The application relates to an L-pantoic acid lactone dehydrogenase, an expression vector thereof and application of the L-pantoic acid lactone dehydrogenase and the expression vector thereof in D-pantoic acid lactone synthesis

Abstract

The present application provides an L-pantoic acid lactone dehydrogenase derived from Skermania sp. ID1734, a nucleic acid molecule encoding the L-pantoic acid lactone dehydrogenase, a vector for expressing the L-pantoic acid lactone dehydrogenase in a host cell, and an engineered bacterium expressing the enzyme. The present application further provides an engineered bacterium co-expressing the L-pantoic acid lactone dehydrogenase and any one or more selected from the group consisting of a ketopantoate reductase and a glucose dehydrogenase. The present application further provides a method for producing D-pantoic acid lactone using an engineered bacterium expressing the L-pantoic acid lactone dehydrogenase derived from Skermania sp. ID1734. One of the technical solutions provided by the present application is to provide a recombinant vector or a combination of recombinant vectors, wherein the recombinant vector or the combination of recombinant vectors comprises a gene expressing the L-pantoic acid lactone dehydrogenase. The present application also provides the use or application of the L-pantoic acid lactone dehydrogenase, the nucleic acid molecule, the engineered bacterium, the vector or the combination of vectors in the synthesis of D-pantoic acid lactone.

Description

Technical Field

[0001] It belongs to the field of bio-enzyme catalysis synthesis, specifically involving the preparation of L-pantolactone to ketopantolactone and the synthesis of D-pantolactone. Background Technology

[0002] Pantothenic acid (PA), also known as vitamin B5 or D-calcium pantothenate, is a water-soluble vitamin composed of pantothenic acid and β-alanine, with only the D-form (D-PA) possessing biological activity. In living cells, pantothenic acid serves as an important precursor to coenzyme A (CoA) and acyl carrier protein (ACP), participating in the metabolism of carbohydrates, fatty acids, proteins, and energy. Currently, pantothenic acid is widely used in the pharmaceutical, food, feed, and cosmetic industries. In industrial production, the synthesis of D-calcium pantothenate begins with the chemical synthesis of a mixture of DL-pantothenic acid lactones, followed by separation and cyclization. The cyclized D-pantothenic acid lactone then reacts with calcium 3-aminopropionate to synthesize D-calcium pantothenate. This process is relatively complex and energy-intensive. Enzymatic redox synthesis of D-pantothenic acid lactone from L-pantothenic acid lactone reduces process complexity, saves costs, and is more environmentally friendly.

[0003] The bioenzymatic method utilizes the synergistic action of multiple enzymes to dehydrogenate L-pantolactone. The resulting ketopantolactone is then subjected to the action of ketopantolactone reductase to obtain the target product, D-pantolactone. Compared to current industrial methods for preparing D-pantolactone, this method eliminates steps such as separation, cyclization, and racemization, offering advantages such as energy saving and reduced consumption. The specific pathway diagram is as follows:

[0004]

[0005] Bioenzymatic pathway

[0006] L-Pantothenic acid lactone dehydrogenase oxidizes L-pantothenic acid lactone to keto-pantothenic acid lactone, which is then further reduced to D-pantothenic acid lactone by keto-pantothenic acid lactone reductase. L-Pantothenic acid lactone dehydrogenase is a crucial step in the redox process for preparing D-pantothenic acid lactone, yet research on it is currently limited. Si, D, et al. expressed the L-pantoyl lactone dehydrogenase (LPLDH) gene from *Rhodococcus erythropolis*, achieving conversion rates of 92% and 80% at substrate concentrations of 0.768 M and 1.15 M, respectively. (*Rhodococcus erythropolis* (Si, D., 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. *Appl Microbiol Biotechnol* 95, 431–440 (2012)). Hangzhou Xinfu Technology Co., Ltd. expressed *Nocardia farcinica* (CN201910366852.5) ​​and *Cnuibacter*. L-pantolactone dehydrogenases derived from *physcomitrellae* (CN201910366923.1) and others have been overexpressed and their catalytic systems constructed, both showing certain catalytic activity, indicating the feasibility of this route. Currently, there are no reports on L-pantolactone hydroxyoxidases derived from *Skermania sp. ID1734*. Summary of the Invention

[0007] In order to obtain a new L-pantolactone hydroxy oxidase, the inventors screened a large number of genes using the Uniprot gene database and finally discovered that the flavin dehydrogenase gene (A0A554V9U8) from Skermania sp. ID1734 (hereinafter referred to as MsfDH gene) expresses a protein with L-pantolactone hydroxy oxidase activity, thus completing the present invention. The function of MsfDH remains controversial in the existing technology. For example, in the Uniprot database, this gene is only annotated as heme / flavin dehydrogenase (without actual experiments), while on NCBI (WP_143768573.1), it is annotated as deamination oxidase, and its function in the biosynthesis of the reducing cofactor Mycofactocin has been studied (Ayikpoe, RS, & Latham, JA (2019). MftD Catalyzes the Formation of a Biologically Active Redox Center in the Biosynthesis of the Ribosomally Synthesized and Post-translationally Modified Redox Cofactor, Mycofactocin. Journal of the American Chemical Society. doi:10.1021 / jacs.9b06102).

[0008] The inventors of this invention, through analysis, screening, and verification of a large number of genes, including the MsfDH gene, discovered that not all genes annotated as flavin dehydrogenases possess L-pantolactone dehydrogenase activity. Only the protein encoded by the MsfDH gene (MsfDH protein is sometimes referred to as MsfDH below) exhibits L-pantolactone dehydrogenase activity. The inventors further screened the vector and host cells used and modified the expression sequence of the MsfDH gene based on the screening results, obtaining an engineered bacterium that stably expresses L-pantolactone dehydrogenase activity. The inventors further modified the engineered bacterium by expressing ketopantolactone reductase and glucose dehydrogenase in addition to L-pantolactone dehydrogenase, thus obtaining a multi-enzyme co-expression engineered bacterium with higher efficiency in D-pantolactone synthesis. Furthermore, in an aqueous reaction system, L-pantolactone spontaneously undergoes partial hydrolysis to produce L-pantolic acid as pH changes, which leads to a decrease in the yield of the target product, D-pantolactone. However, the inventors of this invention unexpectedly discovered that MsfDH not only has oxidative dehydrogenation capabilities for L-pantolactone but also oxidizes L-pantolic acid (i.e., it has dual oxidative capabilities for both L-pantolic acid and L-pantolactone). This is beneficial for ultimately improving the efficiency of the D-type pantolic acid / lactone synthesis reaction and reducing product impurities.

