Method for synthesizing acetyl coenzyme A by multi-enzyme cascade catalysis of calcium pantothenate

Abstract

The invention discloses a method for synthesizing acetyl coenzyme A by multi-enzyme cascade catalysis of calcium pantothenate, and belongs to the technical field of biological catalysis engineering. According to the invention, a new way for efficiently and catalytically synthesizing the acetyl coenzyme A by adopting a bifunctional enzyme of pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase, phosphopantetheine adenylyltransferase, dephosphorylated coenzyme A kinase, malonyl coenzyme A synthetase and malonyl coenzyme A decarboxylase in a cascade manner is adopted; a coupling system is preferably selected, D-calcium pantothenate, L-cysteine hydrochloride and malonic acid are used as substrates, and the yield of the synthesized acetyl coenzyme A reaches up to 8.3 g / L after 90 min of reaction. According to the method disclosed by the invention, the compound which is cheap and easy to obtain is used as a precursor to construct the multi-enzyme coupled recombinant escherichia coli, and the acetyl coenzyme A is catalytically synthesized, so that the synthesis cost is greatly reduced, and a solid foundation is laid for industrially, green and efficiently preparing the acetyl coenzyme A.

Description

Technical Field

[0001] The invention relates to a method for synthesizing acetyl-CoA by calcium pantothenate through multi-enzyme cascade catalysis, and belongs to the technical field of biocatalytic engineering. Background Art

[0002] Acetyl-CoA is a key metabolic intermediate in cells. It is the product of oxidative decarboxylation of pyruvate produced after fatty acid β-oxidation and glycolysis. It is directly involved in the metabolic processes of tricarboxylic acid cycle (TCA), fatty acids, sugars and amino acids. Acetyl-CoA is widely used in the industry for the production of various high-value products such as fatty acids, polyketides, terpenoids and flavonoids, and is of great significance in medicine, antibiotics and disease treatment.

[0003] The main source of intracellular acetyl-CoA is the conversion of pyruvate. Pyruvate dehydrogenase directly catalyzes the conversion of pyruvate into acetyl-CoA. Pyruvate is catalyzed by pyruvate decarboxylase, acetaldehyde dehydrogenase, and acetyl-CoA synthetase to produce acetaldehyde, acetate, and acetyl-CoA. Pyruvate is oxidized by pyruvate ferredoxin oxidoreductase, pyruvate NADP + Oxidoreductase or pyruvate formate lyase catalyzes the conversion to acetyl-CoA. ATP-dependent citrate lyase consumes one ATP molecule to convert citrate into acetyl-CoA and oxaloacetate. Phosphoketolase converts 5'-phospho-D-xylulose into acetyl phosphate, which is then converted into acetyl-CoA under the catalysis of phosphotransacetylase. Acetoacetaldehyde dehydrogenase directly converts acetaldehyde into acetyl-CoA. There are also non-natural acetyl-CoA synthesis pathways, such as directed evolution of glycolaldehyde synthase to condense two formaldehyde molecules into one glycolaldehyde, converting glycolaldehyde into acetyl phosphate through phosphokinase, and finally using PTA to catalyze the synthesis of acetyl-CoA. Luo et al. designed and constructed a new natural CO 2 The fixed pathway, i.e., the reductive tricarboxylic acid branch / 4-hydroxybutyryl-CoA / ethylmalonyl-CoA / acetyl-CoA cycle, can synthesize 1150 μM (0.93 g / L) acetyl-CoA from 200 μM pyruvate, and the yield and synthesis efficiency need to be further improved.

[0004] It is difficult to accumulate acetyl-CoA in high concentrations by methods such as metabolic pathway modification. The efficient synthesis of the high-value product acetyl-CoA from the cheap substrate calcium pantothenate through enzymatic reactions is of great significance for its industrial production. Summary of the invention

[0005] As an intermediate metabolite, acetyl-CoA has the problems of being difficult to accumulate at high concentrations in cells, low specificity in chemical synthesis, and difficulty in obtaining donors for biological synthesis. The present invention provides a method for synthesizing acetyl-CoA by multi-enzyme cascade catalysis, which is applied to the production of acetyl-CoA.

[0006] The invention provides a method for synthesizing coenzyme A. The method comprises the following steps: adding calcium pantothenate, cysteine, CTP or a mixture of CTP and ATP to an enzyme reaction system containing pantothenate kinase derived from Staphylococcus aureus, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme derived from Escherichia coli, phosphopantothenylthiothioate adenylate transferase derived from Corynebacterium ammoniagenes and dephospho-coenzyme A kinase derived from Escherichia coli, and then catalytically preparing coenzyme A.

