L-pantoic acid lactone dehydrogenase and application of co-expressed engineering bacteria thereof in synthesis of D-pantoic acid lactone

Abstract

The present application provides a L-pantoic acid lactone dehydrogenase derived from Rhodococcus erythropolis, and a mutant thereof, a nucleic acid molecule encoding the L-pantoic acid lactone dehydrogenase or the mutant thereof, a vector for expressing the L-pantoic acid lactone dehydrogenase or the mutant thereof in a host cell, and an engineered bacterium expressing the enzyme or the mutant thereof. The present application further provides an engineered bacterium co-expressing the L-pantoic acid lactone dehydrogenase or the mutant thereof and any one or more selected from the group consisting of a chaperone, a D-ketopantoic acid lactone reductase, and a glucose dehydrogenase. The present application further provides a method for producing D-pantoic acid lactone using the engineered bacterium expressing the L-pantoic acid lactone dehydrogenase derived from Rhodococcus erythropolis or the mutant thereof.

Description

Technical Field

[0001] This falls under the field of biotechnology, specifically involving the co-expression of pantothenic acid lactone dehydrogenase from Rhodococcus erythropolis and its applications. Background Technology

[0002] D-Pantothenic acid lactone is a key chiral intermediate in the synthesis of D-calcium pantothenate (vitamin B5), D-panthenol, and D-panthenylthiols, which are widely used in food, feed additives, pharmaceuticals, and cosmetics.

[0003] Currently, the main synthetic method for D-pantolactone begins with aldol condensation, using inexpensive isobutyraldehyde and formaldehyde to synthesize hydroxypentanal, adding hydrogen cyanide under acidic conditions to form cyanohydrin, followed by hydrolysis and cyclization to obtain DL-pantolactone. Alternatively, a bioenzymatic method (D-pantolactone hydrolase) can be used to selectively synthesize D-pantolic acid, followed by solvent extraction to separate D-pantolic acid and L-pantolactone, and acidification of D-pantolactone to obtain D-pantolactone. Although biocatalytic resolution has been applied to commercial production, the maximum theoretical yield of D-pantolactone is only 50%, and an additional racemic step is required to recover unreacted substrate to improve the yield. Therefore, the method still suffers from problems such as numerous repetitive steps and high acid-base consumption.

[0004] The asymmetric synthesis of D-pantolactone using oxidoreductases demonstrates significant application potential due to its high enantiomeric purity and high theoretical yield. One approach involves the selective oxidation of L-pantolactone with L-pantolactone dehydrogenase to ketopantolactone, followed by selective reduction with D-ketopantolactone reductase to prepare D-pantolactone. Another alternative route similarly involves the selective oxidation of L-pantolactone with L-pantolactone dehydrogenase to ketopantolactone, which then spontaneously hydrolyzes to ketopantoacid. This ketopantoacid is then converted to D-pantoacid using D-ketopantoacid reductase, and finally, acid is added to close the ring of D-pantoacid, synthesizing D-pantolactone. The first route is simpler and directly yields a chiral, pure final product. Through coenzyme regeneration and engineering modification, only a small amount or no cofactor needs to be added.

[0005] As mentioned above, L-pantolactone dehydrogenase is a key enzyme in the redox asymmetric synthesis of D-pantolactone, responsible for catalyzing the crucial first step of the reaction. Currently, there are very few reported L-pantolactone dehydrogenases. While the L-pantolactone dehydrogenase from *Nocardia asteroides*, discovered in 1992, has some enzymatic characterization data, it is mainly expressed as inclusion bodies in *Escherichia coli*, and the encoding gene remains unknown (Kataoka M, et al. *European Journal of Biochemistry* 1992, 204, 799-806). Furthermore, an L-pantolactone dehydrogenase from *Rhodococcus rubrum* was reported in 2012. In terms of its properties, if the expression of the *Rhodococcus rubrum*-derived L-pantolactone dehydrogenase is enhanced in its original host, using 0.768 M of L-pantolactone as a substrate, the conversion rate can reach 91.9% after 144 hours of whole-cell reaction. However, the generated ketopantolate lactone spontaneously hydrolyzes to ketopantolate, requiring additional expression of ketopantolate reductase to convert it into D-pantolate (SiD, Urano N, Nozaki S, et al. L-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of L-pantoyl lactone. Applied Microbiology and Biotechnology, 2012, 95: 431-440). However, when expressed in *E. coli*, the L-pantolate lactone dehydrogenase exhibits low solubility, resulting in lower activity than wild-type *Rhodococcus erythropolis*. Therefore, it is not possible to co-express ketopantolate reductase with the L-pantolate lactone dehydrogenase derived from *Rhodococcus erythropolis* in *E. coli*, necessitating the construction of multiple engineered bacteria for stepwise reactions. Escherichia coli, as a biological platform for fermentation engineering, is currently the most extensively studied universal host. The inability to apply E. coli significantly reduces the operability of the enzyme. Furthermore, if different hosts are used, such as E. coli cells expressing ketopantolate reductase and Rhodococcus rubrum cells respectively, for the biotransformation of L-pantolate lactone, the conversion rate will be reduced, and the process will be more complex and costly. These drawbacks hinder its further application in the biotransformation of L-pantolate lactone. Summary of the Invention

[0006] In view of the above-mentioned technical problems, the inventors of this invention optimized the expression system of L-pantolactone dehydrogenase derived from Rhodococcus erythropolis, significantly improving the solubility of L-pantolactone dehydrogenase from Rhodococcus erythropolis in Escherichia coli, resulting in significant L-pantolactone dehydrogenase activity in Escherichia coli. Furthermore, the inventors of this invention constructed various engineered bacteria expressing this enzyme based on Escherichia coli and verified the efficiency of the biosynthesis method of D-pantolactone using these engineered bacteria. The results showed that the biosynthesis method of D-pantolactone has high conversion rate and fast speed, and has great commercial application prospects. Building upon this, in order to further enhance the activity of the L-pantolactone dehydrogenase derived from Rhodococcus erythropolis in Escherichia coli, the inventors of this invention further mutated the L-pantolactone dehydrogenase derived from Rhodococcus erythropolis, and screened out two mutants of the L-pantolactone dehydrogenase derived from Rhodococcus erythropolis with further enhanced activity in Escherichia coli.

[0007] As described above, the present invention provides an L-indo-lactone dehydrogenase or a mutant thereof derived from *Rhodococcus erythropolis*, a nucleic acid molecule encoding the L-indo-lactone dehydrogenase or a mutant thereof, a vector for expressing the L-indo-lactone dehydrogenase or a mutant thereof in host cells, and an engineered bacterium expressing the enzyme or a mutant thereof. The present invention further provides an engineered bacterium co-expressing the L-indo-lactone dehydrogenase or a mutant thereof and any one or more selected from the group consisting of: molecular chaperones, D-keto-indo-lactone reductase, and glucose dehydrogenase. The present invention further provides a method for producing D-indo-lactone using an engineered bacterium expressing the L-indo-lactone dehydrogenase or a mutant thereof derived from *Rhodococcus erythropolis*.

[0008] Specifically, the present invention includes the following aspects:

[0009] 1. An isolated L-pantolactone dehydrogenase comprising an amino acid sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0010] 2. An isolated nucleic acid molecule encoding L-indohydrin dehydrogenase as described in item 1.

[0011] 3. The nucleic acid molecule as described in item 2, wherein the nucleic acid molecule comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 10 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0012] 4. A recombinant vector or combination of recombinant vectors comprising (a) of the following and optionally (b) of the following:

[0013] (a) A nucleic acid sequence encoding an L-pantolactone dehydrogenase as described in item 1, said nucleic acid sequence comprising a nucleic acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 10 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence;

[0014] (b) A nucleic acid sequence encoding a molecular chaperone, said molecular chaperone being selected from one or more of the following: glutathione thiotransferase (GST), disulfide oxidoreductase (dsbA), protein disulfide isomerase (dsbC), glutathione reductase (gor), thioredoxin-1 (trxA), thioredoxin reductase (trxB), and nucleotide exchange factor (grpE). Preferably, dsbA, GST, or TrxA.

