Enzyme composition and its use in the preparation of ceramide np

Abstract

The present application relates to the field of biotechnology, and particularly to an enzyme composition and its application in preparing ceramide NP. The present application provides a new method for synergistically catalyzing the synthesis of ceramide NP by multiple enzymes; the method is based on enzyme directed evolution, and uses propenal and glycine as starting substrates, and under the action of mutant glycine aldolase, enaminoketol reductase, pentenoate ligase, olefin reductase, phosphite dehydrogenase and polyphosphate kinase, and in cooperation with a metal catalyst Grubbs Catalyst, to realize the synthesis of high-efficiency and high-purity ceramide NP; compared with the traditional microbial fermentation method, the synthesis path of the present application has the advantages of mild reaction condition, convenience for continuous production, short conversion period (<8h), low cost and high product purity (byproduct content <0.5%), and is suitable for the demand of industrial large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an enzyme composition and its application in the preparation of ceramide NP. BACKGROUND

[0002] Ceramide is an amphipathic lipid molecule with long-chain amino alcohol and fatty acid combined by amide bond. Its structural diversity (such as differences in carbon chain length, saturation and hydroxyl number) leads to the identification of more than 1000 subtypes. As a key mediator of cell signal regulation, ceramide has significant effects on maintaining skin barrier, promoting collagen generation, antioxidant and anti-aging, and inhibiting melanin deposition. Its molecular polarity is highly compatible with the stratum corneum of the skin, which can enhance absorption efficiency and synergistically enhance the penetration of skin care products with ingredients such as linolenic acid and hyaluronic acid. In the field of cosmetics, ceramide has become one of the three core beauty materials along with collagen and hyaluronic acid, due to its long-lasting moisturizing (reducing water loss by more than 30%), repairing damaged barrier (accelerating epidermal regeneration), and anti-UV damage properties. Based on its multifunctional biological activity and cross-field application potential, ceramide shows breakthrough commercial value in precision skincare, functional food and targeted drug development.

[0003] The production of ceramide is mainly a microbial-chemical combined preparation method. Through microbial fermentation to produce sphingolipid precursors, and then chemical modification (such as acylation, purification) to synthesize high-purity ceramide. For example, Huaxi Biotech induces the spores of Wickerhamiella sp. and combines screening to obtain a TAPS high-yield strain, uses supercritical CO2 extraction method to extract TAPS, and then generates ceramide through enzyme-catalyzed acylation reaction; Chongqing Zhihe Biotech develops a TAPS production method without specific strain disclosure through fermentation process parameter adjustment, uses ion exchange chromatography to purify TAPS, and then synthesizes ceramide through palmitoyl chloride esterification reaction; Wincreate Diversa uses gene editing technology to knock out the serine degradation and phosphorylation related genes of Pichia sp., and strengthens the expression of key enzymes, so that the TAPS yield is increased to 2 g / L, TAPS is enriched through membrane separation technology, and then the long-chain structure of ceramide is constructed through solid-phase synthesis method; Ajinomoto Co., Inc. inhibits the activity of Saccharomyces cerevisiae LCB4 and CKA2 genes, enhances the activity of serine palmitoyltransferase and other enzymes, so that the TAPS yield reaches 2164 mg / L, free fatty acids are removed through high-temperature molecular distillation, and then the double bond structure of ceramide is stabilized through hydrogenation reaction. Although the chemical synthesis method has low cost, its efficiency is insufficient, and the plant extraction method is limited by high cost and low efficiency, so it is crucial to develop a new ceramide NP synthesis method with high energy efficiency, low cost and high conversion rate. SUMMARY

[0004] Therefore, the present application provides an enzyme composition and its application in the preparation of ceramide NP.

[0005] The present application provides a glycine aldolase variant (BaAldo), the amino acid sequence of which is shown as SEQ ID NO: 1.

[0006] The present application provides a combination enzyme, which comprises at least one of: an enamine pentanol reductase or a variant thereof (WsCarRed), a pentenoate ligase or a variant thereof (OsEAPLigase), an olefin reductase or a variant thereof (EreneRed), a phosphite dehydrogenase or a variant thereof (PspPTDH), and / or a polyphosphate kinase or a variant thereof (KdPPK), and the glycine aldolase variant of claim 1.

[0007] The Uniprot ID of the enamine pentanol reductase is A0A157S7Y1;

[0008] The Uniprot ID of the pentenoate ligase is H2FZR9;

[0009] The Uniprot ID of the olefin reductase is A0A7K7GRB6;

[0010] The Uniprot ID of the phosphite dehydrogenase is F2YGD2;

[0011] The Uniprot ID of the polyphosphate kinase is F0F2Z3.

[0012] The amino acid sequence of the variant of the enamine pentanol reductase is shown as SEQ ID NO: 1;

[0013] The amino acid sequence of the variant of the pentenoate ligase is shown as SEQ ID NO: 2;

[0014] The amino acid sequence of the variant of the olefin reductase is shown as SEQ ID NO: 3;

[0015] The amino acid sequence of the variant of the phosphite dehydrogenase is shown as SEQ ID NO: 4;

[0016] The amino acid sequence of the variant of the polyphosphate kinase is shown as SEQ ID NO: 5.