[0009] This invention provides an L-pantolactone dehydrogenase derived from Skermania sp. ID1734, a nucleic acid molecule encoding the L-pantolactone dehydrogenase, a vector for expressing the L-pantolactone dehydrogenase in host cells, and an engineered bacterium expressing the enzyme. This invention further provides an engineered bacterium co-expressing the L-pantolactone dehydrogenase and any one or more strains selected from the group consisting of ketopantolactone reductase and glucose dehydrogenase. This invention further provides a method for producing D-pantolactone using an engineered bacterium expressing the L-pantolactone dehydrogenase derived from Skermania sp. ID1734. One of the technical solutions provided by this invention is to provide a recombinant vector or combination of recombinant vectors containing a gene expressing the L-pantolactone dehydrogenase. This invention also provides the L-pantolactone dehydrogenase, the nucleic acid molecule, the engineered bacterium, and the application or use of the vector or vector combination in the synthesis of D-pantolactone.

[0010] Specifically, the present invention includes the following aspects:

[0011] 1. An L-pantolactone dehydrogenase comprising an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with said sequence.

[0012] 2. A nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding the L-indohydrin dehydrogenase described in item 1.

[0013] 3. The nucleic acid molecule as described in item 2, wherein the nucleic acid molecule comprises a nucleic acid sequence as shown in SEQ ID NO:1 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence.

[0014] 4. A nucleic acid molecule as described in item 2 or 3, which is a carrier or a combination of carriers.

[0015] 5. A nucleic acid molecule as described in any one of items 2 to 4, further comprising a nucleic acid sequence expressing keto-universalactone reductase and / or a nucleic acid sequence encoding glucose dehydrogenase.

[0016] 6. A nucleic acid molecule as described in any one of items 2 to 5, which is a combination of vectors and comprises:

[0017] (a) A first vector: which contains the nucleic acid sequence encoding the L-pantolactone dehydrogenase described in item 1; and

[0018] (b) Second carrier: which contains a nucleic acid sequence encoding keto-indoglutamate reductase and / or a nucleic acid sequence encoding glucose dehydrogenase.

[0019] 7. The nucleic acid molecule as described in any one of items 4 to 6, wherein:

[0020] The first and second carriers in the carrier or the combination of carriers are obtained by editing any one of the following carriers: pET28a, pCDFDuet1, pACYCDuet-1, pETDuet-1, pRSFDuet-1.

[0021] 8. A nucleic acid molecule as described in any one of items 5 to 7, wherein the nucleic acid sequence encoding ketopantolate reductase is the nucleic acid sequence shown in SEQ ID NO:3 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with said sequence; and / or

[0022] The nucleic acid sequence encoding glucose dehydrogenase is the nucleic acid sequence shown in SEQ ID NO:5 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence.

[0023] 9. A nucleic acid molecule as described in any one of items 6 to 8, wherein the first vector comprises a nucleic acid sequence as shown in SEQ ID NO:7 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with said sequence.

[0024] 10. A nucleic acid molecule as described in any one of items 6 to 9, wherein the second vector comprises a nucleic acid sequence as shown in SEQ ID NO:20 or a nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with said sequence.

[0025] 11 An engineered bacterium, wherein the engineered bacterium expresses L-pantolactone dehydrogenase as described in item 1; or comprises a nucleic acid molecule as described in any one of items 2 to 10.

[0026] 12. The engineered bacteria as described in item 11, wherein the engineered bacteria are obtained by processing any one or more host cells selected from the group consisting of: Escherichia coli, Bacillus subtilis, yeast cells, or Aspergillus.

[0027] 13. The engineered bacteria as described in item 11 or 12, wherein the host cell is Escherichia coli BL21.

[0028] 14. A method for producing D-pantolactone, comprising:

[0029] (a) The step of catalyzing L-indohydrin dehydrogenase to generate ketoindohydrin by L-indohydrin dehydrogenase as described in item 1.

[0030] (b) The steps for forming D-indohydrin from ketone-indohydrin lactone.

[0031] 15. The method of claim 14, wherein step (a); or steps (a) and (b) are performed using engineered bacteria as described in any one of claims 11 to 13.

[0032] 16. The method as described in item 15, wherein step (a); or the specific implementation steps of step (a) and step (b) include:

[0033] (i) The step of reacting the engineered bacteria as described in any one of items 11 to 13 in a reaction environment containing L-pantolactone at a final concentration of 10-65 g / L, wherein the final concentration of L-pantolactone is preferably 40-65 g / L.

[0034] 17. The method of claim 16, wherein the reaction environment further comprises: glucose and / or NADP. + ,

[0035] The final concentration of glucose is 10-100 g / L, preferably 16-100 g / L, and NADP... + The final concentration is 5-20 g / L, preferably 10 g / L.

[0036] 18. The method as described in item 16 or 17, wherein step (i) is performed at 25-35°C and 200-350 rpm, preferably at 30°C and 200 rpm.

[0037] 19. Use of the L-indolactone dehydrogenase as described in item 1, the nucleic acid molecule as described in any one of items 2 to 10, or the engineered bacteria as described in any one of items 11 to 13 in the synthesis of D-indolactone.

[0038] 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.

[0039] In this specification, "comprises" not only means "to include" but also means "to be composed of".

[0040] 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% identity with the specific sequence disclosed in this invention.

[0041] In this specification, the term "L-indo-indo-lactone dehydrogenase" generally refers to a protein enzyme with the activity of dehydrogenating L-indo-lactone to form keto-indo-lactone.

[0042] 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, aniline phosphates (phosphoranilidate), and phosphoramide esters.

[0043] 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.

[0044] In this specification, the term "isolated protein" generally refers to a protein that has been isolated 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. B 848 (2007) 79-87.

[0045] 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 (e.g., the modified commercial strain E. coli BL21) or Bacillus subtilis, and fungal cells such as yeast cells or Aspergillus.

[0046] In this specification, the term "engineered bacteria" refers to a bacterial cell line in which a foreign gene is efficiently expressed in a host cell using genetic engineering methods.

[0047] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transferring an inserted nucleic acid molecule into a host cell and / or between host cells; or for introducing a target sequence into a host cell and / or between host cells as a template for editing. 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 function of the species or cell type, but typically includes 5' non-transcriptional sequences 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 include 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.