[0015] 5. The recombinant vector or combination of recombinant vectors as described in item 4, wherein the molecular chaperone has any sequence selected from the group consisting of: any sequence shown in any one of SEQ ID NO: 11 to SEQ ID NO: 17 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the above sequences.

[0016] 6. The recombinant vector or combination of recombinant vectors as described in item 4 or 5, further comprising:

[0017] (c) Nucleic acid sequence encoding glucose dehydrogenase.

[0018] 7. The recombinant vector or combination of recombinant vectors as described in any one of items 4 to 6, wherein the nucleic acid sequence encoding glucose dehydrogenase comprises the nucleic acid sequence shown in SEQ ID NO: 4 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0019] 8. The recombinant vector or combination of recombinant vectors as described in any one of items 4 to 7, further comprising:

[0020] (d) Nucleic acid sequence encoding D-keto-indohydranolate reductase.

[0021] 9. The recombinant vector or combination of recombinant vectors as described in item 8, wherein the nucleic acid sequence encoding D-keto-universalactone reductase comprises the nucleic acid sequence shown in SEQ ID NO: 6 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0022] 10. The recombinant vector or combination of recombinant vectors as described in any one of items 4 to 9, wherein the combination of recombinant vectors comprises a first recombinant vector and a second recombinant vector.

[0023] The first recombinant vector contains:

[0024] (a) A nucleic acid sequence encoding the L-pantolactone dehydrogenase as described in item 1, said nucleic acid sequence comprising a nucleic acid sequence as shown in any one of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 10 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence; and

[0025] (b) A nucleic acid sequence encoding a molecular chaperone, said molecular chaperone having any sequence selected from the group consisting of: sequences shown in any one of SEQ ID NO: 11 to SEQ ID NO: 17 or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with any of the above sequences.

[0026] The second recombinant vector contains:

[0027] (c) The nucleic acid sequence encoding glucose dehydrogenase; and

[0028] (d) Nucleic acid sequence encoding D-keto-indohydranolate reductase.

[0029] 11. The recombinant vector or combination of recombinant vectors as described in any one of items 4 to 10, wherein the first recombinant vector is a recombinant vector obtained by editing any of the following vectors: pCDFDuet1, pACYCDuet-1, pETDuet-1, pRSFDuet-1.

[0030] 12. The recombinant vector or combination of recombinant vectors as described in any one of items 4 to 11, wherein the second recombinant vector is a recombinant vector obtained by editing the pET28a vector.

[0031] 13. A protein combination comprising (a) of the following and one or more selected from (b), (c) and (d):

[0032] (a) L-indohydrin dehydrogenase as described in item 1;

[0033] (b) Molecular chaperones selected from GST, dsbA, dsbC, gor, TrxA, TrxB or grpE;

[0034] (c) D-keto-pantolysin reductase;

[0035] (d) Glucose dehydrogenase.

[0036] 14. The protein combination as described in item 13, wherein the molecular chaperone is GST, TrxA, or dsbA.

[0037] 15. The protein combination as described in item 13 or 14, wherein the glucose dehydrogenase comprises an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0038] 16. The protein combination of any one of items 13 to 16, wherein the D-keto-universalactone reductase comprises an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with said sequence.

[0039] 17. An engineered bacterium expressing L-pantolactone dehydrogenase as described in item 1, or expressing a combination of proteins as described in any one of items 13 to 16, or containing a nucleic acid molecule as described in item 2 or 3, or containing a recombinant vector or a combination of recombinant vectors as described in any one of items 4 to 12.

[0040] 18. The engineered bacteria as described in item 17, wherein the engineered bacteria are obtained by processing any one of the host cells selected from: Escherichia coli, Bacillus subtilis, yeast cells, or Aspergillus.

[0041] 19. The engineered bacteria as described in item 17 or 18, wherein the host cell is Escherichia coli E. coli BL21(DE3).

[0042] 20. A method for preparing D-pantolactone, comprising the following steps:

[0043] (a) The step of dehydrogenating L-pantolactone by L-pantolactone dehydrogenase as described in item 1 to generate ketopantolactone.

[0044] (b) The step of reducing ketone-indohydrin to form D-indohydrin.

[0045] 21. The method for preparing D-pantolactone as described in item 20, wherein step (a) is carried out by engineered bacteria as described in any one of items 17 to 19; or both steps (a) and (b) are carried out by engineered bacteria as described in any one of items 17 to 19.

[0046] 22. The method for preparing D-pantolytic lactone as described in item 20 or 21, wherein the specific steps of the method include:

[0047] (i) The step of processing the host cells and screening for engineered bacteria as described in any one of items 17 to 19;

[0048] (ii) The step of inducing the engineered bacteria to express the required L-pantolactone dehydrogenase, chaperone protein, D-ketopantolactone reductase, glucose dehydrogenase or protein combination.

[0049] (iii) The step of adding the engineered bacteria to a substrate containing L-indohydrin for reaction.

[0050] 23. The method for preparing D-pantolytic lactone as described in any one of items 20 to 22, wherein step (ii) is carried out at 20°C to 28°C, preferably 25°C.

[0051] 24. The method for preparing D-pantolactone as described in any one of items 20 to 23, wherein the final concentration of L-pantolactone is 10-65 g / L, preferably 50-65 g / L.

[0052] 25. The method for preparing D-pantolactone as described in item 22, wherein the substrate in step (iii) is further selected from one or more of the following: glucose, NADP+ or CaCO3.

[0053] 26. The method for preparing D-pantolactone as described in item 25, wherein the final concentration of glucose is 10-100 g / L, preferably 16-100 g / L.

[0054] 27. The method for preparing D-pantolactone as described in item 25, wherein the final concentration of NADP+ is 0.02-0.15 mg / mL, preferably 0.05-0.1 mg / mL.

[0055] 28. The method for preparing D-pantolactone as described in item 25, wherein the final concentration of CaCO3 is 0.1-0.3 g / mL, preferably 0.15-0.25 g / mL.

[0056] 29. The method for preparing D-pantolactone as described in any one of items 22 to 28, wherein the reaction temperature in step (iii) is 25°C to 30°C, preferably 30°C.

[0057] 30. The method for preparing D-pantolytic lactone as described in any one of items 22 to 29, wherein step (a3) ​​is carried out at 200-350 rpm, preferably 200 rpm.

[0058] In this specification, unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. When a term is provided in the singular, the plural form of that term is also included unless otherwise stated. Unless otherwise stated, nucleic acid sequences in the text of this specification are given in a 5′ to 3′ orientation relative to the promoter. Unless otherwise stated, amino acid sequences in the text of this specification are given in an N-terminal to C-terminal orientation.

[0059] In this specification, "comprising" means not only "to include" but also "to consist of".

[0060] In this specification, the term "enzyme" refers to any substance that catalyzes or promotes one or more chemical or biochemical reactions, generally including enzymes composed entirely or partially of polypeptides, but may also include enzymes composed of different molecules comprising polynucleotides. In this invention, the amino acid sequence of the enzyme can be modified within a certain range, provided that the specific catalytic activity is not significantly inhibited; for example, it can be an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the specific sequence disclosed in this invention. In this specification, the terms "protein," "proteinate," or "enzyme" are not limited to proteins expressed in a host body, but can also refer to purified proteins or derivatives thereof.

[0061] In this specification, the term "L-indo-indo-lactone dehydrogenase" generally refers to a protein, polypeptide, or derivative thereof that has the activity of catalyzing the dehydrogenation of L-indo-indo-lactone to form keto-indo-lactone.