[0017] The combination enzyme of the present application can be free or immobilized, and the immobilized enzyme comprises the combination enzyme of the present application and a carrier; the carrier comprises an epoxy resin.

[0018] The present application provides a biological material, which comprises at least one of I)~V) as follows:

[0019] I), a nucleic acid encoding the glycine aldolase variant of the present application or the combination enzyme of the present application.

[0020] II) an expression unit comprising the nucleic acid as defined in I);

[0021] III) a recombinant vector comprising the nucleic acid as defined in I) or the expression unit as defined in II);

[0022] IV) a host cell into which the nucleic acid as defined in I) or the expression unit as defined in II) has been genomically integrated, or into which the recombinant vector as defined in III) has been transfected or transformed;

[0023] V) a culture obtained by culturing the host cell as defined in IV).

[0024] The nucleic acid of the present application can be DNA, RNA, cDNA or PNA. In the embodiments of the present application, the nucleic acid is in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The nucleic acid can include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g. promoters or transcription terminators). The nucleic acid can be linear or circular in topology. The nucleic acid can be part of a vector (such as an expression or cloning vector), or a fragment. The nucleic acid can be obtained directly from natural sources, or can be prepared with the aid of recombination, enzymatic or chemical techniques. The RNA form is mRNA and the like obtained by transcription of a gene.

[0025] In the present application, the nucleic acid can be optimized or not. Specifically, the nucleic acid of the present application is optimized. The optimization includes but is not limited to: codon usage bias, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, alteration of GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splice sites, early polyadenylation sites, internal ribosome entry sites and binding sites, negative CpG islands, RNA instability regions, repeat sequences (direct repeats, inverted repeats, etc.) and restriction sites that can affect cloning.

[0026] The present application also provides an expression unit, which refers to a DNA sequence from the beginning of a promoter to the end of a terminator. The promoter and the terminator can also include regulatory fragments between or on both sides, which can include promoters, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, and homologous recombination sites, such as enhancers of promoters, poly (A) signals, etc.

[0027] The present application provides a recombinant vector, which includes at least one of the nucleic acid or the expression unit of the present application and a vector backbone.

[0028] Further, the source of the vector backbone of the present application includes plant, animal, bacteria, fungi, bacteriophage, or virus, which are not limited by the present application. The animal includes mammal and non-mammal, and the mammal expression vector includes but is not limited to pcDNA 3.1, pIRES, pTT3, pCEP4, pATX1, or pCHO1.0. The bacterial vector includes but is not limited to pET28a, pET16b, pET26b, pET28a, pET31b, pBAD, pBADHis, pTrc99a, pTrcHis, pACYCduet-1, pET duet-1, pCDFduet-1, pColdI, pColdII, etc. The fungal vector includes but is not limited to pYES2, pYES3, pYES6, pAUR23, etc.

[0029] The recombinant vector of the present application refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule comprising a desired coding sequence and suitable nucleic acid sequences or elements necessary for the expression of the operably linked coding gene in a specific host organism. In the present specification, "plasmid" and "vector" can be used interchangeably sometimes, because plasmid is the most commonly used form of vector at present. However, the present application is intended to include such other forms of expression vectors which perform equivalent functions, which are known in the art or will become known, including but not limited to: plasmid, bacteriophage particle, viral vector and / or only potential genomic insert. In specific embodiments, the nucleic acid encoding the fusion protein provided by the present application can be constructed in various prokaryotic expression vectors. For example, the backbone vector thereof can be pET28a.

[0030] The present application provides a host cell transformed or transfected with the recombinant vector. The source of the host cell includes plant, animal, bacteria, fungi, bacteriophage or virus, which are not limited by the present application. In specific embodiments of the present application, the host cell is derived from bacteria; specifically, E. coli, more specifically, E. coli (BL21) and / or E. coli DH5α.

[0031] The transformation method includes chemical transformation and electroporation; the transfection method includes calcium phosphate coprecipitation, artificial liposome method, viral transfection. The viral transfection includes adenovirus transfection, adeno-associated virus transfection, lentivirus transfection, etc.

[0032] The present application provides at least one of the following I)~III) in the preparation of ceramide NP:

[0033] I), the glycine aldolase variant of claim 1;

[0034] II), the combined enzyme of claim 2 or 3;

[0035] III) the biomaterial according to any one of claims 4 to 6.

[0036] The present application provides a product containing ceramide NP, characterized in that the raw material comprises at least one of i) to iii) as follows:

[0037] i) the glycine aldolase variant according to the present application;

[0038] ii) the combination enzyme according to the present application;

[0039] iii) the biomaterial according to the present application.

[0040] The present application provides a preparation method of ceramide NP, characterized in that it comprises synthesizing ceramide NP by using at least one of A) to C) as follows:

[0041] A) the glycine aldolase variant according to the present application;

[0042] B) the combination enzyme according to the present application;

[0043] C) the biomaterial according to the present application.

[0044] Further, the preparation method is characterized in that it comprises the following steps:

[0045] synthesizing the ceramide NP by using propenal and glycine as substrates in the presence of the combination enzyme according to claim 2 or 3, a cofactor and a catalyst;

[0046] the cofactor comprises NAD +、 ATP and magnesium ions;

[0047] the catalyst comprises Hoveyda-Grubbs.