[0048] 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 then briefly heat-shocking them to allow the plasmid to enter the cells. Electroporation is another method for allowing plasmids to enter cells.

[0049] In this specification, MsfDH may refer to the MsfDH gene itself or the protein expressed by the MsfDH gene. The MsfDH gene includes the naturally occurring MsfDH gene and the processed mRNA or cDNA sequence used to express the MsfDH protein. The specific meaning should be unambiguous based on the context. Attached Figure Description

[0050] Figure 1 The image shows an SDS-PAGE electrophoresis pattern of proteins such as MsfDH.

[0051] Figure 2 A characterization diagram showing the L-pantolytic acid / lactone oxidative dehydrogenation activity of engineered bacteria expressing MsfDH protein.

[0052] Figure 3 The results show the purification of raw material DL mixed lactone and the analysis of L-pantolytic acid lactone purity by extractive liquid chromatography.

[0053] Figure 4 This image shows the characterization of L-pantolactone conversion activity of engineered bacteria expressing a three-enzyme combination. Detailed Implementation

[0054] The present invention will be further illustrated by specific embodiments below. 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.

[0055] 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 under the conditions recommended in the manufacturer's instructions.

[0056] This invention provides an L-pantolactone dehydrogenase (MsfDH) derived from Skermania sp. ID1734, a nucleic acid molecule encoding this L-pantolactone dehydrogenase, a vector for expressing the L-pantolactone dehydrogenase in host cells, and an engineered bacterium expressing the enzyme. This invention further provides an engineered bacterium co-expressing the L-pantolactone dehydrogenase and any one or more strains selected from the group consisting of ketopantolactone reductase and glucose dehydrogenase. This invention further provides a method for producing D-pantolactone using an engineered bacterium expressing the L-pantolactone dehydrogenase derived from Skermania sp. ID1734. One of the technical solutions provided by this invention is to provide a recombinant vector or a combination of recombinant vectors containing a gene expressing the L-pantolactone dehydrogenase. This invention also provides the L-pantolactone dehydrogenase, the nucleic acid molecule, the engineered bacterium, and the application or use of the vector or vector combination in the synthesis of D-pantolactone. In one embodiment, the application of the L-pantolactone dehydrogenase in the synthesis of D-pantolactone can be carried out by engineered bacteria expressing the L-pantolactone dehydrogenase, or by isolating the protein of the L-pantolactone dehydrogenase, a derivative of the protein, or a preparation of the protein (e.g., an enzyme preparation). In this invention, an enzyme preparation refers to various biological products with catalytic function after purification and / or processing of the enzyme.

[0057] The amino acid sequence of the L-indo-lactone dehydrogenase described above is shown in SEQ ID NO:2. The nucleic acid sequence encoding the L-indo-lactone dehydrogenase is shown in SEQ ID NO:1.

[0058] Furthermore, those skilled in the art should understand that when the aforementioned amino acid sequence or nucleic acid sequence still possesses 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. In one embodiment of the invention, a recombinant vector or combination of recombinant vectors is provided, the recombinant vector or combination of recombinant vectors comprising a nucleic acid sequence expressing the L-pantolactone dehydrogenase. The recombinant vector or combination of recombinant vectors may also optionally comprise nucleic acid sequences expressing ketopantolactone reductase and / or glucose dehydrogenase. In one embodiment, the nucleic acid sequence expressing the L-pantolactone dehydrogenase and the nucleic acid sequence expressing ketopantolactone reductase and / or glucose dehydrogenase may be contained in one recombinant vector, or may be contained separately in multiple recombinant vectors as a combination of recombinant vectors. The recombinant vector can be any vector capable of normal expression in host cells. From the perspective of artificially controlling the timing of gene expression to effectively avoid adverse effects of gene expression products on early host growth, the preferred recombinant vector is one with an inducible promoter. The preferred recombinant vector is obtained by editing any one of the following vectors: pET28a, pCDFDuet1, pACYCDuet-1, pETDuet-1, and pRSFDuet-1. More preferably, pET28a is selected as the recombinant vector expressing L-pantothenic acid lactone dehydrogenase, and pCDFDuet1 is selected as the recombinant vector expressing ketopantothenic acid lactone reductase and / or glucose dehydrogenase.

[0059] In one specific embodiment, the present invention provides the application of the L-pantolactone dehydrogenase, the nucleic acid molecule encoding the L-pantolactone dehydrogenase, a vector, a combination of vectors, and an engineered bacterium expressing the L-pantolactone dehydrogenase in the synthesis of D-pantolactone. Specifically, it provides the application of the L-pantolactone dehydrogenase, the nucleic acid molecule encoding the L-pantolactone dehydrogenase, the vector, a combination of vectors, and the engineered bacterium expressing the L-pantolactone dehydrogenase for catalyzing the production of ketopantolactone from L-pantolactone. In a more preferred embodiment, the present invention provides the application of the L-pantolactone dehydrogenase, the nucleic acid molecule encoding the L-pantolactone dehydrogenase, the vector, a combination of vectors, and the engineered bacterium expressing the L-pantolactone dehydrogenase in the biosynthesis of D-pantolactone.

[0060] In one specific embodiment, the present invention also constructs an engineered bacterium for the synthesis of D-pantolactone, wherein the engineered bacterium expresses the L-pantolactone dehydrogenase. In one embodiment, the L-pantolactone dehydrogenase is expressed in a host cell through artificial processing. In a preferred embodiment, by simultaneously expressing a multi-enzyme system including the L-pantolactone dehydrogenase in a single engineered bacterium, 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 in the art, such as *Escherichia coli*, *Bacillus subtilis*, yeast cells, or *Aspergillus*, 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).

[0061] 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, ketopantolactone reductase, and glucose dehydrogenase. A specific method for the single-engineered bacterial expressing the multi-enzyme cascade catalytic system to catalyze the generation of D-pantolactone can be as follows: The engineered bacteria expressing the L-pantolactone dehydrogenase (MsfDH), ketopantolactone reductase, and / or glucose dehydrogenase are induced and cultured at an induction temperature of 22-28°C, preferably 25°C. After 20 hours of induction, the wet bacterial cells are collected as a catalyst. Next, the induced expression wet bacterial cells (engineered bacteria) are reacted with L-pantolactone as a substrate and glucose as a coenzyme cycle substrate, for example in a 50 mM pH 8.0 Tris buffer solution. The reaction conditions can be 25-35°C and 200-350 rpm. From the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell, the reaction conditions are preferably 30°C and 200 rpm.