[0062] In this specification, the term "molecular chaperone," also known as a molecular chaperone, generally refers to a class of proteins that assist in the assembly of molecules within cells and in the folding of proteins. They typically play a crucial role in the folding of peptide chains, promoting the correct folding and assembly of proteins. Molecular chaperones facilitate the efficient folding of newly synthesized proteins, prevent their aggregation, and ensure protein homeostasis within the cell.

[0063] In this specification, the term "nucleic acid" or "nucleic acid molecule" generally refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., the corresponding forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have alterations to the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications include, for example, replacing one or more hydroxyl groups with halogens, alkyl groups, amines, and azides, or functionalizing the sugar into an ether or ester. Furthermore, the entire sugar moiety may be replaced by stereochemically and electronically similar structures such as aza-sugars and carbocyclic sugar analogs. Examples of base moiety modifications include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester bonds or similar linkages. Phosphodiester-linked analogs include thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, aniline thiophosphates, phosphoranilidates, and phosphoramide esters. It should be noted that, in this application, the term "nucleic acid molecule" includes not only nucleic acid molecules with a single sequence but also combinations of multiple nucleic acid molecules.

[0064] In this specification, "isolated nucleic acid molecule" generally refers to a nucleic acid molecule that is not integrated into the genomic DNA of an organism. For example, a DNA molecule encoding a receptor that has been isolated from the genomic DNA of a cell is an isolated DNA molecule. Another non-limiting example of an isolated nucleic acid molecule is a chemically synthesized nucleic acid molecule that is not integrated into the genome of an organism. Yet another non-limiting example of an isolated nucleic acid molecule is a nucleic acid molecule that has been isolated from a particular species and is smaller than a complete DNA molecule from the chromosome of that species.

[0065] In this specification, the term "isolated protein" generally refers to a protein that has been separated from its components in its native environment. In some embodiments, the protein is purified to a purity greater than 95% or 99%, which is determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). A review of methods for evaluating protein purity can be found in Flatman, S. et al., J. Chrom. B848 (2007) 79-87.

[0066] In this specification, the term "host cell" refers to a cell that can be used to introduce the vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, and fungal cells such as yeast cells or Aspergillus.

[0067] In this specification, the term "engineered bacteria" refers to a bacterial cell line that uses genetic engineering methods to achieve efficient expression of exogenous genes in host cells.

[0068] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transferring inserted nucleic acid molecules into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product. In this application, the vector may contain one or more of the aforementioned nucleic acid molecules. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements regulating gene transcription or mRNA translation. In some embodiments, the expression control sequence is a tunable element. The specific structure of the expression control sequence may vary depending on the species or cell type and its function, but it typically includes a 5' non-transcriptional sequence and 5' and 3' non-translational sequences, respectively, involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, etc. For example, the 5' non-transcriptional expression control sequence may contain a promoter region, which may contain a promoter sequence for transcriptionally controlling the functional linker nucleic acid. The vector described in this application may be selected from plasmids, retroviral vectors, and lentiviral vectors.

[0069] In this specification, the term "transformation" generally refers to a change in the genetic characteristics of a cell, which is transformed when the cell is modified to contain new DNA or RNA. For example, a cell is transformed when its native state is genetically modified by introducing new genetic material via transfection, transduction, or other techniques. In bacteria, "competent" refers to a state capable of uptake DNA. Competent cells can be produced by laboratory procedures known in the art, such as cooling the cells in the presence of divalent cations (e.g., CaCl2) to make the cell wall permeable to plasmid DNA; or culturing the cells with a plasmid and subsequently subjecting them to brief heat shock to allow the plasmid to enter the cell. Electroporation is another method for allowing plasmids to enter cells.

[0070] In this specification, the term "final concentration" refers to the concentration in the reaction system. Attached Figure Description

[0071] Figure 1 . Figure 1 This diagram illustrates the reaction catalyzed by the coupling of three enzymes—L-indo-indo-lactone dehydrogenase ReLDH, D-keto-indo-lactone reductase CglCPR, and glucose dehydrogenase BmGDH—to synthesize D-indo-indo-lactone from L-indo-indo-lactone.

[0072] Figure 2 . Figure 2 SDS-PAGE electrophoresis image of L-pantoacid dehydrogenase solublely expressed in Escherichia coli (ReLDH: 35.7 kDa).

[0073] Figure 3 . Figure 3 A is the liquid chromatogram of L-indophosphatidylcholine and D-indophosphatidylcholine. Figure 3 B is the liquid chromatogram of the reaction solution of L-pantolactone dehydrogenase coupled with CglCPR and BmGDH three enzymes.

[0074] Figure 4 . Figure 4 The conversion rates of 50 g / L L-pantolactone catalyzed by L-pantolactone dehydrogenase ReLDH coupled with CglCPR and BmGDH multi-enzyme co-expression with different molecular chaperones were measured.

[0075] Figure 5 . Figure 5 The time progression of the conversion of 50 g / L L-pantolactone catalyzed by L-pantolactone dehydrogenase ReLDH coupled with CglCPR and BmGDH at different expression temperatures is shown in the figure.

[0076] Figure 6 . Figure 6The conversion rates of 65 g / L L-pantolactone catalyzed by wild-type and mutant L-pantolactone dehydrogenase ReLDH coupled with CglCPR and BmGDH multi-enzymes under the co-expression of molecular chaperone GST were determined. Detailed Implementation

[0077] This invention provides an L-indo-lactone dehydrogenase (ReLDH) derived from *Rhodococcus erythropolis* and its mutants, a nucleic acid molecule encoding the L-indo-lactone dehydrogenase or its mutants, a vector for expressing the L-indo-lactone dehydrogenase or its mutants in host cells, and an engineered bacterium expressing the enzyme or its mutants. This invention further provides an engineered bacterium co-expressing the L-indo-lactone dehydrogenase or its mutants and any one or more of the following: molecular chaperones, D-keto-indo-lactone reductase, and glucose dehydrogenase. This invention further provides a method for producing D-indo-lactone using engineered bacteria expressing the L-indo-lactone dehydrogenase derived from *Rhodococcus erythropolis* or its mutants. One of the technical solutions provided by this invention is to provide a recombinant expression vector comprising a gene expressing the L-indo-lactone dehydrogenase or its mutants and optionally a molecular chaperone gene.

[0078] The amino acid sequence of the L-pantolactone dehydrogenase (ReLDH) described above is shown in SED ID NO: 1. The nucleic acid sequence encoding the L-pantolactone dehydrogenase is shown in SED ID NO: 2. The amino acid sequences of the mutants of the L-pantolactone dehydrogenase (ReLDH) screened in this invention are shown in SED ID NO: 7 and SED ID NO: 9. The nucleic acid sequences encoding the mutants of the L-pantolactone dehydrogenase (ReLDH) screened in this invention are shown in SED ID NO: 8 and SED ID NO: 10.

[0079] Furthermore, those skilled in the art should understand that when the above-mentioned amino acid sequence or nucleic acid sequence still has the technical effects described in this invention after substitution and / or deletion and / or addition of one or more residues, it is also within the scope of protection intended by this application.

[0080] The molecular chaperone is selected from any one or more of the following proteins: glutathione thiotransferase (GST), disulfide oxidoreductase (dsbA), protein disulfide isomerase (dsbC), glutathione reductase (gor), thioredoxin-1 (trxA), thioredoxin reductase (trxB), and nucleotide exchange factor (grpE). From the perspective of improving the catalytic activity of L-pantolactone dehydrogenase, the molecular chaperone is preferably selected from any one or more of the following proteins: dsbA, GST, and TrxA.