[0048] Further, the preparation method according to the present application comprises the following steps:

[0049] Step 1, synthesizing enaminopentanol by using propenal and glycine as substrates in the presence of BaAldo, WsCarRed, PspPTDH and NAD +

[0050] Step 2, synthesizing dienamide intermediate by using enaminopentanol, pentenoic acid and partial hexaphosphoric acid as substrates in the presence of OsEAPLigase, KdPPK, ATP, magnesium chloride;

[0051] Step 3, generating the ceramide NP by using dienamide intermediate and C15 olefin as substrates in the presence of a catalyst. ​

[0052] The application develops a new process for preparing ceramide by combining a multi-enzyme synergistic catalytic system and a metal coenzyme. Through the synergistic design of multi-enzyme cascade reaction and coenzyme regeneration system, efficient and green synthesis of high-value ceramide is realized from bulk raw materials such as propenal and glycine. The process uses glycine aldolase to catalyze the condensation of propenal and glycine to form enaminopentanoic acid, which is converted into enaminopentanol by enaminopentanol reductase WsCarRed, and then acylated and linked with pentenoic acid by pentenoic acid ligase Oseapligase. The fatty acid chain extension is completed by Grubb's catalyst mediated olefin metathesis reaction, and the ceramide product is obtained by stereoselective reduction driven by the NADH regeneration system. The process can be selected by step-by-step reaction, and all the enzymes are optimized by directed evolution and exhibit excellent catalytic compatibility. By precisely regulating the activity ratio of each enzyme, a straight-through conversion from propenal to ceramide can be realized. The process has the core advantages of environmental friendliness of the whole biological catalytic path, operational flexibility of the modular design, and economy and ease of continuous production of the coenzyme regeneration system, providing an efficient and sustainable ceramide production solution for the cosmetics, pharmaceutical and other fields.

[0053] In addition, the application found that Grubbs metal catalyst is not well compatible with other step enzyme catalysis by trying to prepare ceramide NP in one pot.

[0054] The application provides a new method for synthesizing ceramide NP by multi-enzyme synergistic catalysis. Based on enzyme directed evolution, the method uses propenal and glycine as starting substrates, and realizes the synthesis of high-efficiency and high-purity ceramide NP under the action of mutant glycine aldolase, enaminopentanol reductase, pentenoic acid ligase, olefin reductase, phosphite dehydrogenase and polyphosphate kinase, and in cooperation with metal catalyst Grubbs Catalyst. Compared with the traditional microbial fermentation method, the synthesis path of the application has the advantages of mild reaction conditions, easy continuous production, short conversion period (<8h), low cost and high product purity (byproduct content <0.5%), which is suitable for industrial large-scale production demand. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 The SDS-PAGE gel detection diagram after enzyme purification is shown in the figure, wherein M is a protein standard marker; 1 is EreneRed; 2 is WsCarRed; 3 is KdPPK; 4 is PspPTDH; 5 is BaAldo; 6 is OsEAPLigase;

[0056] Figure 2The purified liquid chromatography chromatogram of ceramide NP is shown. Test conditions: Thermo Hypersil GOLD™ C18 column (4.6×250mm, 5μm); mobile phase: acetonitrile-isopropanol mixture (7:3 v / v) and 0.1% trifluoroacetic acid aqueous solution, flow rate 1.0mL / min, 40°C, 210nm.

[0057] Figure 3 Indications of ceramide NP 1 H-NMR, D2O as solvent, Varian 600 M NMR;

[0058] Figure 4 Indications of ceramide NP 13 C-NMR, D2O as solvent, Varian 600 M NMR;

[0059] Figure 5 Mass spectrum of ceramide NP, Agilent Ultivo LC / TQ. Detailed Implementation

[0060] This invention provides an enzyme composition and its application in the preparation of ceramide NP. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0061] In the preparation method described in this invention, acrolein and glycine are condensed by glycine aldolase BaAldo, followed by reduction to enaminopentanol catalyzed by enaminopentanol reductase WsCarRed. Then, enaminopentanol and pentenoic acid form a dienamide intermediate through pentenoic acid ligase Oseapligase. Subsequently, this intermediate undergoes C-C linkage with two C15 olefin molecules mediated by Grubbs catalyst Grubbs Catalyst. Finally, the ligation product is reduced by ErEnRed reductase to obtain the ceramide product. After directed evolution optimization, the activity, selectivity, and stability of the enzymes are significantly improved, catalytic efficiency is greatly enhanced, and the product has high purity, meeting the needs of industrial production. This technology breaks through and reduces raw material costs to 38% of fermentation methods, possessing industrial application value in the precise production of pharmaceutical-grade ceramides and the green and continuous production of cosmetic-grade ceramides.

[0062] Glycine aldolase (BaAldo): derived from Bordetella ansorpii (Uniprot ID: A0A157S7Y1), the native enzyme (WT BaAldo) has weak activity on propenal and glycine, and after sequence structure design and modification (BaAldo), its activity on the two substrates is significantly improved, and the specific mutation sites are: C9R, Q49N, P122S, N123D, L189V, E204F and M287I.