[0062] In the illustrated reaction system, the amount of L-pantolactone can be 10-65 g / L, and preferably 40-65 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 is 10-100 g / L, and preferably 16-100 g / L, more preferably 40 g / L, from the perspective of using the engineered bacteria of the present invention constructed with Escherichia coli as the host cell; NADP... +The final concentration is 5-20 g / L, preferably 10 g / L from the perspective of using the engineered bacteria of the present invention constructed with *E. coli* as the host cell. The final concentration of wet bacterial cells expressing L-pantolactone dehydrogenase (MsfDH), ketopantolactone reductase, glucose dehydrogenase and / or molecular chaperone is 5-40 g / L, preferably 10 g / L. The ketopantolactone reductase and glucose dehydrogenase can be derived from any species, provided they can be normally expressed and active in the engineered bacteria. From the perspective of using the engineered bacteria of the present invention constructed with *E. coli* as the host cell, the nucleic acid sequence of the 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:5. The nucleic acid sequence of the ketopantolactone reductase derived from Candida glabata is shown in SEQ ID NO:3.

[0063] Example 1: Screening and activity characterization of L-pantolactone dehydrogenase

[0064] 1.1 Multiple sequence alignment and bioinformatics analysis were performed using literature review and enzymatic information provided by the BRENDA database. Six candidate gene sequences were mined from databases such as Uniprot and NCBI, and the AmHao sequence from patent (CN110423717B) was used as a positive control. The gene abbreviations are shown in Table 1. The sequences were synthesized by Genscript and then onto the pET28a vector after codon optimization. The sequences are shown in the appendix.

[0065] The pET-28a plasmid containing the candidate gene, the positive control gene, and the empty pET-28a plasmid (negative control) were transformed into the BL21 host and cultured overnight at 37°C. Three to four single colonies were selected for PCR verification. The PCR-verified single colonies were inoculated into 5 ml of LB liquid seed medium containing 50 mg / mL kanamycin and cultured overnight at 37°C and 200 rpm. 2% seed culture was then inoculated into 50 ml of LB liquid medium containing kanamycin, and the culture was allowed to proceed. 600To a concentration of 0.6-0.8, add 0.1 mM IPTG (isopropyl β-D-Thiogalactoside), and induce for 20 h at 25°C and 200 rpm. Centrifuge at 8000 rpm and 4°C for 10 min, discard the supernatant; add 10 mL of buffer to resuspend and wash twice, centrifuge at 8000 rpm and 4°C for 10 min, discard the supernatant, resuspend and lyse a portion of the precipitate, and preserve the remaining wet cells for later use. Centrifuge the lysed bacterial culture, and perform SDS-PAGE verification on the supernatant. The results are as follows: Figure 1 As shown.

[0066] 1.2 Characterization of L-pantolactone oxidative dehydrogenase activity from different gene sources:

[0067] Reaction conditions: 50 mM pH 8.0 Tris buffer, 10 g / L of DL-pantolytic acid lactone mixture (the content of each component is shown in Control in Table 1), 10 g / L of the above-mentioned wet bacterial cells from different gene sources, with a control without bacterial cells, reacted at 30℃ and 200 rpm for 20 h. After the reaction was completed, centrifuged at 8000 rpm for 10 min, the supernatant was collected, and the content and area percentage of D, L-pantolytic acid and its lactone were determined. The results are shown in Table 1.

[0068] Chromatographic detection conditions:

[0069] Column: Daicel CHIRALPAK IG; 4.6 mm × 250 mm × 5 μm

[0070] Mobile phase A: 0.1% formic acid aqueous solution

[0071] Mobile phase B: Methanol

[0072] Flow rate: 0.5 mL / minute

[0073] Detection wavelength: 214nm

[0074] Column temperature: 30℃

[0075] Injection volume: 1uL

[0076] Running time: 20 minutes

[0077] Elution gradient: A:B = 60:40, isogradient elution

[0078] Formula for calculating the result:

[0079] ee% = (A D -A L ) / (A D +A L )

[0080] In the formula:

[0081] ee%: Enantiomeric excess of D-pantolactone (hereinafter the same).

[0082] A D : Percentage of D-pantothenic acid lactone peak area, %.

[0083] A L : Percentage of L-pantothenic acid lactone peak area, %.

[0084] Table 1. Comparison of L-pantolactone oxidative dehydrogenase activities from different gene sources

[0085] Source genes D-acid% L-acid% D-lactone% L-lactone% Control 8.04％ 8.07％ 41.70％ 42.20％ HMD 7.53％ 6.72％ 43.43％ 42.32％ SLD 6.96％ 7.48％ 42.34％ 43.22％ AmHao 2.34％ 4.37％ 65.21％ 28.08％ MsfDH 9.77％ 2.51％ 68.69％ 23.04％ PmLLDH 5.59％ 4.86％ 49.33％ 40.23％ SpoLao 6.76％ 5.27％ 46.68％ 41.28％ RanHao-1 5.60％ 5.16％ 45.94％ 43.31％

[0086] The activity data of the screened genes are shown in Table 1. Compared with the negative control (Control), only AmHao and MsfDH showed a significant reduction in L-pantolytic acid and L-pantolytic lactone in their reaction solutions. Further comparison of the pantolytic acid oxidation data of AmHao and MsfDH revealed that MsfDH was more specific than AmHao in oxidizing L-pantolytic acid substrates.

[0087] Figure 2 The liquid chromatogram of the MsfDH reaction solution shows a significant decrease in the proportion of L-pantolactone and a noticeable reduction in L-pantolactone compared to the blank control (Con). Conversely, the proportions of D-pantolactone and D-pantolactone remain almost unchanged. This indicates that MsfDH is an L-type specific dehydrogenase, exhibiting oxidative dehydrogenation activity towards both L-pantolactone and L-pantolactone. Since raw materials from factories are typically not single pure substances, containing not only mixed L-lactones but also some mixed L-acids, this L-type specificity for L-lactones and L-acids is particularly important for high conversion rates of raw materials and obtaining high purity D-type products when relatively high levels of L-acids are present.