[0081] In one specific embodiment, the present invention also provides a co-expression vector for expressing the L-pantolactone dehydrogenase and the molecular chaperone. Regarding the construction method of the co-expression vector, examples include the following:

[0082] The nucleic acid sequence of GST, dsbA, dsbC, gor, TrxA, TrxB or grpE is inserted into the multiple cloning site 1 of the vector, and the nucleic acid sequence of the L-pantolactone dehydrogenase is inserted into the multiple cloning site 2 of the same vector (hereinafter also referred to as the first vector) to construct the recombinant vector (hereinafter also referred to as the first recombinant vector).

[0083] The first vector can be any vector capable of normal expression in host cells. From the perspective of being able to artificially control the timing of gene expression in order to effectively avoid adverse effects of gene expression products on the early growth of the host, the first vector is preferably a vector with an inducible promoter. From the viewpoint of improving the solubility of the L-pantolactone dehydrogenase in Escherichia coli, the first recombinant vector is preferably obtained by editing any one of the following vectors: pCDFDuet1, pACYCDuet-1, pETDuet-1, pRSFDuet-1.

[0084] In one specific embodiment, the present invention provides the application of the L-indohydrin dehydrogenase in the synthesis of D-indohydrin; in a more preferred specific embodiment, the present invention provides the application of the L-indohydrin dehydrogenase in the biosynthesis of D-indohydrin.

[0085] In one specific embodiment, the present invention also constructs a single engineered bacterium multi-enzyme cascade catalytic system. By simultaneously expressing a protein combination including the L-pantolactone dehydrogenase in a single engineered bacterium and maintaining the activity of the protein combination, it is possible to directly generate chiral pure D-pantolactone from L-pantolactone using a single engineered bacterium, with a simple procedure. The host cell of the engineered bacterium is not particularly limited; for example, commonly used prokaryotic or eukaryotic bacteria can be used. From the perspective of production efficiency, cost, and operability in synthetic biology, the host cell of the engineered bacterium is preferably *Escherichia coli*, more preferably *Escherichia coli* BL21(DE3).

[0086] Specifically, to reduce the cost of using NADPH in large-scale industrial production, the single-engineered bacterial multi-enzyme cascade catalytic system constructed in this invention further includes glucose dehydrogenase for coenzyme regeneration. In a preferred embodiment, the single-engineered bacterial multi-enzyme cascade catalytic system comprises: the L-pantolactone dehydrogenase, D-ketopantolactone reductase, and glucose dehydrogenase. The specific method for the single-engineered bacterial expressing the multi-enzyme cascade catalytic system to catalyze the generation of D-pantolactone is as follows: the engineered bacteria expressing the L-pantolactone dehydrogenase (ReLDH), D-ketopantolactone reductase, glucose dehydrogenase, and / or molecular chaperone protein are induced and cultured, and the wet bacterial cells are collected as a catalyst. Next, the induced wet bacterial cells (engineered bacteria) are reacted with L-pantolactone as a substrate and glucose as a coenzyme cycle substrate, for example, in a buffer solution of pH 7.0 and 0.2M KH2PO4 buffer. CaCO3 is added to control the pH to be no lower than 5. The reaction conditions can be 25-35℃ and 200-350 rpm. From the perspective of using the engineered bacteria of this invention constructed with Escherichia coli as the host cell, the preferred reaction conditions are 30℃ and 200 rpm. After the reaction, the purity and yield of D-pantolactone can be detected. Specifically, for example, the reaction solution can be diluted to an appropriate concentration, and then the supernatant can be collected by centrifugation for high-performance liquid chromatography (HPLC) detection. For example, a chiral HPLC column can be used to detect L-pantolactone and D-pantolactone. For example, the liquid chromatography conditions can be as follows: a CHIRALPAK IG column (250*4.6mm), an organic phase of methanol, an aqueous phase of 0.1% formic acid, isocratic elution (60% A phase, flow rate of 0.5mL / min), and a detection wavelength / UV of 210nm; the retention times of L-indohydrin and D-indohydrin are 15.8min and 14.6min, respectively (the detection conditions used in the embodiments of this invention).

[0087] In the illustrated reaction system, the amount of L-pantolactone can be 10-65 g / L, and preferably 50-65 g / L from the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell; the final concentration of glucose can be 10-100 g / L, and preferably 16-100 g / L from the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell; the concentration of CaCO3 can be 0.1-0.3 g / ml, and preferably 0.15-0.25 g / ml from the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell; NADP... + The final concentration can be 0.02-0.15 mg / mL, and preferably 0.05-0.1 mg / mL from the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell. The final concentration of wet bacterial cells expressing L-pantolactone dehydrogenase (ReLDH), D-ketopantolactone reductase, glucose dehydrogenase and / or molecular chaperone can be 20-40 g / L. As long as they can be normally expressed and active in the engineered bacteria, the D-ketopantolactone reductase and glucose dehydrogenase can be derived from any species. From the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell, the nucleic acid sequence of the D-ketopantolactone reductase is preferably derived from Candida glabata, and the glucose dehydrogenase is preferably derived from Bacillus megaterium IWG3. In a preferred embodiment, the nucleic acid sequence of the glucose dehydrogenase derived from Bacillus megaterium IWG3 is shown in SEQ ID NO.4. The nucleic acid sequence of the D-keto-universalactone reductase derived from Candida glabata is shown in SEQ ID NO. 6.

[0088] In one embodiment, the nucleic acid sequences encoding D-keto-universalanolate reductase and glucose dehydrogenase are respectively inserted into a second vector to obtain a second recombinant vector. The second vector can be any vector that can be normally expressed in host cells. From an operational point of view, it is preferred that the second vector is a vector with an inducible expression promoter, and it is preferred that the second vector is pET28a.

[0089] In one embodiment, the recombinant engineered bacteria expressing the multi-enzyme cascade catalytic system are obtained by introducing a first recombinant vector and a second recombinant vector into a host bacterium for transformation.

[0090] In one embodiment, the wet cell induction expression method is as follows: the cultured seed culture is inoculated at a rate of 1% into a lysate medium containing a final concentration of 50 μg / mL kanamycin and 50 μg / mL streptomycin, and cultured at 37°C and 200 rpm until OD... 600 =0.6-0.8, then add 0.1mM isopropyl thiogalactoside (IPTG), incubate at 28℃ for 20h, and collect wet bacterial cells by centrifugation at 4℃ and 4000rpm for 10min. From the viewpoint of improving the efficiency of the multi-enzyme cascade catalytic system, it is more preferable to treat the recombinant engineered bacteria at 25℃ when inducing expression.

[0091] The main beneficial effects of this invention are as follows: This invention provides a highly catalytically active L-pantolactone dehydrogenase and its mutant, including the encoding gene, vector, and engineered bacteria. Notably, when co-expressed with a molecular chaperone, the conversion rate is increased to over 99% when catalyzing a substrate of 50 g / L compared to engineered bacteria not co-expressed with a molecular chaperone. Engineered bacteria induced at 25°C show higher catalytic efficiency compared to those induced at 28°C, reaching a conversion rate of over 99% after 10 hours. The mutant pCDFDuet-GST-ReLDH-F183L shows a 22% increase in conversion rate compared to the wild type after 12 hours of reaction, reaching over 99%. The co-expression engineered bacteria provided by this invention can achieve the catalytic redox asymmetric synthesis of D-pantolactone from L-pantolactone using a single engineered bacteria. Compared to the chiral resolution method, the process is simpler, reduces the use of acids and bases, and further improves catalytic efficiency.

[0092] Example

[0093] The present invention will be further illustrated by the following embodiments. However, it should be noted that the following embodiments and comparative examples are only used to illustrate the technical effects of the present invention and do not limit the present invention in any way.

[0094] Unless otherwise specified, experimental procedures not described below shall be performed under conventional experimental conditions that should be understood by those skilled in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or as recommended by the manufacturer’s instructions.