[0063] Enamine reductase (WsCarRed): derived from Williamsia sp. (Uniprot ID: A0A0Q5QR09), the native enzyme (WT WsCarRed) has certain activity on enamine valeric acid, but its expression is not ideal, and after systematic rearrangement and modification (WsCarRed), its expression and activity are improved, and the specific mutation sites are: V65D, P66M, D97S, V138T, N235Q, N314I, D315C, E316L, G452M, Y453S, G520N, H580P and D672A.

[0064] Pentenoic acid ligase (OsEAPLigase): derived from Oceanimonas sp. (Uniprot ID: H2FZR9), the native enzyme (WT OsEAPLigase) has weak activity on the substrate, and after optimization of the enzyme sequence (OsEAPLigase), its activity is significantly improved, and the mutation sites are: W17N, R18H, R19L, F26V, R64E, P94M, E95S, D127L, R197F, E202V and D243G.

[0065] Olefin reductase (EreneRed): derived from Erithacus rubecula (Uniprot ID: A0A7K7GRB6), the native enzyme (WT EreneRed) has weak activity on the substrate, and the expression is poor; after systematic modification and optimization of the enzyme, the mutant (EreneRed) activity and expression are significantly improved, and the specific mutation sites are: T37S, R38D, W68L, F82A, F92N, L93A, I114T, D126H, A150L, E151C, V178M, N189S, S253G, H256Q and F258V.

[0066] Phosphite dehydrogenase (PspPTDH): derived from Pseudomonas sp. (Uniprot ID: F2YGD2), the native enzyme (WTPspPTDH) itself has a good ability to regenerate NADH with phosphite, but it has not yet reached the requirements of industrial application, and its expression is not ideal. Through systematic mutation modification, a mutant enzyme (PspPTDH) with better activity and expression is finally obtained, and its specific mutation sites are: P35D, R120I, D123F, D180K, E196S, R252I, G253L and N286H.

[0067] Polyphosphate kinase (KdPPK): derived from Kingella denitrificans (Uniprot ID: F0F2Z3), the native enzyme has good ATP regeneration function, but through certain modification, its stability and expression are improved, and its specific mutation sites are: Q18T, K37D, F131L, E235T and A236L.

[0068] Amino acid sequence of BaAldo: MGSSDTPTRPDGTPYLPPPVLPGQPLASVDTPALAIDLDAFEANLRTMNDWADQHGVALRPHAKAHKCPQVSLRQLALGARGICCQKVSETLPFIAAGILDIHISNQVVGPAKLALLGRLASDAMLSVCVDHPGNVDALSAAMVQAGAHLTVLVEVDVGQGRCGVADSGQAVALAQRIQAAPGLTFGGVQAYHGTVQHLRTRAFRAAVCAQAAERAAAFAQALRDAGIVCERITGGGTGSAEFDAASGIYTELQAGSYAFMDGDYGANDWDGAMAFRHSLFLMSTVISTAKPGRAVLDAGLKSTTIECGLPSVYERPDLKYIAANDEHGVVTVREGDGPALGEVLKLVPSHVDPTFNLHDELVAYRGGMVAEIWPIAARGLSR (SEQ ID NO: 1);

[0069] Amino acid sequence of WsCarRed: MTTTRTLVPRFDTVTYGELADRIAAVAAGWAEFVRPGQFVTTLGFTSVDYAVIDLACAHLGAVSDMLQTSATPAALAPIITETEPTVFAVSIDHLASAVELITTGFAPSRLVVFDNFTDFRSQVDHRLDADATDVGATEDAVKAELREELLGGRVLSAMCGTAPLAPESMFPGYDDDGFYLTGDIMAETEPGHLVYLDRRKNVLKLSQGEFVAVSRLESIFVTADGVAQIFVYGQSSRAFLLAVVVPTDEVSAEIDSDAQLRTRLLDAIGESARGAGLNSYEIPRDIIVERSPFTREAGLLSALTEHQLADGQICLLRDLRRHGNDRPVLETVTRAAMATVGGSGQVEGSVRFSDLGGDSLSALTFSNLLTDIFDVEIPVGVLMSAATDLSAIAEHIERARAGAVRPTFTSVHFAEQLTLDKFIDAETLSAAPSLPRAPEKPGTVLLTGANMSLGRFLCLEWLERLDASDGAGDIADTDLGLDASTWQRLADEVDLIVHPAALVNHVLPYDQLFGPNVVNTAELIALALTSRVKPITYLSTVGVAADGGPASARRSVDETYANGYGNSKWAGEVLLREAPDLCELPVAVFRSDMILAHSTFGGQLNVPDMFTRLVLSVMASGVAPGSFYRSDDGSTDADFTATAITDLGSSVHKGYETYNVVNPHDDGISLATVVDWLIDSGVAVTRIDDYASWVARFETSIRALPEAQRRASLLPLLHAFAEPDVAIAGSAIPSRRFAAAVAEARIGDNGEIPHLSAELIAKYVADLRSLGLVDAAR (SEQ ID NO: 2);