[0088] Example 2: Construction of engineered bacteria for multi-enzyme co-expression and activity characterization of L-pantolactone to D-pantolactone conversion

[0089] 2.1 To further evaluate the ability of the engineered bacteria expressing MsfDH to transform L-indophosphoprolone, it is necessary to obtain L-indophosphoprolone with relatively high purity as a raw material. The inventors used ethyl acetate to perform a simple extraction and purification of L-indophosphoprolone from a mixture of DL lactones derived from Guang'an Mojia Biotechnology Co., Ltd., and analyzed the purity of L-indophosphoprolone by liquid chromatography.

[0090] Extraction liquid phase results as follows Figure 3As shown, the enantiomeric excess (ee) of L-pantolactone was 93.27%, making it the absolute major component. It can be considered as a relatively pure L-pantolactone material and used to evaluate the transformation activity of engineered bacteria on it.

[0091] 2.2 The one-step enzymatic formation of D-pantolactone from L-pantolactone requires the synergistic action of pantolactone dehydrogenase, ketopantolactone reductase, and NADP / NADPH. Therefore, it is necessary to construct a multi-enzyme synergistic system to achieve this scheme. The construction process is as follows:

[0092] Based on the ketopantolactone reductase gene CgCPR from Candida glabata (GenBank ID: CAG61069.1) and the glucose dehydrogenase gene BmGDH from Bacillus megaterium IWG3 (WP_028407571), after codon optimization, the nucleic acid sequences encoding the ketopantolactone reductase from Candida glabata (GenBank ID: CAG61069.1) and the glucose dehydrogenase from Bacillus megaterium IWG3 (WP_028407571) were designed (sequences shown in the appendix), and Genscript synthesized them into the specified pCDFDuet-1 vector.

[0093] The pCDFDuet-1-CgCPR-BmGDH plasmid and the pET-28a-MsfDH plasmid were co-transformed into E. coli BL21(DE3) host cells. Single colonies were picked and inoculated into 5 ml of LB liquid seed medium containing 50 mg / mL (the same below) of streptomycin and kanamycin, and cultured overnight at 37°C and 200 rpm. 2% seed culture was then inoculated into 50 mL of LB liquid medium and cultured until OD (outcome limit). 600 To a final concentration of 0.1 mM IPTG, add to a volume of 0.6-0.8 and induce for 20 h at 25 °C and 200 rpm. Centrifuge the bacterial culture at 8000 rpm and 4 °C for 10 min, discard the supernatant, wash twice with buffer, centrifuge again and discard the supernatant to obtain wet bacterial cells, and store at -80 °C for later use.

[0094] 2.3 Characterization of the activity of the obtained multi-enzyme co-expression engineered bacteria

[0095] Reaction conditions: 50 mM pH 8.0 Tris buffer, 40 g / L extracted L-pantolactone (ee value 93.27%), 40 g / L glucose, 10 g / L wet E. coli BL21(DE3)pET28a-MsfDH / pCDFDuet-1-CgCPR-BmGDH, 10 g / L NADP + The reaction was carried out at 30℃ and 200 rpm for 20 hours. After the reaction was completed, the mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. The content and area percentage of D,L-pantolactone were determined. The results are as follows: Figure 4 As shown. According to Figure 4 The results showed that D-indophosphatidylcholine increased significantly, while L-indophosphatidylcholine decreased significantly and was almost completely consumed. This indicates that the constructed MsfDH / CgCPR-BmGDH multi-enzyme reaction system can successfully convert L-indophosphatidylcholine into D-indophosphatidylcholine in one step, with a conversion rate of up to 99%.

[0096] sequence list

[0097] The main sequences used in this invention will be described below:

[0098] MsfDH nucleic acid sequence (SEQ ID NO:1)

[0099]

[0100] MsfDH amino acid sequence (SEQ ID NO: 2)

[0101] MAKNAWFESVAEAQRRAKKRLPKSVYGALIAGSERGTTLDDNTGAFAELGFAPHVAGLSAKRELATTVLGQNISMPVLISPTGVQAVHPDGEVAVARAAAARGVAMGLSSFASKPVEEVIAANPQTFYQIYWLGDRDSITKRLERARAAGAVGIILTLDWSFSHGRDWGSPVIPERMNLRTMVQFAPEAITRPRWAAQFAKARSIPDLTAPNLVEPGEQAPTFFGAYGQWMATPPPSWDDVAWVCSQWDGPVMLKGVMRLDDAKRAVDAGVSAISVSNHGGNNLDGTPASIRALPAVAKAVGNDIEVLLDGGIRRGSDVVKAVALGARAVMIGRAYLWGLAANGQAGVENVLDILRGGIDSALLGLGHSSVADLSPADVLVPSGFERVLGA

[0102] AmHao nucleic acid sequence

[0103]

[0104] AmHao amino acid sequence

[0105] MSNGWFETVAEAQRRARKRLPKSVYGALVAGSERGITVDDNIAAFAELGFAPHVAGLSDKRELGTTVMGQPISLPVVISPTGVQAVHPDGEVAVARAAAARGTAMGLSSFASKSIEEVAAANPQTFFQMYWVGSRDVLVQRMERARAAGAVGLIMTLDWSFSTGRDWGSPVIPEKLDLKAMARFAPEGITRPKWLWDFAKTRKLPDLTTPNLTPPGGTAPTFFGAYGEWMQTPLPTWEDVAWLREQWGGPFMLKGVMRVDDAKRAVDAGVTAISVSNHGGNNLDGTPAPIRALPAIADAVGGDVEVLLDGGIRRGSDVVKAIALGAKAVLIGRAYLWGLAANGQAGVENVLDILRGGIDS AVLGLGKTSIHELTRDDVVIPPGFERALGVPKS

