing benzyl alcohol yield.

[0034] Example 2: Fermentation medium and seed culture method This embodiment uses the recombinant engineered strain HYSW-BA01 constructed in Example 1 for fermentation culture. Seed culture medium, fermentation medium, and fed culture medium are used during the fermentation process. The seed culture medium consists of: phenylalanine 0.2 g / L, tyrosine 0.4 g / L, sodium citrate 4 g / L, KH₂PO₄ 3 g / L, Na₂HPO₄ 6.78 g / L, yeast extract 10 g / L, soybean peptone 15 g / L, glucose 10 g / L, with pH adjusted to 7.0±0.1. The fermentation medium consists of: phenylalanine 0.1 g / L, tyrosine 0.2 g / L, sodium citrate 2 g / L, KH₂PO₄ 3 g / L, (NH₄)₂SO₄ 4 g / L, Na₂HPO₄ 6.78 g / L, yeast extract 6 g / L, corn steep liquor 6 g / L, glucose 25 g / L, TES stock solution 4 mL / L, with pH adjusted to 7.0±0.1. The TES is a trace element mother liquor containing 0.15 g / L CuSO4·5H2O, 1 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L MnCl2·4H2O, and 0.5 g / L CoCl2·6H2O. The fed culture medium comprises stock solution 1 and stock solution 2. Stock solution 1 consists of: phenylalanine 1 g / L, tyrosine 2 g / L, sodium citrate 20 g / L, (NH4)2SO4 4 g / L, yeast extract 50 g / L, corn steep liquor powder 80 g / L, and TES stock solution 10 mL / L, with the pH adjusted to 7.0±0.1. Stock solution 2 consists of: sodium citrate 20 g / L, (NH4)2SO4 4 g / L, yeast extract 50 g / L, corn steep liquor powder 80 g / L, and sodium formate 20 g / L, with the pH adjusted to 7.0±0.1. During the culture process, ampicillin and kanamycin are added to the culture medium to maintain the dual plasmid selection pressure according to the plasmid resistance requirements. For seed culture, HYSW-BA01 cultured in glycerol was first streaked onto LB agar plates containing kanamycin and ampicillin. After incubation at 37°C overnight, single colonies were picked and inoculated onto slant agar plates containing the corresponding antibiotics. After 12-16 hours of incubation, a suitable amount of bacterial sludge was transferred to seed culture medium and cultured with shaking at 37°C and 220 rpm to obtain seed culture. The seed culture was then inoculated into fermentation medium for further culture. In a preferred embodiment, the seed culture was cultured with shaking in 1000 mL flat-bottomed shake flasks (15% volume) for approximately 10-12 hours before being used to inoculate the fermenter.

[0035] Example 3: Staged fermentation control and reducing force coupled feeding method during production period The recombinant engineered strain HYSW-BA01 obtained in Example 1 was used to prepare seed culture according to the method described in Example 2, and then inoculated into a fermenter for fed-batch fermentation. The fermentation process included a strain growth stage, a protein expression stage, and a benzyl alcohol production stage. During the strain growth stage, the fermentation temperature was controlled at 37℃, the pH was controlled at 7.0±0.1 using 5 M sodium hydroxide, the aeration rate was 1 vvm, and the stirring speed was 200-600 rpm, which was adjusted according to the dissolved oxygen level to maintain dissolved oxygen above 60%. At the same time, 600 g / L glucose mother liquor was used for feeding to maintain the glucose concentration in the fermentation broth above 5 g / L, so as to promote rapid cell growth and biomass accumulation. During the protein expression stage, when the cell growth reaches an OD600 of 10⁻¹⁵, IPTG (isopropylthio-β-D-galactoside) is added to the fermentation system to a final concentration of 1 mM to initiate the expression of the exogenous synthesis pathway and the FDH reducing power regeneration module. During this stage, the fermentation temperature is controlled at 26℃, pH at 7.0±0.1, dissolved oxygen at 30%-60%, and glucose concentration at 0-2 g / L. Simultaneously, "Feeding Medium Stock Solution 1" is used for feeding at a rate of 5-10 mL / L / h, dynamically adjusted according to residual sugar, dissolved oxygen, and cell growth status. The benzyl alcohol production stage begins 6-8 hours after the addition of IPTG. During this stage, the fermentation temperature is adjusted to 30℃, pH at 7.0±0.1, dissolved oxygen at 20%-40%, and glucose concentration at 1-5 g / L. Simultaneously, "Feeding Medium Stock Solution 2" is used for dynamic feeding to ensure that the concentration of formic acid or formate in the fermentation broth does not exceed 2 g / L. The formic acid or formate salt works synergistically with the NAD+-dependent formate dehydrogenase module introduced into the strain to promote intracellular NADH regeneration, thereby enhancing the reduction conversion of benzaldehyde to benzyl alcohol and inhibiting the accumulation of intermediate S-MA. During fermentation, the stirring speed, aeration rate, and feed rate can be adjusted in a timely manner based on dissolved oxygen feedback to achieve differentiated control of cell amplification, exogenous protein expression, and benzyl alcohol production at different stages.