[0095] The expression host *Escherichia coli* BL21(DE3) used in this invention was purchased from Shanghai Weidi Biotechnology Co., Ltd.; the plasmids pCDFDuet and pET28a used in the embodiments of this invention were purchased from Novagen, Inc., USA; the LB liquid culture medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0, sterilized at 115℃ for 30 min.

[0096] Example 1: Construction and expression of L-pantolactone dehydrogenase engineered bacteria based on Escherichia coli

[0097] Based on the gene of L-pantolactone dehydrogenase from Rhodococcus erythropolis (accession number AB689131), after codon optimization, the whole genome was synthesized by Suzhou Genewise Biotechnology Co., Ltd. to obtain a nucleic acid fragment for expressing L-pantolactone dehydrogenase from Rhodococcus erythropolis. The nucleic acid sequence is shown in SED ID NO: 2. This fragment was inserted into the NdeI and XhoI sites of the vector pCDFDuet to construct the recombinant plasmid pCDFDuet-ReLDH.

[0098] The recombinant plasmid pCDFDuet-ReLDH was introduced into competent E. coli cells BL21(DE3) for transformation. Positive strains BL21(DE3) / pCDFDuet-ReLDH were screened by PCR and their DNA was sequenced to verify the correct construction of the recombinant plasmid.

[0099] Positive strains were inoculated into 5 mL of LB liquid medium containing 50 μg / mL streptomycin and cultured overnight at 37°C and 200 rpm with shaking. Then, 1% of the culture was inoculated into 80 mL of LB liquid medium containing 50 μg / mL streptomycin and cultured at 37°C and 200 rpm with shaking until the OD600 was between 0.6 and 0.8. 0.1 mM IPTG was added, and the culture was induced for 18 h at 28°C and 180 rpm. Cells were collected by centrifugation at 8000 rpm for 5 min, and washed twice with phosphate buffer solution (pH 7.0) to obtain wet cells. A portion of the cells was sonicated to disrupt the cell lysis, and the cell lysis buffer was centrifuged at 8000 rpm for 5 min. The resulting lysate and supernatant were subjected to SDS-PAGE verification. The SDS-PAGE gel images are shown below. Figure 2 As shown, high soluble expression of L-pantolactone dehydrogenase from Rhodococcus erythropolis was achieved in Escherichia coli.

[0100] Example 2: Investigation of substrate specificity of L-pantolactone dehydrogenase in Escherichia coli

[0101] Using L-indo-indo-lactone, D-indo-indo-lactone, and DL-indo-lactone as substrates, and the BL21(DE3) / pCDFDuet-ReLDH-induced wet cell culture obtained in Example 1 as a whole-cell catalyst, the substrate specificity of L-indo-indo-lactone dehydrogenase derived from *Rhodococcus erythropolis* was investigated. The reaction was carried out for 20 h in 0.2 M phosphate buffer (pH 7.0), with a total concentration of 20 g / L for both wet cell culture and substrate (10 mL). The reaction temperature and rotation speed were 28 °C and 200 rpm, respectively. After the reaction, the solution was diluted to an appropriate concentration, centrifuged, and the supernatant was transferred to a sample vial for high-performance liquid chromatography (HPLC) analysis. The substrate specificity results of the L-indo-indo-lactone dehydrogenase derived from *Rhodococcus erythropolis* are shown in Table 1. It can be seen that this L-indo-indo-lactone dehydrogenase exhibits high specificity for L-indo-indo-lactone.

[0102] Table 1. Substrate specificity of L-pantolactone dehydrogenase from Rhodococcus erythropolis.

[0103] Substrate active D-Putolactone none L-Putolactone have DL-Pantothenic Acid none

[0104] Example 3: Construction and expression of engineered bacteria co-expressing L-pantolactone dehydrogenase

[0105] 3.1 Construction of pCDFDuet-Molecular Chaperone-ReLDH Recombinant Plasmid

[0106] The laboratory used seven chaperone proteins derived from *E. coli* K12 (MG1655)—GST (GenBank accession number 945758), dsbA (GenBank accession number 948353), dsbC (GenBank accession number 947363), gor (GenBank accession number 948014), grpE (GenBank accession number 947097), TrxA (GenBank accession number 948289), or TrxB (GenBank accession number 948289)—to study these proteins. The plasmid (GenBank accession number 949054) was double-digested with BamHI and NotI, and the digested fragments were recovered. These fragments were then ligated with the recombinant plasmid pCDFDuet-ReLDH, which was also double-digested with BamHI and NotI. The ligation products were then transformed into E. coli BL21(DE3) competent cells, and single colonies were selected for culture. The plasmids were extracted and sequenced for verification, thus constructing 7 recombinant plasmids pCDFDuet-molecular chaperone-ReLDH.

[0107] 3.2pET28a-CglCPR-rbs Construction of BmGDH recombinant plasmid

[0108] Based on the publicly available D-ketopantolactone reductase CglCPR from Candida glabata (GenBank accession number: KM817194.1) and glucose dehydrogenase BmGDH from Bacillus megaterium IWG3 (NCBI accession number: WP_028407571), expression sequences were designed through codon optimization and other methods. These sequences were then synthesized by Suzhou Genewise Biotechnology Co., Ltd. and recombined into the pET-28a(+) plasmid. After successful sequencing, the sequences were introduced into the expression host E. coli BL21(DE3).

[0109] First, using the pET28a-BmGDH recombinant plasmid as a template, primers BmGDH-rbs-F1 and BmGDH-rbs-R1 were designed to amplify the BmGDH fragment, which was then excised and recovered from the gel. Similarly, using the pET28a-CglCPR recombinant plasmid as a template, primers pET28a-F1 and pET28a-R1 were designed to amplify the plasmid backbone, which was then excised and recovered from the gel. The relevant primer sequences are shown in Table 2. Then, the two fragments were ligated using the recombinase Exnase II at 37℃ for 30 min. The ligation system is shown in Table 3, yielding the pET28a-CglCPR- rbs The BmGDH recombinant plasmid was introduced into the expression host E. coli BL21(DE3) and verified by sequencing.

[0110] Table 2 Constructs pET28a-CglCPR- rbs Primers related to the BmGDH recombinant plasmid

[0111] name Primer sequence (5'-3') BmGDH-rbs-F1 GTCAGAAAGCGTAAtttgtttaactttaagaaggagaATGTATAAAGATCTGGAAGGC BmGDH-rbs-R1 caactcagcttcctttcggg pET28a-F1 cgaaaggaagctgagttggc pET28a-R1 caaaTTACGCTTTCTGACTTTCGGCTGTTATATTTG

[0112] Table 3 Enzyme ligation system

[0113] Components Volume (μL) 5xCE buffer 4 PCR product of target gene 2 Linearized pET28a vector 2 Recombinase Exnase II 2 <![CDATA[ddH2o]]> 10

[0114] 3.3 Construction and expression of co-expressed engineered bacteria

[0115] The seven constructed recombinant plasmids pCDFDuet-molecular chaperone-ReLDH and pCDFDuet-ReLDH were respectively coupled with the recombinant plasmid pET28a-CglCPR- rbsBmGDH was mixed at a 1:1 ratio and co-transformed into competent *E. coli* BL21(DE3) cells. The cells were plated on LB agar containing 50 μg / mL kanamycin and 50 μg / mL streptomycin and incubated overnight at 37°C. Single colonies were selected and incubated overnight at 37°C and 200 rpm. Plasmids were extracted to verify successful transformation of both plasmids. The culture was then inoculated into LB agar at a 1:100 ratio and cultured at 37°C and 200 rpm until the OD600 reached approximately 0.6-0.8. 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce culture for approximately 18 h at 28°C and 180 rpm. The cells were collected by centrifugation at 8000 rpm for 5 min at 4°C and washed twice with buffer solution to obtain eight induced genetically engineered bacteria.