[0106] HMD nucleic acid sequence

[0107] ATGCACATAGAGAGGCTAGCTGTAGATGAATCCGTGGGCCGCGCAATGCCACCACAACGTTTCATCGAGGCGCTGAGCGATCTCGGGGTGCCGGTGGAGTTCGCTGGCGAGGACGAGCAGTTTGGTCCGGGTGACGCGGTTGCGAGCTTCGGTCATCGTGACGCGTTTCTGGATGCGGATTGGGTTCACTGCATTCGTGCAGGTTACGACGAATTCCCGGTTGGCGTCTACGAGGAAGCGGGCACGTACCTGACCAACAGCACCGGTATTCATGGTACAACCGTTGGTGAGACGGTGGCGGGCTATATGCTGACCTTCGCCCGCCGCCTGCACGCATATCGTGATGCACAGCACGATCACGCGTGGGATTTACCGCGTTATGAAGAGCCGTTTACCCTGGCGGGCGAGCGCGTCTGTGTTGTCGGCCTGGGTACGTTGGGACGTGGTGTGGTGGATCGTGCCGCTGCGTTGGGCATGGAAGTTGTAGGCGTTCGTCGTTCCGGTGACCCGGTTGACAACGTGTCGACCGTGTACACCCCGGATCGTCTGCACGAGGCGATTGCGGATGCTCGCTTCGTGGTGCTGGCAACCCCTTTGACCGATGAGACTGAAGGTATGGTTGCCGCACCGGAGTTCGAGACTATGCGTGAGGACGCTAGCCTGGTTAATGTTGCGAGAGGTCCGGTGGTGGTGGAATCTGATCTGGTAGCCGCTTTGGACTCCGGCGACATCGCCGGCGCTGCGTTGGACGTCTTTAGCGAAGAACCGCTGCCGGAAGACTCTCCGCTTTGGGATTTTGAAGACGTGCTGATCACCCCGCATGTTAGCGCGGCTACCAGCAAGTACCATGAAGATGTGGCCGCGCTTATCCGCGAAAATATTGAAAAAATCGCGACCGGTGACGAGCTGACGAACCGTGTTGTT

[0108] Amino acid sequence of HMD

[0109] MHIERLAVDESVGRAMPPQRFIEALSDLGVPVEFAGEDEQFGPGDAVASFGHRDAFLDADWVHCIRAGYDEFPVGVYEEAGTYLTNSTGIHGTTVGETVAGYMLTFARRLHAYRDAQHDHAWDLPRYEEPFTLAGERVCVVGLGTLGRGVVDRAAALGMEVVGVRRSGDPVDNVSTVYTPDRLHEAIADARFVVLATPLTDETEGMVAAPEFETMREDASLVNVARGPVVVESDLVAALDSGDIAGAALDVFSEEPLPEDSPLWDFEDVLITPHVSAATSKYHEDVAALIRENIEKIATGDELTNRVV

[0110] SLD nucleic acid sequence

[0111] ATGGCAGAACTATTTTATGACGCTGATGCGGACCTGTCTATCATTCAGGGCCGTAAAGTTGCTGTGATCGGATACGGTAGCCAGGGCCACGCGCACGCGCTGTCTCTGCGTGATAGCGGTGTGGACGTGCGTGTTGGTCTGCATGAAGGCTCGAAGTCCAAAGCCAAGGCTGAGGAACAAGGTCTGAGAGTTGTTCCGGTAGCGGAGGCTGCCGCGGAGGCGGATGTCATCATGGTGTTGATCCCGGATCCGATTCAAGGCGACGTTTACGAGAAAGATATCAAAGACAACCTGAAGGACGGCGACGCGCTTTTCTTCGGTCACGGCTTGAACATCCGCTACGGCTTCGTGAAGCCACCGGCAGGTGTTGATGTGTGCATGGTCGCTCCGAAAGGTCCGGGTCACCTGGTACGCCGTCAGTATGAGGAGGGCCGTGGTGTCCCGTGTTTAGTGGCAGTTGAACAAGATGCCACCGGTAATGCATTTGCACTGGCGTTGTCCTATGCAAAGGGCATTGGCGGCACCCGTGCCGGCGTTATCCGCACCACATTTACCGAAGAGACTGAGACGGACTTGTTTGGCGAACAGGCAGTGCTGGCTGGTGGCGTCACCGCGCTGGTGAAAGCGGGTTTCGAAACGCTGACCGAAGCTGGTTATCAGCCGGAAATCGCGTATTTCGAGTGCCTGCACGAATTGAAGCTGATTGTTGACCTGATGTACGAGGGCGGTTTGGAGAAAATGCGTTGGAGCATCAGCGAAACGGCTGAGTGGGGTGACTATGTTACCGGTCCGCGTATTATTACCGACGCGACCAAAGCGGAGATGCGTAAGGTGCTGGCAGAGATTCAAGATGGTACGTTTGCGAAGAACTGGATGGATGAGTACCATGGTGGTCTTAAGAAGTACAATGAATACAAAAAACAGGACAGCGAACATTTGCTGGAAACCACCGGCAAAGAACTCCGCAAGCTGATGAGCTGGGTTGATGAAGAGGCG

[0112] SLD amino acid sequence

[0113] MAELFYDADADLSIIQGRKVAVIGYGSQGHAHALSLRDSGVDVRVGLHEGSKSKAKAEEQGLRVVPVAEAAAEADVIMVLIPDPIQGDVYEKDIKDNLKDGDALFFGHGLNIRYGFVKPPAGVDVCMVAPKGPGHLVRRQYEEGRGVPCLVAVEQDATGNAFALALSYAKGIGGTRAGVIRTTFTEETETDLFGEQAVLAGGVTALVKAGFETLTEAGYQPEIAYFECLHELKLIVDLMYEGGLEKMRWSISETAEWGDYVTGPRIITDATKAEMRKVLAEIQDGTFAKNWMDEYHGGLKKYNEYKKQDSEHLLETTGKELRKLMSWVDEEA

[0114]

[0115] PMLLDH amino acid sequence

[0116] MSNGWFETVAEAQRRAKRRLPRSVYGALVAGSERGQTIEDNMGAFAELGFAPHVAGLSDQRDLATTVMMGQPVSMPVLISPTGVQAVHPEGEVAVARAAAARGVAMGLSSFASKSVEEVAAANPQTFFQMYWVGSRDVLVQRMERAKAAGAVGLIMTLDWSFSNGRDWGSPNIPEKMDFKTMVKFAPEGVTRPKWLWDF AKTRRLPDLTTPNLTPPGGTAPTFFGAYGEWIQTPLPTWDDVAWLRQQWDGPFMLKGVTRVDDAKRAVDAGVSAISVSNHGGNNLDGTPATIRALPPIADAVGEQIEVLLDGGVRRGSDVVKALALGAKAVMIGRAYLWGLAANGQAGVENVLDILRGGIDSAVLGLGLTSVHDISRGDVVIPPGFIRELGVHEAAEL

[0117] SpoLao nucleic acid sequence

[0118]