[0007] In one embodiment of the present invention, the enzyme reaction system is obtained by mixing the obtained pantothenate kinase, phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme, phosphopantheine adenylyl transferase, and dephospho-CoA kinase; or by using recombinant Escherichia coli to overexpress any one or more of pantothenate kinase, phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme, phosphopantheine adenylyl transferase, and dephospho-CoA kinase. After culturing, the bacterial cells are collected, and the crude enzyme solution is obtained by crushing the bacterial cells or purifying them to obtain a pure enzyme solution.

[0008] In one embodiment of the present invention, the NCBI number of the pantothenate kinase is: WP_000862728.1, the NCBI number of the phosphopanthenoylcysteine ​​synthetase-phosphopanthenoylcysteine ​​decarboxylase bifunctional enzyme is: WP_000050139.1, the NCBI number of the phosphopanthenoylthiothioate adenylate transferase is: WP_040356089.1, and the NCBI number of the dephospho-CoA kinase is: WP_001269520.1.

[0009] In one embodiment of the present invention, the added amount of calcium pantothenate is 2.5-10 mM, the added amount of cysteine ​​is 15-60 mM, the added amount of CTP is 20-80 mM, and the added amount of ATP is 35-140 mM.

[0010] In one embodiment of the present invention, the reaction system further contains metal ions magnesium ions or manganese ions, and the amount of the metal ions added is: 0-10 mM;

[0011] In one embodiment of the present invention, the reaction conditions are: temperature: 25-50° C., pH: 5.0-10.5.

[0012] In one embodiment of the present invention, the amount of pantothenate kinase added is 0.1-100 μM; the amount of phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme added is 0.1-100 μM; the amount of phosphopantotheine adenylate transferase added is 0.1-100 μM; the amount of dephospho-CoA kinase added is 0.1-100 μM; preferably, the amount of genetically engineered bacteria added is 5-25 g / L.

[0013] The invention provides a method for synthesizing acetyl-CoA. The method comprises the following steps: adding calcium pantothenate, cysteine, malonate, CTP or a mixture of CTP and ATP to an enzyme reaction system containing pantothenate kinase derived from Staphylococcus aureus, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme derived from Escherichia coli, phosphopantothenylthiothioylamine adenylate transferase derived from Corynebacterium ammoniagenes, dephospho-CoA kinase derived from Escherichia coli, acyl-CoA synthetase derived from Streptomyces sp. and malonyl-CoA decarboxylase derived from Rhizobium sp., and then catalytically preparing acetyl-CoA.

[0014] In one embodiment of the present invention, the amino acid sequence of the pantothenate kinase SaCoaA is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of the phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme CoaBC is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of the phosphopantetheine adenylate transferase CoaD is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; the amino acid sequence of the dephospho-CoA kinase CoaE is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8; the amino acid sequence of the malonyl-CoA synthetase SsACS is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10; the amino acid sequence of the malonyl-CoA decarboxylase RsMCD is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0015] SEQ ID NO.1

[0016] MKVGIDAGGTLIKIVQEQDNQRTFKTELTKNIDQVVEWLNQQQIEKLCLTGGNAGVIAENINIPAQIF

[0017] VEFDAASQGLGILLKEQGHDLADYIFANVGTGTSLHYFDGQSQRRVGGIGTGGGMIQGLGYLLSQIT

[0018] DYKQLTDMAQHGDRNTIDLKVRHIYKDTEPPIPGDLTAANFGHVLHHLDADFTPSNKLAAVIGVVG

[0019] EVVTTMAITVAREFKTENIVYIGSSFHNNALLRKVVEDYTVLRGCKPYYVENGAFSGAIGALYLGKSEQID NO.2

[0020] atgaaagtaggtatcgacgctggcggcactctgattaaaatcgttcaagaacaggacaatcagcgcacctttaagaccgagctgaccaaaaacatcgaccag

[0021] gtagtcgaatggctgaaccaacagcagatcgaaaaactgtgcctgaccggtggtaacgcaggtgttatcgcggaaaacatcaacatcccggcccagatcttc

[0022] gtggagttcgacgcggcaagccagggtctgggtatcctgctgaaagaacaaggtcatgatctggctgactacatcttcgcgaacgttggcacgggtacttccc

[0023] tgcactacttcgatggtcagagccagcgtcgtgtcggtggcattggcacgggcggtggtatgatccaaggcctgggttacctgctgtcccagatcactgacta