[0116] Example 4: Synthesis of D-pantolactone from L-pantolactone using a single engineered bacterial multi-enzyme cascade system

[0117] Eight co-expressed engineered bacteria prepared in Example 3 were used as biocatalysts. In a 10 mL reaction system, the bacteria were first resuspended in 0.2 M phosphate buffer (pH 7.0) to a final concentration of 20 g / L, containing L-indophosphoprol, glucose, and NADP. + The L-pantolactone was reacted with CaCO3 at concentrations of 50 g / L, 66 g / L, 0.1 mg / mL, and 0.2 g / mL at 30 °C and 200 rpm for 15 h. The conversion rate of L-pantolactone was detected by high-performance liquid chromatography (HPLC) with a chiral column. The results showed that among the eight co-expressed engineered bacteria, those co-expressed with molecular chaperones dsbA, GST, or TrxA achieved a conversion rate of over 99% for 15 h, with an enantiomeric excess ee value greater than 99% and a D-pantolactone yield greater than 99%.

[0118] The formula for calculating the ee value is: (L peak area of ​​indomethacin - D peak area of ​​indomethacin) / (L peak area of ​​indomethacin + D peak area of ​​indomethacin)

[0119] Example 5: Activity evaluation of engineered bacteria induced to express D-pantolactone in a multi-enzyme cascade system during the synthesis of D-pantolactone from L-pantolactone.

[0120] Following the method in Example 3, the co-expressed engineered bacteria (pCDFDuet-GST-ReLDH and pET28a-CglCPR-) were subjected to... rbsBmGDH was used to induce expression, the only difference being that the expression temperature was set to 25℃ or 20℃, while all other conditions remained the same. The collected wet bacterial cells were used to catalyze the synthesis of D-pantolactone from L-pantolactone. The reaction conditions were as follows: in a 10 mL reaction system, the cells were first resuspended in 0.2 M phosphate buffer (pH 7.0) to a final concentration of 20 g / L, containing the substrate L-pantolactone, glucose, and NADP. + The bacteria were reacted with CaCO3 at concentrations of 50 g / L, 66 g / L, 0.1 mg / mL, and 0.2 g / mL at 30 °C and 200 rpm. Samples were taken at different time points for analysis. The reduction of L-pantolactone was detected using a high-performance liquid chromatography (HPLC) chiral column. The results showed that the co-expression engineered bacteria induced at 25 °C achieved a conversion rate of over 99% after 10 h, and had almost completely converted to D-pantolactone (yield greater than 99%). The conversion rates at 20 °C and 28 °C were 93% and 89%, respectively, and only reached over 99% after 15 h.

[0121] Example 6: Construction of L-pantolactone dehydrogenase mutant and comparison of its catalytic activity with wild type

[0122] Construction of 6.1L-Pantothenic acid lactone dehydrogenase mutant

[0123] L-pantolactone dehydrogenase derived from *Rhodococcus erythropolis* exhibits good catalytic activity when co-expressed with the molecular chaperone GST. To further enhance its potential for industrial application, the inventors used EVcouplings (https: / / v2.evcouplings.org / ) to analyze the amino acid sequence of ReLDH, obtaining results such as multiple sequence alignment, de novo structural prediction, and mutation effects. Substitutions showing beneficial mutation coefficients greater than 2.5 were explored, and combined with conserved sequence analysis, positions 183, 156, 372, and 55 of the amino acid sequence of SED ID NO: 1 were identified as mutation targets.

[0124] The ReLDH mutant was constructed using site-directed mutagenesis. Using the vector pCDFDuet-GST-ReLDH from the original strain in Example 3 as a template, the plasmid containing the mutation site was amplified by polymerase chain reaction (PCR) using the primers listed in Table 4. After digesting the template with DpnI, the plasmid was transformed into E. coli BL21(DE3) competent cells. Single clones were picked and cultured overnight at 37°C in 4 mL LB liquid medium. Sequencing was performed by Suzhou Genewiz Technology Co., Ltd., yielding four plasmids: pCDFDuet-GST-ReLDH-F183L, pCDFDuet-GST-ReLDH-I156V, pCDFDuet-GST-ReLDH-T372H, and pCDFDuet-GST-ReLDH-A55V. These plasmids were stored at -20°C.

[0125] Table 4

[0126]

[0127]

[0128] 6.2 Induction of expression of L-pantolactone dehydrogenase co-expressing engineered bacteria

[0129] Four mutant plasmids were constructed and mixed 1:1 with the recombinant plasmid pET28a-CglCPR-rbsBmGDH from Example 3. These were then co-transformed into competent *E. coli* BL21(DE3) cells and plated on LB solid medium containing 50 μg / mL kanamycin and 50 μg / mL streptomycin, and cultured overnight at 37°C. Single colonies were selected and cultured overnight at 37°C and 200 rpm. Plasmids were extracted to verify successful transformation with both plasmids. The culture solution was inoculated into LB liquid medium at a 1:100 ratio and cultured at 37°C and 200 rpm until the OD600 reached approximately 0.6-0.8. 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to induce culture for approximately 18 h at 25°C and 180 rpm. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min at 4℃, and washed twice with buffer solution to obtain four induced genetically engineered bacteria.

[0130] 6.3 Comparison of the activities of wild-type and mutant L-pantolactone dehydrogenase co-expressing engineered bacteria

[0131] Using the engineered bacteria prepared above, containing wild-type and four mutant co-expression strains, as biocatalysts, the reaction system was first resuspended in 0.2M phosphate buffer (pH 7.0) to a final concentration of 20 g / L. The concentrations of substrate L-indoglutamate, glucose, NADP+, and CaCO3 were 65 g / L, 85.8 g / L, 0.1 mg / mL, and 0.3 g / mL, respectively. The reaction was carried out at 30°C and 200 rpm for 12 h. The conversion rate of wild-type pCDFDuet-GST-ReLDH was 77%, while the conversion rate of mutant pCDFDuet-GST-ReLDH-F183L was greater than 99% after 12 h, and the calculated yield of D-indoglutamate was over 98%. The conversion rate of pCDFDuet-GST-ReLDH-I156V was 91%. It is evident that the catalytic activity of the engineered bacteria is further enhanced through the mutation, and even when the concentration of L-indolactone as the substrate reaches an upper limit of 65 g / L, a conversion rate of over 90% can be achieved within 12 hours.

[0132] Sequence List:

[0133] The following provides information on the main sequences used in this invention.

[0134] SED ID NO: 1∶

[0135] amino acid sequence of Rhodococcus erythropolis L-pantolactone dehydrogenase

[0136] MAKNAFFETVAEAQRRAKKRLPKSVYAALVAGSEKGLTVDDNVAAFSELGFAPHAAGLSDKREMSTTIMGQDISLPVMISPTGVQAVHPDGEVAVARAAAARGTAIGLSSFASKSIEEVAAANPQVFFQMYWVGSRDVLLQRMERARAAGAKGLIITTDWSFSYGRDWGSPSIPEKMDLKAMFQFAPEGIMRPKWL LEFAKTGKIPDLTTPNLAAPGQPAPTFFGAYGEWMQTPLPTWEDIAWLREQWGGPFMLKGIMRIDDAKRAVDAGVSAISVSNHGGNNLDGTPAPIRVLPGIAEAVGDQVEVVLDGGIRRGGDVVKALALGAKAVMLGRAYLWGLSANGQAGVENVLDLMRMGIDSGLMGLGHSSITELSPADLVIPEGFTRTLGAS*

[0137] SED ID NO: 2:

[0138] DNA sequence encoding Rhodococcus erythropolis L-pantolactone dehydrogenase

[0139]

[0140] SED ID NO: 3∶

[0141] Amino acid sequence of glucose dehydrogenase from Bacillus megaterium IWG3

[0142] MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG*

[0143] SED ID NO: 4:

[0144] DNA sequence encoding Bacillus megaterium IWG3

[0145] ATGTATAAAGATCTGGAAGGCAAAGTGGTTGTGATTACCGGCAGCAGCACCGGCCTGGGCAAAAGCATGGCGATTCGCTTTGCGACCGAAAAAGCGAAAGTTGTGGTTAATTATCGCAGCAAAGAAGATGAAGCGAACAGCGTGCTGGAAGAAATTAAAAAAGTGGGCGGCGAAGCGATTGCGGTGAAAGGCGATGTGACCGTGGAAAGCGATATTATTAACCTGGTGCAGAGCGCGATTAAAGAATTTGGCAAACTGGATGTGATGATTAACAACGCGGGCCTGGAAAACCCGGTGCCGAGCCATGAAATGAGCCTGAGCGATTGGAACAAAGTGATTGATACCAACCTGACCGGCGCGTTTCTGGGCAGCCGCGAAGCGATTAAATATTTTGTGGAAAACGATATTCGCGGCACCGTGATTAACATGAGCAGCGTGCATGAAAAAATTCCGTGGCCGCTGTTTGTGCATTATGCGGCGAGCAAAGGCGGCATGCGCCTGATGACCAAAACCCTGGCGCTGGAATATGCGCCGAAAGGCATTCGCGTGAACAACATTGGCCCGGGCGCGATTAACACCCCGATTAACGCGGAAAAATTTGCCGATCCGGAACAGCGCGCGGATGTGGAAAGCATGATTCCGATGGGCTATATTGGCGAACCGGAAGAAATTGCGGCGGTGGCGGCGTGGCTGGCGAGCAGCGAAGCGAGCTATGTGACCGGCATTACCCTGTTTGCGGATGGCGGCATGACCCTGTATCCGAGCTTTCAAGCGGGCCGCGGCTAA

[0146] SEQ ID NO: 5:

[0147] Amino acid sequence of Candida glabata D-ketopantolactone reductase

[0148] MVKQEFFKLNNGHEMPGVAIVGTGTKWHKVNETDENFSQTLVDQLKYALSLPGVVHLDAAEFYMTYREVGRALAETSKPRDEIFITDKYWTLSKVTENPIVGLETGLKRLGLEYVDLYLLHSPFISKETNGFSLEEAWGMMEELYHSGKAKNIGVSNFAKEDLERVLKVCKVKPQVNQIEFNAFLQNQTPGIYNFCKQNDIQLAAYSPLGPLQKKPADGNSQPFYSYINKLAQHYNKTPGQVLLRWVTKRGVVAVTTSEKKERIKQAQEIFEFDLKDDEVTEITKLGLDHEPLRLYWHDQYNKYNSESQKA*

[0149] Accession ID No.: 6:

[0150] DNA sequence encoding Candida glabata D-ketopantolactone reductase

[0151] ATGGTGAAACAAGAATTTTTTAAACTGAACAACGGCCATGAAATGCCGGGCGTGGCGATTGTGGGCACCGGCACCAAATGGCATAAAGTGAACGAAACCGATGAAAACTTTAGTCAGACCCTGGTGGATCAGCTGAAATATGCGCTGAGCCTGCCGGGCGTGGTGCATCTGGATGCGGCGGAATTTTATATGACCTATCGCGAAGTGGGCCGCGCGCTGGCGGAAACGAGCAAACCGCGCGATGAAATTTTTATTACCGATAAATATTGGACCCTGAGCAAAGTGACCGAGAACCCGATTGTGGGCCTGGAAACCGGCCTGAAACGCCTGGGCCTGGAATATGTGGATCTGTATCTGCTGCATAGCCCGTTTATTAGCAAAGAAACCAACGGCTTTAGCCTGGAAGAAGCGTGGGGCATGATGGAAGAACTGTATCATAGCGGCAAAGCGAAAAACATTGGCGTGAGCAACTTTGCGAAAGAAGATCTGGAACGCGTGCTGAAAGTGTGCAAAGTGAAACCGCAAGTGAATCAGATTGAATTTAACGCGTTTCTGCAGAATCAGACCCCGGGCATTTATAACTTTTGCAAACAGAACGATATTCAGCTGGCGGCGTATAGCCCGCTGGGCCCGCTGCAGAAAAAACCGGCGGATGGCAACAGTCAGCCGTTTTATAGCTATATTAACAAACTGGCGCAGCATTATAACAAAACCCCGGGCCAAGTGCTGCTGCGCTGGGTGACCAAACGCGGCGTGGTGGCGGTGACCACGAGCGAAAAAAAAGAACGCATTAAACAAGCGCAAGAAATTTTTGAATTTGATCTGAAAGATGATGAAGTGACCGAAATCACGAAACTGGGCCTGGATCATGAACCGCTGCGCCTGTATTGGCATGATCAGTATAACAAATATAACAGCGAAAGTCAGAAAGCGTAA

[0152] SED ID NO:7:

[0153] The amino acid sequence of Rhodococcus erythropolis L-pantolactone dehydrogenase mutant F183L

[0154] MAKNAFFETVAEAQRRAKKRLPKSVYAALVAGSEKGLTVDDNVAAFSELGFAPHAAGLSDKREMSTTIMGQDISLPVMISPTGVQAVHPDGEVAVARAAAARGTAIGLSSFASKSIEEVAAANPQVFFQMYWVGSRDVLLQRMERARAAGAKGLIITTDWSFSYGRDWGSPSIPEKMDLKAMLQFAPEGIMRPKWL LEFAKTGKIPDLTTPNLAAPGQPAPTFFGAYGEWMQTPLPTWEDIAWLREQWGGPFMLKGIMRIDDAKRAVDAGVSAISVSNHGGNNLDGTPAPIRVLPGIAEAVGDQVEVVLDGGIRRGGDVVKALALGAKAVMLGRAYLWGLSANGQAGVENVLDLMRMGIDSGLMGLGHSSITELSPADLVIPEGFTRTLGAS*

[0155] SED ID NO: 8:

[0156] Base sequence of Rhodococcus erythropolis L-pantolactone dehydrogenase mutant F183L

[0157]

[0158] Rhodococcus erythropolis L-pantolactone dehydrogenase mutant I156V

[0159] amino acid sequence

[0160] MAKNAFFETVAEAQRRAKKRLPKSVYAALVAGSEKGLTVDDNVAAFSELGFAPHAAGLSDKREMSTTIMGQDISLPVMISPTGVQAVHPDGEVAVARAAAARGTAIGLSSFASKSIEEVAAANPQVFFQMYWVGSRDVLLQRMERARAAGAKGLIVTTDWSFSYGRDWGSPSIPEKMDLKAMFQFAPEGIMRPKWL LEFAKTGKIPDLTTPNLAAPGQPAPTFFGAYGEWMQTPLPTWEDIAWLREQWGGPFMLKGIMRIDDAKRAVDAGVSAISVSNHGGNNLDGTPAPIRVLPGIAEAVGDQVEVVLDGGIRRGGDVVKALALGAKAVMLGRAYLWGLSANGQAGVENVLDLMRMGIDSGLMGLGHSSITELSPADLVIPEGFTRTLGAS*

[0161] SED ID NO: 10:

[0162] The base sequence of the 1156V mutant of Rhodococcus erythropolis L-pantolactone dehydrogenase

[0163]

[0164] SED ID NO: 11:

[0165] GST amino acid sequence

[0166] MKLFYKPGACSLASHITLRESGKDFTLVSVDLMKKRLENGDDYFAVNPKGQVPALLLDDGTLLTEGVAIMQYLADSVPDRQLLAPVNSISRYKTIEWLNYIATELHKGFTPLFRPDTPEEYKPTVRAQLEKKLQYVNEALKDEHWICGQRFTIADAYLFTVLRWAYAVKLNLEGLEHIAAFMQRMAERPEVQDALSAEGLK