[0119] SpoLao amino acid sequence

[0120] MEITNVNEYEAIAKQKLPKMVYDYYASGAEDQWTLAENRNAFSRILFRPRILIDVTNIDMTTTILGFKISMPIMIAPTAMQKMAHPEGEYATARAASAAGTIMTLSSWATSSVEEVASTGPGIRFFQLYVYKDRNVVAQLVRRAERAGFKAIALTVDTPRLGRREADIKNRFVLPPFLTLKNFE GIDLGKMDKANDSGLSSYVAGQIDRSLSWKDVAWLQTITSLPILVKGVITAEDARLAVQHGAAGIIVSNHGARQLDYVPATIMALEEVVKAAQGRIPVFLDGGGVRRGTDVFKALALGAAGVFIGRPVVFSLAAEGEAGVKKVLQMMRDEFELTMALSGCRSLKEISRSHIAADWDGPSSRAVARL

[0121] RanHao-1 nucleic acid sequence

[0122]

[0123] RanHao-1 amino acid sequence

[0124] MPLVCLADFKAHAQKQLSKTSWDFIEGEADDGITYSENIAAFKRIRLRPRYLRDMSKVDTRTTIQGQEISAPICISPTAFHSIAWPDGEKSTARAAQEANICYVISSYASYSLEDIVAAAPEGFRWFQLYMKSDWDFNKQMVQRAEALGFKALVITIDTPVLGNRRRDKRNQLNLEANILLKDLRALKEEKPTQSVPVSFPKASFCWNDLSLLQSITRLPIILKGILTKEDAELAMKHNVQGIVVSNHGGRQLDEVSASIDALREVVAAVKGKIEVYMDGGVRTGTDVLKALALGARCIFLGRPILWGLACKGEDGVKEVLDILTAELHRCMTLSGCQSVAEISPDLIQFSRL

[0125] CgCPR nucleic acid sequence (SEQ ID NO:3)

[0126] ATGGTGAAACAAGAATTTTTTAAACTGAACAACGGCCATGAAATGCCGGGCGTGGCGATTGTGGGCACCGGCACCAAATGGCATAAAGTGAACGAAACCGATGAAAACTTTAGTCAGACCCTGGTGGATCAGCTGAAATATGCGCTGAGCCTGCCGGGCGTGGTGCATCTGGATGCGGCGGAATTTTATATGACCTATCGCGAAGTGGGCCGCGCGCTGGCGGAAACGAGCAAACCGCGCGATGAAATTTTTATTACCGATAAATATTGGACCCTGAGCAAAGTGACCGAGAACCCGATTGTGGGCCTGGAAACCGGCCTGAAACGCCTGGGCCTGGAATATGTGGATCTGTATCTGCTGCATAGCCCGTTTATTAGCAAAGAAACCAACGGCTTTAGCCTGGAAGAAGCGTGGGGCATGATGGAAGAACTGTATCATAGCGGCAAAGCGAAAAACATTGGCGTGAGCAACTTTGCGAAAGAAGATCTGGAACGCGTGCTGAAAGTGTGCAAAGTGAAACCGCAAGTGAATCAGATTGAATTTAACGCGTTTCTGCAGAATCAGACCCCGGGCATTTATAACTTTTGCAAACAGAACGATATTCAGCTGGCGGCGTATAGCCCGCTGGGCCCGCTGCAGAAAAAACCGGCGGATGGCAACAGTCAGCCGTTTTATAGCTATATTAACAAACTGGCGCAGCATTATAACAAAACCCCGGGCCAAGTGCTGCTGCGCTGGGTGACCAAACGCGGCGTGGTGGCGGTGACCACGAGCGAAAAAAAAGAACGCATTAAACAAGCGCAAGAAATTTTTGAATTTGATCTGAAAGATGATGAAGTGACCGAAATCACGAAACTGGGCCTGGATCATGAACCGCTGCGCCTGTATTGGCATGATCAGTATAACAAATATAACAGCGAAAGTCAGAAAGCGTAA

[0127] CgCPR amino acid sequence (SEQ ID NO:4)

[0128] MVKQEFFKLNNGHEMPGVAIVGTGTKWHKVNETDENFSQTLVDQLKYALSLPGVVHLDAAEFYMTYREVGRALAETSKPRDEIFITDKYWTLSKVTENPIVGLETGLKRLGLEYVDLYLLHSPFISKETNGFSLEEAWGMMEELYHSGKAKNIGVSNFAKEDLERVLKVCKVKPQVNQIEFNAFLQNQTPGIYNFCKQNDIQLAAYSPLGPLQKKPADGNSQPFYSYINKLAQHYNKTPGQVLLRWVTKRGVVAVTTSEKKERIKQAQEIFEFDLKDDEVTEITKLGLDHEPLRLYWHDQYNKYNSESQKA

[0129] BmGDH nucleic acid sequence (SEQ ID NO: 5)

[0130] ATGTATAAAGATCTGGAAGGCAAAGTGGTTGTGATTACCGGCAGCAGCACCGGCCTGGGCAAAAGCATGGCGATTCGCTTTGCGACCGAAAAAGCGAAAGTTGTGGTTAATTATCGCAGCAAAGAAGATGAAGCGAACAGCGTGCTGGAAGAAATTAAAAAAGTGGGCGGCGAAGCGATTGCGGTGAAAGGCGATGTGACCGTGGAAAGCGATATTATTAACCTGGTGCAGAGCGCGATTAAAGAATTTGGCAAACTGGATGTGATGATTAACAACGCGGGCCTGGAAAACCCGGTGCCGAGCCATGAAATGAGCCTGAGCGATTGGAACAAAGTGATTGATACCAACCTGACCGGCGCGTTTCTGGGCAGCCGCGAAGCGATTAAATATTTTGTGGAAAACGATATTCGCGGCACCGTGATTAACATGAGCAGCGTGCATGAAAAAATTCCGTGGCCGCTGTTTGTGCATTATGCGGCGAGCAAAGGCGGCATGCGCCTGATGACCAAAACCCTGGCGCTGGAATATGCGCCGAAAGGCATTCGCGTGAACAACATTGGCCCGGGCGCGATTAACACCCCGATTAACGCGGAAAAATTTGCCGATCCGGAACAGCGCGCGGATGTGGAAAGCATGATTCCGATGGGCTATATTGGCGAACCGGAAGAAATTGCGGCGGTGGCGGCGTGGCTGGCGAGCAGCGAAGCGAGCTATGTGACCGGCATTACCCTGTTTGCGGATGGCGGCATGACCCTGTATCCGAGCTTTCAAGCGGGCCGCGGCTAA

[0131] Amino acid sequence of BmGDH (SEQ ID NO: 6)

[0132] MYKDLEGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDIINLVQSAIKEFGKLDVMINNAGLENPVPSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDIRGTVINMSSVHEKIPWPLFVHYAASKGGMRLMTKTLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG*

[0133] pET-28a-MsfDH vector sequence (SEQ ID NO:7)

[0134]

[0135] pCDFDuet-1-CgCPR-BmGDH sequence (SEQ ID NO:20)

[0136]

Claims

1. A nucleic acid molecule, wherein, The nucleic acid molecule encodes L-indoglutamate dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2, and encodes ketoindoglutamate reductase.