[0024] caaacagctgaccgacatggcgcagcatggcgaccgtaatactatcgatctgaaagtgcgccacatttacaaagatactgaaccgccaatcccgggtgatct

[0025] gaccgcagccaacttcggtcacgttctgcaccacctggatgccgacttcaccccatccaacaaactggccgccgtgatcggcgtagttggcgaagtggttact

[0026] accatggccattaccgttgctcgcgaattcaagaccgagaacatcgtctacattggttccagcttccacaataacgcgctgctgcgtaaagtggtggaagattac

[0027] accgtgctgcgtggttgtaaaccgtactacgtcgaaaacggcgctttctctggcgcgattggcgcactgtacctgggcaaa

[0028] SEQ ID NO.3

[0029] MSLAGKKIVLGVSGGIAAYKTPELVRRLRDRGADVRVAMTEAAKAFITPLSLQAVSGYPVSDSLLDP

[0030] AAEAAMGHIELGKWADLVILAPATADLIARVAAGMANDLVSTICLATPAPVAVLPAMNQQMYRAAA

[0031] TQHNLEVLASRGLLIWGPDSGSQACGDIGPGRMLDPLTIVDMAVAHFSPVNDLKHLNIMITAGPTRE

[0032] PLDPVRYISNHSSGKMGFAIAAAAARRGANVTLVSGPVSLPTPPFVKRVDVMTALEMEAAVNASVQ

[0033] QQNIFIGCAAVADYRAATVAPEKIKKQATQGDELTIKMVKNPDIVAGVAALKDHRPYVVGFAAETNN

[0034] VEEYARQKRIRKNLDLICANDVSQPTQGFNSDNNALHLFWQDGDKVLPLERKELLGQLLLDEIVTR

[0035] YDEKNRR

[0036] SEQ ID NO.4

[0037] atgagcctggcgggtaaaaaaattgttctgggtgtttccggtggcatcgcagcctacaaaacgccggaactggtacgtcgcctgcgtgatcgtggtgcggatg

[0038] tacgtgtagctatgacggaagcggcaaaagcgttcatcaccccgctgtccctgcaggcagtttctggttatccggtatccgatagcctgctggacccggctgct

[0039] gaagctgctatgggtcacattgagctgggtaagtgggcggatctggttatcctggcgccagctacggccgatctgatcgcccgtgtagcggcaggcatggct

[0040] aatgacctggtgtctaccatttgcctggcaaccccggcaccagtagcagtgctgccggctatgaatcagcaaatgtaccgtgctgctgcgactcagcacaatct

[0041] ggaagttctggcatcccgtggcctgctgatctggggtccagatagcggttctcaggcatgtggtgatatcggcccaggtcgcatgctggacccactgaccatc

[0042] gttgacatggccgtcgctcatttctccccagtgaacgacctgaaacacctgaacattatgattaccgcgggtccaacccgtgaaccactggacccggttcgtta

[0043] catctccaaccactcttctggtaaaatgggcttcgctatcgcagcagcagcagctcgccgtggtgctaacgttacgctggtttctggtccagtctccctgccgac

[0044] cccaccattcgtaaagcgcgttgacgtgatgaccgcgctggaaatggaagcagcggtgaacgcttctgtgcagcagcagaacatctttattggctgtgctgcg

[0045] gtggctgattaccgtgccgctaccgtagctccggagaagatcaagaaacaggcgactcagggtgacgaactgaccatcaaaatggtaaaaaacccagacat

[0046] tgtagcgggtgtggcggccctgaaagaccaccgtccgtacgtggtaggttttgcggcggagaccaacaacgtggaagaatacgctcgtcagaagcgtatcc

[0047] gcaaaaatctggacctgatctgcgcaaacgatgtctctcagccgactcaaggtttcaatagcgacaacaacgctctgcacctgttttggcaagatggtgacaaa

[0048] gtactgccgctggaacgtaaagagctgctgggtcaactgctgctggatgaaatcgtgacccgctacgatgaaaaaaaccgccgtSEQ ID NO.5

[0049] MTKAVCPGSFDPMTLGHLDIFNRSAALFDEVTILVTGNPNKQTGLFSIDERVDLINQVIDQPNIHVDT

[0050] WAGLLVDYTTENNIDAIVKGLRSSLDYEYELPMAQMNKRLSGIDTVLLLTDEKYGYISSSLCKEVAK

[0051] YGGDISGFFPEPVGQAVLEKYRA

[0052] SEQ ID NO.6

[0053] atgaccaaagcggtttgtccgggtagcttcgatccgatgaccctgggtcacctggatatcttcaaccgtagcgcggcgctgttcgatgaagttaccatcctggtt