[0070] Amino acid sequence of OsEAP Ligase: MQLVSTIEQLRATITENHLAGLRIGVVPTMGNLHDGHLELVRRARERADKVVVSIFVNPLQFDEAADLDAYPRTLAQDCERLGELAELVFTPTMSLMYPNGQQQQTRVSVPGVSEVLEGALRPGHFLGVSTVVCKLFNLVQPDLACFGEKDYQQLALIRKMTADLNLPVEIIGVPTVRAEDGLALSSRNGYLTDAEFALAPVLAATMNWLAERLAAGERDLEQLAAEGRARLDNAGFRTDGIGMVDADTLTGLTEHSRAVVILMAAFLGKARLIDNQVVRLG (SEQ ID NO: 3);

[0071] Amino acid sequence of PspPTDH: MLPKLVITHRVHDEILQLLAPHCELMTNQTDSTLQREEILRRCRDAQAMMAFMPDRVDADFLQACPELRVVGCALKGFDNFDVDACTARGVWLTFVPDLLAVPTAELAIGLAVGLGRHLIAAFAFVRSGKFQGWQPQFYGTGPDNATVGILGMGAIGLAMADRLQGWGATLQYHEAKALKTQTEQRLGLRRVACSSLFASSDFILLALPLNADTQHLVNAELLALVRPGALLVNPCRGSVVDEAAVLAALEILQLGGYAADVFEMEDWARADRPRLIDPALLAHPHTLFTPHIGSAVRAVRLEIERCAAQNIIQALAGARPINAANRLPKAEPAAC (SEQ ID NO: 4);

[0072] Amino acid sequence of KdPPK: MSEKQLQPFEKIELGDQTSSLKIFEEMVLKYQGQVSDEDSDSAPLPADYPYKTRMSRRVYEKEKKKLQIELLKVQSWVKESGQRIVSLFEGRDAAGKGGTIKRYMEHLNPRGARVVALEKPTETERGQWYLQRYIQNLPTAGEMVFFDRSWYNRAGVERVMGFCDPQEYLLFMRQAPELERMLVASGVHLFKFWFSVSREEQLRRFVSRYSDPLKHWKLSPVDVQSLDRWDAYTTLKNAMFFHTHTKDAPWAIIKSDDKKRARLNCIRYFLHHLDYPNKDVKAIGPVDELIVLQPNMNAVAQEGG (SEQ ID NO: 5);

[0073] Amino acid sequence of ErneRed: TAWSLLAATWSLLATAWSLLASAFLGALALLQQGPASDRGGGLEISELLQDLLRYGKTKRGSEQLPALLRLLQVPKRWFTHAYVVSVLWNGNALICLFRAEFLGESLPSWIQDTHHALGRDSHRKHKDGEHFSALLVLLLLWLHSCRRLLCCLWTSVFSNGVIHIVQYCFGLGYYIAMGSTVLCQVPTSGRNGKKLSVQICWHHIIGVMMYIWASLHQHRCLAILANLRKSRSGKVVSLSHSVPFGDWFESVGCPQYVAELLIYVSMAITLGIHNVTWWCVVMYVLFNQALAAVLCHEFYQKNFSSYPKHRKAFIPLVF (SEQ ID NO: 6);

[0074]

[0075]

[0076] Nucleotide sequence of OsEAP Ligase: atgcagctggtgagcaccattgaacagctgcgcgcgaccattaccgaaaaccatctggcgggcctgcgcattggcgtggtgccgaccatgggcaacctgcatgatggccatctggaactggtgcgccgcgcgcgcgaacgcgcggataaagtggtggtgagcatttttgtgaacccgctgcagtttgatgaagcggcggatctggatgcgtatccgcgcaccctggcgcaggattgcgaacgcctgggcgaactggcggaactggtgtttaccccgaccatgagcctgatgtatccgaacggccagcagcagcagacccgcgtgagcgtgccgggcgtgagcgaagtgctggaaggcgcgctgcgcccgggccattttctgggcgtgagcaccgtggtgtgcaaactgtttaacctggtgcagccggatctggcgtgctttggcgaaaaagattatcagcagctggcgctgattcgcaaaatgaccgcggatctgaacctgccggtggaaattattggcgtgccgaccgtgcgcgcggaagatggcctggcgctgagcagccgcaacggctatctgaccgatgcggaatttgcgctggcgccggtgctggcggcgaccatgaactggctggcggaacgcctggcggcgggcgaacgcgatctggaacagctggcggcggaaggccgcgcgcgcctggataacgcgggctttcgcaccgatggcattggcatggtggatgcggataccctgaccggcctgaccgaacatagccgcgcggtggtgattctgatggcggcgtttctgggcaaagcgcgcctgattgataaccaggtggtgcgcctgggctaa (SEQ ID NO: 9);

[0077]