2. The nucleic acid molecule as described in claim 1, wherein, The nucleotide sequence encoding the L-indohydrin dehydrogenase is shown in SEQ ID NO:

1.

3. The nucleic acid molecule as described in claim 1, wherein, The amino acid sequence of the keto-indohydranolate reductase is shown in SEQ ID NO:

4.

4. The nucleic acid molecule as described in claim 3, wherein, The nucleotide sequence encoding the keto-indohydranolate reductase is shown in SEQ ID NO:

3.

5. The nucleic acid molecule of claim 1, further encoding glucose dehydrogenase.

6. The nucleic acid molecule as described in claim 5, wherein, The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:

6.

7. The nucleic acid molecule of claim 6, wherein, The nucleotide sequence encoding the glucose dehydrogenase is shown in SEQ ID NO:

5.

8. A carrier assembly comprising: (a) First vector: encoding L-indohydrin dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2; and, (b) Second carrier: which encodes keto-indohydrin reductase.

9. The carrier assembly as described in claim 8, wherein, The nucleotide sequence encoding the L-indohydrin dehydrogenase is shown in SEQ ID NO:

1.

10. The carrier assembly as described in claim 8, wherein, The amino acid sequence of the keto-indohydranolate reductase is shown in SEQ ID NO:

4.

11. The carrier assembly as claimed in claim 10, wherein, The nucleotide sequence encoding the keto-indohydranolate reductase is shown in SEQ ID NO:

3.

12. The carrier assembly as described in claim 8, wherein, The second carrier also encodes glucose dehydrogenase.

13. The carrier assembly as described in claim 12, wherein, The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:

6.

14. The carrier assembly as described in claim 13, wherein, The nucleotide sequence encoding the glucose dehydrogenase is shown in SEQ ID NO:

5.

15. The carrier assembly as described in claim 8, wherein, The first and second carriers are each obtained independently by editing any one of the following carriers: pET28a, pCDFDuet1, pACYCDuet-1, pETDuet-1, pRSFDuet-1.

16. The carrier assembly as described in claim 15, wherein, The first vector contains a nucleotide sequence as shown in SEQ ID NO:

7.

17. The carrier assembly as described in claim 15, wherein, The second vector contains a nucleotide sequence as shown in SEQ ID NO:

20.

18. An engineered bacterium, wherein, The engineered bacteria express L-pantolactone dehydrogenase and ketopantolactone reductase, with amino acid sequences as shown in SEQ ID NO:

2.

19. The engineered bacteria as described in claim 18, wherein, The engineered bacteria also express glucose dehydrogenase.

20. The engineered bacteria as described in claim 18 or 19, wherein, The engineered bacteria comprise a nucleic acid molecule as described in any one of claims 1-7 or a vector combination as described in any one of claims 8-17.

21. The engineered bacteria as described in claim 18, wherein, The engineered bacteria are obtained by processing any one or more host cells selected from the group consisting of: Escherichia coli, Bacillus subtilis, yeast cells, or Aspergillus.

22. The engineered bacteria as described in claim 21, wherein, The host cell was Escherichia coli BL21.

23. A method for producing D-pantolactone, comprising: (a) The step of catalyzing the production of keto-universalactone from L-universalactone by an L-universalactone dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 2; and, (b) The step of forming D-indohydrin from the ketone-indohydrin lactone.

24. The method of claim 23, wherein, Step (a) is performed using engineered bacteria that express L-pantolactone dehydrogenase with an amino acid sequence as shown in SEQ ID NO:

2.

25. The method of claim 24, wherein, The specific implementation steps of step (a) include: (i) The step of reacting the engineered bacteria expressing the L-indolactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2 in a reaction environment containing L-indolactone at a final concentration of 10-65 g / L.

26. The method of claim 23, wherein, The method is carried out by using engineered bacteria as described in any one of claims 18-22.

27. The method of claim 26, wherein, The specific implementation steps of the method include: (i) The step of reacting the engineered bacteria as described in any one of claims 18-22 in a reaction environment containing L-indohydrin at a final concentration of 10-65 g / L.

28. The method of claim 25 or 27, wherein, The final concentration of the L-indohydrin is 40-65 g / L.

29. The method of claim 25 or 27, wherein, Step (i) is performed at 25-35 °C and 200-350 rpm.

30. The method of claim 29, wherein, Step (i) is performed at 30 °C and 200 rpm.

31. The method of claim 25 or 27, wherein, The reaction environment further includes: glucose and NADP. + , The final concentration of glucose is 10-100 g / L, and NADP... + The final concentration is 5-20 g / L.

32. The method of claim 31, wherein, The final concentration of glucose is 16-100 g / L, and the NADP... + The final concentration is 10 g / L.

33. The method of claim 31, wherein, Step (i) is performed at 25-35 °C and 200-350 rpm.

34. The method of claim 33, wherein, Step (i) is performed at 30 °C and 200 rpm.

35. The use of L-pantolactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2, the nucleic acid molecule as described in any one of claims 1-7, the vector combination as described in any one of claims 8-17, or the engineered bacteria as described in any one of claims 18-22 in the synthesis of D-pantolactone.

36. The use of L-indo-indo-lactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2, the nucleic acid molecule encoding L-indo-indo-lactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2, the vector encoding L-indo-indo-lactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2, or the engineered bacteria expressing L-indo-indo-lactone dehydrogenase with the amino acid sequence shown in SEQ ID NO: 2 in catalyzing the production of keto-indo-indo-lactone from L-indo-indo-lactone.

37. A method for producing keto-indoparatide, comprising: a step of catalyzing L-indoparatide to produce keto-indoparatide via an L-indoparatide dehydrogenase having an amino acid sequence as shown in SEQ ID NO:

2.

38. The method of claim 37, wherein, The method is carried out using engineered bacteria that express L-pantolactone dehydrogenase with an amino acid sequence as shown in SEQ ID NO: 2.

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