[0054] accggtaacccgaacaaacagaccggcctgttcagcatcgatgaacgtgttgatctgatcaaccaggttatcgatcagccgaacatccacgttgatacctggg

[0055] cgggcctgctggttgattacaccaccgaaaacaacatcgatgcgatcgttaaaggcctgcgtagcagcctggattacgaatacgaactgccgatggcgcaga

[0056] tgaacaaacgtctgtctggcatcgataccgttctgctgctgaccgatgaaaaatacggctacatctctagcagcctgtgcaaagaagttgcgaaatacggcggt

[0057] gatatcagcggcttcttcccggaaccggttggccaggcggttctggaaaaataccgtgcg

[0058] SEQ ID NO.7

[0059] MRYIVALTGGIGSGKSTVANAFADLGINVIDADIIARQVVEPGAPALHAIADHFGANMIAADGTLQRR

[0060] ALRERIFANPEEKNWLNALLHPLIQQETQHQIQQATSPYVLWVVPLLVENSLYKKANRVLVVDVSPE

[0061] TQLKRTMQRDDVTREHVEQILAAQATREARLAVADDVIDNNGAPDAIASDVARLHAHYLQLASQFV

[0062] SQEKP

[0063] SEQ ID NO.8

[0064] atgcgttacatcgtggctctgactggcggcatcggttctggtaaatctacggtggctaacgctttcgctgatctgggcatcaacgtcatcgacgcagacatcatt

[0065] gcacgtcaggtcgtcgaaccaggcgcaccggctctgcacgcaatcgcagaccactttggtgcgaacatgattgcggcggatggtaccctgcagcgtcgtgc

[0066] gctgcgcgaacgtatctttgctaacccggaggaaaagaactggctgaacgcgctgctgcacccgctgatccagcaggagacccagcaccagattcagcag

[0067] gccacctctccgtacgttctgtgggttgttccgctgctggtggagaattctctgtataaaaaggcgaaccgcgtgctggtcgttgacgtaagcccggagaccca

[0068] gctgaagcgtacgatgcaacgcgatgacgtaacccgcgaacacgttgagcagatcctggctgcacaggcaacccgtgaagctcgtctggcagtcgccgac

[0069] gatgttatcgacaacaacggtgctccggatgcgattgcctccgacgtggctcgtctgcatgctcattatctgcagctggcgtcccaattcgtttcccaagaaaaa

[0070] cca

[0071] SEQ ID NO.9

[0072] MSFLFPALVGGPGERSGTGVPPDEVRGRVALRFGVRSLTYGQLAAAAGAVAGRVRGAERVAVWATPE

[0073] LETAVAVVGTLLAGRAAVPLNPKSGDKELAHILTDSAPTAVLAAPGAELPAAFAGLQRIDVDAAAVG

[0074] ALPEDRTAESDPALVVYTSGTTGPPKGAVIPRRALAATLDALADAWQWTGEDVLVHGLPLFHVHGL

[0075] VLGILGPLRRGGSVRHLGRFDTAGVARELNDGATMLFGVPTMYHRIAEALPTDPALAKALGRARLL

[0076] VSGSAALPVHDHERIAAATGRRVVERYGMTETLMNTSVRADGEARAGTVGLPLPGVELRLVEEDGT

[0077] AIAAYDGETVGEIQVRGPNLFTEYLNRPDATAAAFTPDGWFRTGDMAVREADGYVRIVGRKATDLI

[0078] KSGGYKIGAGEIENALLEHPGVREAAVTGEPDPDLGERIVAWVVPAEPQSPPSAEELADHVAGRLAP

[0079] HKRPRVVRYLDALPRNDMGKIMKRALGA

[0080] SEQ ID NO.10

[0081] atgtcattcttatttcccgctctagtaggaggcccgggtgagcgctcgggcaccggtgtgccgcctgatgaagttcgtggtcgtgttgcgctccgcttcggcgt

[0082] gcgtagcctgacgtacggtcagctggcggcggcggcgggcgcggtggccggtcgtgtgagaggcgctgagcgtgttgctgtttgggcaacgccggaact

[0083] ggaaaccgctgtggcggtcgtgggcactctgctggccggcagagcagcggtgccgctgaacccgaagtccggtgataaagaactggcccatattttgacc

[0084] gacagcgcaccaacggcggtgttagcggctccgggtgcagaattaccggcggcttttgctggccttcagcgtatcgatgtcgatgctgctgcggttggtgcgt