[0078] Nucleotide sequence of KdPPK: atgagcgaaaaacagctgcagccgtttgaaaaaattgaactgggcgatcagaccagcagcctgaaaatttttgaagaaatggtgctgaaatatcagggccaggtgagcgatgaagatagcgatagcgcgccgctgccggcggattatccgtataaaacccgcatgagccgccgcgtgtatgaaaaagaaaaaaaaaaactgcagattgaactgctgaaagtgcagagctgggtgaaagaaagcggccagcgcattgtgagcctgtttgaaggccgcgatgcggcgggcaaaggcggcaccattaaacgctatatggaacatctgaacccgcgcggcgcgcgcgtggtggcgctggaaaaaccgaccgaaaccgaacgcggccagtggtatctgcagcgctatattcagaacctgccgaccgcgggcgaaatggtgttttttgatcgcagctggtataaccgcgcgggcgtggaacgcgtgatgggcttttgcgatccgcaggaatatctgctgtttatgcgccaggcgccggaactggaacgcatgctggtggcgagcggcgtgcatctgtttaaattttggtttagcgtgagccgcgaagaacagctgcgccgctttgtgagccgctatagcgatccgctgaaacattggaaactgagcccggtggatgtgcagagcctggatcgctgggatgcgtataccaccctgaaaaacgcgatgttttttcatacccataccaaagatgcgccgtgggcgattattaaaagcgatgataaaaaacgcgcgcgcctgaactgcattcgctattttctgcatcatctggattatccgaacaaagatgtgaaagcgattggcccggtggatgaactgattgtgctgcagccgaacatgaacgcggtggcgcaggaaggcggctaa (SEQ ID NO: 11);

[0079] EreneRed nucleotide sequence: accgcgtggagcctgctggcggcgacctggagcctgctggcgaccgcgtggagcctgctggcgagcgcgtttctgggcgcgctggcgctgctgcagcagggcccggcgagcgatcgcggcggcggcctggaaattagcgaactgctgcaggatctgctgcgctatggcaaaaccaaacgcggcagcgaacagctgccggcgctgctgcgcctgctgcaggtgccgaaacgctggtttacccatgcgtatgtggtgagcgtgctgtggaacggcaacgcgctgatttgcctgtttcgcgcggaatttctgggcgaaagcctgccgagctggattcaggatacccatcatgcgctgggccgcgatagccatcgcaaacataaagatggcgaacattttagcgcgctgctggtgctgctgctgctgtggctgcatagctgccgccgcctgctgtgctgcctgtggaccagcgtgtttagcaacggcgtgattcatattgtgcagtattgctttggcctgggctattatattgcgatgggcagcaccgtgctgtgccaggtgccgaccagcggccgcaacggcaaaaaactgagcgtgcagatttgctggcatcatattattggcgtgatgatgtatatttgggcgagcctgcatcagcatcgctgcctggcgattctggcgaacctgcgcaaaagccgcagcggcaaagtggtgagcctgagccatagcgtgccgtttggcgattggtttgaaagcgtgggctgcccgcagtatgtggcggaactgctgatttatgtgagcatggcgattaccctgggcattcataacgtgacctggtggtgcgtggtgatgtatgtgctgtttaaccaggcgctggcggcggtgctgtgccatgaattttatcagaaaaactttagcagctatccgaaacatcgcaaagcgtttattccgctggtgttttaa (SEQ ID NO: 12);

[0080] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market. The present application is further described below in combination with examples:

[0081] Example 1 Fermentation production of enzymes

[0082] The gene sequences corresponding to the synthetic glycine aldolase (BaAldo), enaminoketol reductase (WsCarRed), pentenoate ligase (OsEAPLigase), olefin reductase (EreneRed), phosphite dehydrogenase (PspPTDH), and polyphosphate kinase (KdPPK) were cloned into the pET28a plasmid through NdeI / XhoI enzyme cutting sites, and the plasmid was transformed into E. coli (BL21) cells for plate culture, and finally single colonies were selected for liquid step-by-step scale-up culture.

[0083] The basic process of liquid step-by-step scale-up culture is as follows: first, the single colony on the plate is transferred into 5 mL of LB culture solution containing 50 μM kanamycin (37 o C) for culture. When the cells grow to the logarithmic phase, they are inoculated into 250 mL of LB culture solution containing the same antibiotic, and finally transferred into a 5 L culture fermentation tank for culture. When the OD 600 of the cells is 25, 0.25 mM IPTG28 o C is added to induce protein expression for 8 h, and then the wet cells 25-40 g are collected by centrifugation (4000 rpm, 25 min). To verify the expression of the enzyme, first, a small amount of cells is mixed with Tris-HCl buffer (50 mM, pH 7.5), and then the cells are broken by freeze-thawing, and the supernatant is obtained after high-speed centrifugation to run SDS-PAGE protein gel (sodium dodecyl sulfate-polyacrylamide gel) to determine the soluble expression of the protein. After confirming the correct remaining cells, they are mixed with the buffer (10 g of wet cells are mixed with about 200 mL of the above buffer), and then the cells are broken by high pressure and centrifuged at high speed (16000 rpm, 15 min) to remove the cell wall. Finally, the obtained enzyme-containing clear liquid (crude enzyme liquid) is determined for enzyme unit activity (U is the amount of enzyme required to convert 1 μmol of substrate in 1 min at room temperature) (as shown in Table 1) and then directly used for subsequent use, or further purified and used for immobilization (solid enzyme reaction).