[0036] Example 4: Production of benzyl alcohol by high-density fed-batch fermentation using recombinant strain HYSW-BA01 Seed culture of recombinant strain HYSW-BA01 was prepared according to the method described in Example 2. Following the staged fermentation control and reducing force coupled feeding method during the production period described in Example 3, high-density fed-batch fermentation was carried out in a 5 L stainless steel fermenter with a liquid volume of 70%. During fermentation, samples were taken periodically to detect cell concentration, residual glucose concentration, benzyl alcohol concentration, residual sodium formate concentration, and the contents of intermediate S-MA and byproduct PLA, evaluating the product synthesis performance of recombinant strain HYSW-BA01 under high-density fermentation conditions. After 72 h of fermentation, the OD of recombinant strain HYSW-BA01 was... 600The biomass reached 83.1 g / L, with a wet weight of 164.1 g / L, a dry weight of 34.9 g / L, a PLA titer of 4.6 g / L, an S-MA titer of 11.2 g / L, and a benzyl alcohol titer of 18.3 g / L. The results indicate that, by employing the staged fermentation control and production-period reducing power coupled feeding strategy described in this invention, HYSW-BA01 can maintain a high biomass within a relatively short fermentation cycle, achieving efficient accumulation of benzyl alcohol. As a control, under the same fermentation system, high-density fed-batch fermentation was carried out using XBQ31, with a fermentation cycle of 84 h, and the cell OD... 600 The concentrations were 47.3 g / L (wet weight), 105.3 g / L (dry weight), 19.7 g / L (solar alcohol), 3.8 g / L (S-MA), 21.3 g / L (benzyl alcohol), and 11.6 g / L (benzyl alcohol). Compared with XBQ31, HYSW-BA01 showed a shorter fermentation cycle, a significantly increased bacterial concentration, and a 58% increase in benzyl alcohol yield. Simultaneously, the accumulation of intermediate S-MA decreased significantly. This indicates that the present invention, by introducing an FDH reducing power regeneration module and combining it with formate-coupled feeding during the production period, effectively enhances the reduction conversion of benzaldehyde to benzyl alcohol and improves overall fermentation performance.

[0037] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A method for producing benzyl alcohol by staged fermentation of recombinant Escherichia coli based on reducing power regeneration, characterized in that, Includes the following steps: (1) A recombinant Escherichia coli containing a benzyl alcohol biosynthesis metabolism module was used as the starting strain, and a reducing power regeneration module was introduced into the starting strain, the reducing power regeneration module including an NAD+-dependent FDH expression module. (2) The strain obtained in step (1) is inoculated into the fermentation system for fed-batch fermentation culture. The batch fermentation culture includes at least the cell growth stage, the induction expression stage and the benzyl alcohol production stage. (3) During the cell growth stage, the fermentation conditions are controlled to promote cell proliferation; (4) During the induction expression stage, the fermentation conditions are adjusted to promote the expression of the benzyl alcohol biosynthesis metabolism module and the reducing power regeneration module; (5) During the benzyl alcohol production stage, the dissolved oxygen level and carbon supply intensity are controlled, and a feeding system containing formic acid or formate is used for dynamic feeding to drive the FDH to catalyze the oxidation of formic acid and regenerate NADH, thereby promoting the biosynthesis of benzyl alcohol.

2. The method according to claim 1, characterized in that, The benzyl alcohol biosynthetic metabolic module includes an exogenous enzymatic reaction module that converts aromatic precursors into benzaldehyde and / or reduces benzaldehyde to benzyl alcohol and / or an endogenous metabolic module that enhances expression.

3. The method according to claim 1, characterized in that, The starting strain is an engineered strain constructed using Escherichia coli K-12 derivatives or B-line derivatives as the host.

4. The method according to claim 1, characterized in that, The FDH is obtained by optimizing the expression of a gene from a heterologous microorganism through host expression, and is introduced into the starting strain via plasmid expression and / or chromosome integration.

5. The method according to claim 1, characterized in that, During the cell growth stage, the glucose concentration is controlled at above 5 g / L; during the induction expression stage, the glucose concentration is controlled at 0-2 g / L; and during the benzyl alcohol production stage, the glucose concentration is controlled at 1-5 g / L.

6. The method according to claim 1, characterized in that, The fermentation temperature during the cell growth stage is 37℃, pH is 7.0±0.1, aeration rate is 1 vvm, stirring speed is 200-600 rpm, and dissolved oxygen is controlled above 60%.

7. The method according to claim 1, characterized in that, When the bacterial cells grow to an OD600 of 10⁻¹⁵, an inducer is added to the fermentation system to initiate the expression of the benzyl alcohol biosynthesis metabolism module and the reducing power regeneration module.

8. The method according to claim 7, characterized in that, The inducer is IPTG, with a final concentration of 1 mM.

9. The method according to claim 1, characterized in that, The fermentation temperature during the induction expression stage was 26℃, the pH was 7.0±0.1, and the dissolved oxygen was controlled at 30%-60%.

10. The method according to claim 1, characterized in that, The benzyl alcohol production stage is switched in 6-10 hours after induction.

11. The method according to claim 1, characterized in that, The fermentation temperature during the benzyl alcohol production stage is 30℃, the pH is 7.0±0.1, and the dissolved oxygen is controlled at 20%-40%.

12. The method according to claim 1, characterized in that, The dynamic feeding method controls the concentration of formic acid or formate in the fermentation broth to no higher than 2 g / L.

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