[0167] SED ID NO: 12:

[0168] DsbA amino acid sequence

[0169] MKKIWLALAGLVLAFSASAAQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIKGEEYDAAWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKK

[0170] SED ID NO: 13:

[0171] dsbC amino acid sequence

[0172] MKKGFMLFTLLAAFSGFAQADDAAIQQTLAKMGIKSSDIQPAPVAGMKTVLTNSGVLYITDDGKHIIQGPMYDVSGTAPVNVTNKMLLKQLNALEKEMIVYKAPQEKHVITVFTDITCGYCHKLHEQMADYNALGITVRYLAFPRQGLDSDAEKEMKAIWCAKDKNKAFDDVMAGKSVAPASCDVDIADHYALGVQLGVSGTPAVVLSNGTLVPGYQPPKEMKEFLDEHQKMTSGK

[0173] SED ID NO: 14:

[0174] Gor amino acid sequence

[0175] MTKHYDYIAIGGGSGGIASINRAAMYGQKCALIEAKELGGTCVNVGCVPKKVMWHAAQIREAIHMYGPDYGFDTTINKFNWETLIASRTAYIDRIHTSYENVLGKNNVDVIKGFARFVDAKTLEVNGETITADHILIATGGRPSHPDIPGVEYGIDSDGFFALPALPERVAVVGAGYIAVELAGVINGLGAKTHLFVRKHAPLRSFDPMISETLVEVMNAEGPQLHTNAIPKAVVKNTDGSLTLELEDGRSETVDCLIWAIGREPANDNINLEAAGVKTNEKGYIVVDKYQNTNIEGIYAVGDNTGAVELTPVAVAAGRRLSERLFNNKPDEHLDYSNIPTVVFSHPPIGTVGLTEPQAREQYGDDQVKVYKSSFTAMYTAVTTHRQPCRMKLVCVGSEEKIVGIHGIGFGMDEMLQGFAVALKMGATKKDFDNTVAIHPTAAEEFVTMR

[0176] SED ID NO: 15:

[0177] grpE amino acid sequence

[0178] MSSKEQKTPEGQAPEEIIMDQHEEIEAVEPEASAEQVDPRDEKVANLEAQLAEAQTRERDGILRVKAEMENLRRRTELDIEKAHKFALEKFINELLPVIDSLDRALEVADKANPDMSAMVEGIELTLKSMLDVVRKFGVEVIAETNVPLDPNVHQAIAMVESDDVAPGNVLGIMQKGYTLNGRTIRAAMVTVAKAKA

[0179] SED ID NO: 16:

[0180] trxA amino acid sequence

[0181] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA

[0182] SED ID NO: 17:

[0183] trxB amino acid sequence

[0184] MGTTKHSKLLILGSGPAGYTAAVYAARANLQPVLITGMEKGGQLTTTTEVENWPGDPNDLTGPLLMERMHEHATKFETEIIFDHINKVDLQNRPFRLNGDNGEYTCDALIIATGASARYLGLPSEEAFKGRGVSACATCDGFFYRNQKVAVIGGGNTAVEEALYLSNIASEVHLIHRRDGFRAEKILIKRLMDKVENGNIILHTNRTLEEVTGDQMGVTGVRLRDTQNSDNIESLDVAGLFVAIGHSPNTAIFEGQLELENGYIKVQSGIHGNATQTSIPGVFAAGDVMDHIYRQAITSAGTGCMAALDAERYLDGLADAK

Claims

1. A nucleic acid molecule co-expressing L-pantolactone dehydrogenase and a molecular chaperone, comprising (a) and (b) the following: (a) A nucleic acid sequence encoding an L-pantolactone dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 1, and said nucleic acid sequence as shown in SEQ ID NO: 2; (b) A nucleic acid sequence encoding a molecular chaperone, wherein the molecular chaperone is selected from any one of the following groups: glutathione thiotransferase GST, disulfide oxidoreductase dsbA, protein disulfide isomerase dsbC, and thioredoxin-1 trxA.

2. The nucleic acid molecule of claim 1, wherein the molecular chaperone is selected from any one of the following: disulfide oxidoreductase dsbA, glutathione thiotransferase GST, or thioredoxin-1 trxA.

3. The nucleic acid molecule as described in claim 1, wherein, The amino acid sequence of the glutathione thiotransferase GST is shown in SEQ ID NO: 11, the amino acid sequence of the disulfide oxidoreductase dsbA is shown in SEQ ID NO: 12, the amino acid sequence of the protein disulfide isomerase dsbC is shown in SEQ ID NO: 13, and the amino acid sequence of the thioredoxin-1 trxA is shown in SEQ ID NO:

16.

4. A recombinant vector comprising a nucleic acid molecule as described in any one of claims 1 to 3.

5. A combination of recombinant vectors, comprising a first recombinant vector and a second recombinant vector. The first recombinant vector contains: (a) A nucleic acid sequence encoding an L-pantolactone dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 1, and said nucleic acid sequence as shown in SEQ ID NO: 2; and (b) A nucleic acid sequence encoding a molecular chaperone, wherein the molecular chaperone is selected from any one of the following groups: glutathione thiotransferase GST, disulfide oxidoreductase dsbA, protein disulfide isomerase dsbC, and thioredoxin-1 trxA; The first recombinant vector co-expresses L-indolactone dehydrogenase and molecular chaperone; The second recombinant vector contains: (c) The nucleic acid sequence encoding glucose dehydrogenase; and (d) Nucleic acid sequence encoding D-keto-indohydranolate reductase.

6. The recombinant vector combination of claim 5, wherein the amino acid sequence of the glutathione thiotransferase GST is as shown in SEQ ID NO: 11, the amino acid sequence of the disulfide oxidoreductase dsbA is as shown in SEQ ID NO: 12, the amino acid sequence of the protein disulfide isomerase dsbC is as shown in SEQ ID NO: 13, the amino acid sequence of the thioredoxin-1 trxA is as shown in SEQ ID NO: 16, the amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO: 3, and the amino acid sequence of the D-keto-universaloacetyl lactone reductase is as shown in SEQ ID NO:

5.

7. An engineered bacterium comprising a nucleic acid molecule as described in any one of claims 1 to 3, a recombinant vector as described in claim 4, or a combination of recombinant vectors as described in claim 5 or 6; The engineered bacteria are obtained by processing host cells selected from Escherichia coli.

8. The engineered bacteria as described in claim 7, wherein, The host cell was Escherichia coli E.coil BL21(DE3).

9. A method for preparing D-pantolactone, comprising the following steps: (a) L-indohydrin dehydrogenase, with an amino acid sequence as shown in SEQ ID NO: 1, catalyzes the dehydrogenation of L-indohydrin to generate ketoindohydrin. (b) Reduction by ketone-based pantothenic acid lactone to form D-pantothenic acid lactone. in, Step (a) is performed using the engineered bacteria as described in claim 7 or 8; or, both steps (a) and (b) are performed using the engineered bacteria as described in claim 7 or 8.

10. The method for preparing D-pantolytic lactone according to claim 9, wherein, The specific implementation steps of the method include: (i) The step of processing the host cells and screening for engineered bacteria as described in claim 7 or 8; (ii) The step of inducing the engineered bacteria to express the required protein; (iii) The step of adding the engineered bacteria to a substrate containing L-indohydrin for reaction.

11. The method for preparing D-pantolactone as described in claim 10, wherein, Step (ii) involves induction culture at 20℃~28℃.

12. The method for preparing D-pantolactone as described in claim 11, wherein step (ii) is performed by induction culture at 25°C.

13. The method for preparing D-pantolactone according to any one of claims 9 to 12, wherein, The final concentration of L-indohydrin is 10-65 g / L.

14. The method for preparing D-indohydrin according to claim 13, wherein the final concentration of L-indohydrin is 50-65 g / L.

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