[0084] Table 1. Information on the properties of the enzymes used

[0085]

[0086] Example 2 Preparation of enaminoketol with propionaldehyde, glycine and phosphite as raw materials, three mixed liquid enzymes (BaAldo, WsCarRed, PspPTDH)

[0087] In 1 L of 25 mM pH 7.5 Tris-HCl solution, 5.6 g of propionaldehyde (100 mM), 8.3 g of glycine (110 mM), 0.7 g of NAD (1 mM) and 17.4 g of phosphite (220 mM) were added to adjust the pH of the solution to 7.5, and then 2000 U of BaAldo crude enzyme solution, 4000 U of WsCarRed crude enzyme solution and 4000 U of PspPTDH crude enzyme solution were added to start the reaction, and the reaction was carried out for 4 h under 30 + C Light stirring, the pH of the reaction system was maintained between 6.5 and 8.5 by using acid and alkali during the reaction; after 4 h, the enzymes in the reaction solution were precipitated by adding acid, and then the enzymes were removed by rapid centrifugation (10000 rpm, 10 min), and then the solution was adjusted to pH 7.0, and NAD and other phosphorus-containing impurities were removed by using D201 anion exchange resin, and the product-containing effluent was purified by using D101 non-polar resin for collection, and finally the crude product was desalted, concentrated and crystallized (ethanol:H2O=3:1, V:V) by using a reverse osmosis membrane to obtain 10.8 g of enaminoketol white solid (final yield 92%). o C Light stirring, the pH of the reaction system was maintained between 6.5 and 8.5 by using acid and alkali during the reaction; after 4 h, the enzymes in the reaction solution were precipitated by adding acid, and then the enzymes were removed by rapid centrifugation (10000 rpm, 10 min), and then the solution was adjusted to pH 7.0, and NAD and other phosphorus-containing impurities were removed by using D201 anion exchange resin, and the product-containing effluent was purified by using D101 non-polar resin for collection, and finally the crude product was desalted, concentrated and crystallized (ethanol:H2O=3:1, V:V) by using a reverse osmosis membrane to obtain 10.8 g of enaminoketol white solid (final yield 92%).

[0088] Example 3 Synthesis of dienamide intermediate with enaminoketol, pentenoic acid and metaphosphoric acid as raw materials, and liquid enzyme (OsEAPLigase, KdPPK)

[0089] In 1 L of 25 mM pH 7.5 Tris-HCl solution, 11.7 g of enaminoketol (100 mM), 11.0 g of pentenoic acid (110 mM), 22.0 g of sodium hexametaphosphate (36 mM), 0.6 g of ATP (1 mM) and 4.1 g of magnesium chloride hexahydrate (20 mM) were added to adjust the pH of the solution to 7.5, and then 3000 U of OsEAPLigase crude enzyme solution and 2000 U of KdPPK crude enzyme solution were added to start the reaction, and the reaction was carried out for 2 h under 30 o C Light stirring, the pH of the reaction system was maintained between 6.5 and 8.5 by using acid and alkali during the reaction; after 4 h, the enzymes in the reaction solution were precipitated by adding acid, and then the enzymes were removed by rapid centrifugation (10000 rpm, 10 min), and then the solution was adjusted to pH 7.0, and NAD and other phosphorus-containing impurities were removed by using D201 anion exchange resin, and the product-containing effluent was purified by using D101 non-polar resin for collection, and finally the crude product was desalted, concentrated and crystallized (ethanol:H2O=3:1, V:V) by using a reverse osmosis membrane to obtain 10.8 g of enaminoketol white solid (final yield 92%).

[0090] Example 4 Preparation of enaminoketol with enaminoketol and C15 olefin as raw materials, metal catalyst cross-metathesis reaction

[0091] In 1 L of mixed solvent of methanol and toluene (V:V=2:1), 40 g of dienamide intermediate (200 mM) and 19 g of C15 olefin (450 mM) were added, and then 100 mg of Hoveyda-Grubbs (Sigma) metal catalyst was added to start the reaction; the reaction was carried out at 30 o After stirring for 2 h, 500 mL of ice water was added to terminate the reaction; then the mixture was extracted with ethyl acetate 3 times (500 mL each time), the organic solution was reserved and rotary evaporation was performed to remove the solvent, and finally the crude product was crystallized (ethanol:H2O=5:1, V:V) to obtain 82.2 g of olefin ceramide white solid (final yield 71%).

[0092] Example 5 Preparation of ceramide NP using olefin ceramide and sodium phosphite as raw materials and liquid mixed enzyme (EreneRed, PspPTDH)

[0093] In 750 mL of 25 mM pH 7.5 Tris-HCl solution, 250 mL of isopropyl alcohol was added, and then 58 g of olefin ceramide (100 mM), 11.5 g of sodium phosphite (110 mM) and 0.7 g of NAD + (1 mM) were added, and then the pH of the solution was adjusted to 7.5; then 6000 U of EreneRed crude enzyme solution and 4000 U of PspPTDH crude enzyme solution were added to start the reaction; the reaction was carried out at 30 o C with slight stirring, the pH of the reaction system was maintained between 6.5 and 8.5 during the reaction; after 6 h, the reaction was terminated, the enzyme in the reaction solution was precipitated by adding acid, and then the enzyme precipitate was removed by rapid centrifugation (10000 rpm, 10 min), and then the solution was adjusted to pH 7.0 and NAD and other phosphorus-containing impurities were removed by using D201 anion exchange resin, the product-containing effluent was purified and collected by using D101 non-polar resin, and finally the crude product was desalted, concentrated and crystallized (ethanol:H2O=5:1, V:V) by using reverse osmosis membrane to obtain 50 g of ceramide white solid (final yield 85%).

[0094] Example 6 Preparation of ceramide NP using olefin ceramide and sodium phosphite as raw materials and liquid mixed enzyme (WTEreneRed, WTPspPTDH)

[0095] Similar to Example 5 above, the enzyme was replaced by natural enzyme during the preparation process.

[0096] Similarly, in 750 mL of 25 mM pH 7.5 Tris-HCl solution, 250 mL of isopropyl alcohol was added, and then 29 g of olefin ceramide (50 mM), 5.7 g of sodium phosphite (55 mM) and 0.7 g of NAD +(1 mM) and the pH of the solution was adjusted back to 7.5; then 5000 U of EreneRed crude enzyme and 5000 U of PspPTDH crude enzyme were added to initiate the reaction; 30 o C Light stirring, the pH of the reaction system was maintained between 6.5 and 8.5 by using acid and alkali during the reaction; after 12 h, the reaction was terminated, the enzyme in the reaction solution was precipitated by adding acid, and the enzyme precipitate was removed by rapid centrifugation (10000 rpm, 10 min); then the solution was adjusted to pH 7.0, and NAD and other phosphorus-containing impurities were removed by using D201 anion exchange resin; the product-containing effluent was collected by using D101 nonpolar resin for purification; finally, the crude product was purified by using a silica gel column chromatography (eluent n-hexane: ethyl acetate = 3:1 to 1:2) to obtain 7.6 g of ceramide NP brown solid (final yield 13%).

[0097] The above is only the preferred embodiment of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A glycine aldolase variant, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. A combination enzyme, characterized in that, The combined enzymes are: the glycine aldolase variant, enaminopentanol reductase variant, pentenoic acid ligase variant, olefin reductase variant, phosphite dehydrogenase variant, and polyphosphate kinase variant as described in claim 1. The amino acid sequence of the enaminopentyl reductase variant is shown in SEQ ID NO:2; The amino acid sequence of the pentenoic acid ligase variant is shown in SEQ ID NO:3; The amino acid sequence of the olefin reductase variant is shown in SEQ ID NO:6; The amino acid sequence of the phosphorylated dehydrogenase variant is shown in SEQ ID NO:4; The amino acid sequence of the polyphosphate kinase variant is shown in SEQ ID NO:

5.

3. A biomaterial, characterized in that, Includes biomaterial 1 or biomaterial 2; The biomaterial 1 includes one of the following shown in I) to V): I) The nucleic acid encoding the glycine aldolase variant of claim 1; II) Expression units containing nucleic acids as shown in I); III) Recombinant vectors containing nucleic acids as shown in I) or expression units as shown in II); IV) Integrating the genome into the nucleic acid shown in I) or the expression unit shown in II), or transfecting or transforming the host cell with the recombinant vector shown in III); V) Cultures obtained by culturing host cells as shown in IV); The biomaterial 2 includes one of the following i) to v): i) The nucleic acid encoding the combinatorial enzyme of claim 2; ii) Expression units containing nucleic acids as shown in i); iii) Recombinant vectors containing nucleic acids as shown in i) or expression units as shown in ii); iv) Integrating the genome into the nucleic acid shown in i) or the expression unit shown in ii), or transfecting or transforming the host cell with the recombinant vector shown in iii); v) Cultures obtained by culturing host cells as shown in iv) 4. The biomaterial according to claim 3, characterized in that, The recombinant vector's backbone is derived from pET28a.

5. The biomaterial according to claim 3, characterized in that, The host cells include Escherichia coli (BL21) and / or Escherichia coli DH5α.

6. The application of one of the following (I) to (II) in the preparation of ceramide NP: I) The combined enzyme according to claim 2; II) Biomaterial 2 in any one of claims 3 to 5.

7. A method for preparing ceramide NP, characterized in that, The synthesis of ceramide NP using the combined enzyme of claim 2 comprises the following steps: Step 1: Using acrolein and glycine as substrates and NAD+ as a cofactor, and utilizing the glycine aldolase variant, enaminopentanol reductase variant, and phosphite dehydrogenase variant in the combined enzyme described in claim 2, enaminopentanol is synthesized. Step 2: Using enaminopentanol, pentenoic acid, and metahexaphosphate as raw materials, and ATP and magnesium ions as cofactors, diene amide is synthesized using the pentenoic acid ligase variant and polyphosphate kinase variant in the combined enzyme described in claim 2. Step 3: Using diene amide and C15 olefin as raw materials, cross metathesis reaction is carried out using a metal catalyst to obtain olefin ceramide; Step 4: Using olefin ceramide and sodium phosphite as raw materials, and NAD+ as a cofactor, ceramide NP is synthesized using the olefin reductase variant and phosphite dehydrogenase variant in the combined enzyme described in claim 2. The metal catalyst is Hoveyda-Grubbs.

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