[0085] tgccggaagatcgtaccgccgagagcgatccggcactagtcgtgtataccagcggcacgaccggtccgccgaagggtgcggtgattccgcgtcgcgctct

[0086] ggctgcgacgctggatgccttggccgatgcgtggcaatggaccggagaagacgttctggtgcacggcctgccgctttttcatgttcacggtttagttttgggca

[0087] tcctgggtccgctccgtcgcggcggtagcgttcgtcatctgggtcgctttgataccgcaggcgttgcccgtgagctgaacgatggtgcgaccatgctgttcggt

[0088] gttccgaccatgtatcatcgtatcgcggaggcgttgccgacggacccagctcttgccaaagcactgggccgcgcgcgtctgctggtgagcggttctgccgct

[0089] ctgcctgttcacgatcacgaacgtatcgctgcggcgaccggccgtcgtgtagtcgagcgctacggcatgaccgaaaccctgatgaacacctctgttcgagcg

[0090] gacggcgaggcacgtgcgggcactgttggtctgccgttgcctggcgtggaactgcgtctggttgaagaggacggtactgcgattgcggcttatgatgggga

[0091] gacggtgggtgagattcaggtgcgtggtccgaatctgttcaccgaatacctgaaccgtccggacgcgaccgcggcggcgttcaccccggacggctggtttc

[0092] gtaccggtgacatggcagtgcgtgaggcggacggttatgttcgcatcgtcggtcggaaggcgactgacctgattaagtccggcggttacaagattggcgccg

[0093] gtgagatcgaaaatgcattgctggagcacccgggtgttcgcgaagcggccgtgaccggcgaaccggacccggatctgggtgagcgcatcgttgcatgggt

[0094] tgtccccgcggagccgcaaagcccgccaagtgcggaggaactggcagaccacgtggccggccgtctggcaccgcataaacgtccgcgcgtcgtgcgcta

[0095] cctggacgctctgccgcgtaatgacatgggtaaaatcatgaaacgagcgttgggcgct

[0096] SEQ ID NO.11

[0097] MLQSITDRGRQLLFSGSRMPQIAAEADLHTLCEMLLSSRGEASGMALAAEIFDRWSTLGGDGQQAF

[0098] FRMLHEKFGPDTGRLDQAIEIYRSDKSSASIIKLHQAAESRRQELLRRLNHAPNGTAKLVRMREHLL

[0099] ASSDRSEGYHALDADFTHLFGSWFNRGFLTLRPIDWSTPASILEKIIQYEAVHEIAGWEELRRRLAPA

[0100] DRRCFAFFHPRLADEPLVFVEVALTRSMPRAIADVLDEGREQISADQATTAVFYSISNCQDGLRGISFG

[0101] NFLIKQVVDDLRRDFPGLKNFVTLSPVPGFARWLSRVRGVGADGALGDEELETLRLLDDPDWAANE

[0102] DAATEVERILLPLAARYFLVERTPEGRPLDPVARFHLGNGARLERLNFRGDRSSKAMHQAHGLMVN

[0103] YLYELEDIIANHEALAQRGEVIASTGVRSLLKKKDESRGHRPDGSRKFGQIISSTLGGGRKSEQ IDNO.12

[0104] atgctgcagagcattaccgatcgcggccgtcagctgctgtttagcggcagccgcatgccgcagattgcggcggaagcggatctgcataccctgtgcgaaat

[0105] gctgctgagcagccgcggcgaagcgagcggcatggcgctggcggcggaaatttttgatcgctggagcaccttaggcggcgatggtcagcaagcgttttttc

[0106] gcatgctgcatgaaaaatttggcccggataccggccgcctggatcaagcgattgaaatttatcgcagcgataaaagcagcgcgagcattattaaactgcatca

[0107] agcggcggaaagccgccgccaagaactgctgcgccgcctgaaccatgcgccgaacggcaccgcgaaactggtgcgcatgcgcgaacatctgctggcga

[0108] gcagcgatcgcagcgaaggctatcatgcgctggatgcggattttacccatctgtttggcagctggtttaaccgcggctttctgaccctgcgcccgattgattgga

[0109] gcaccccggcgagcattctggaaaaaattattcagtatgaagcggtgcatgaaattgcgggctgggaagaactgcgccgtcgcctggcgccggcggatcgc

[0110] cgctgctttgcgttttttcatccgcgcctggcggatgaaccgctggtgtttgtggaagtggcgctgacccgcagcatgccgcgcgcgattgcggatgtgctgga

[0111] tgaaggccgcgaacagattagcgcggatcaagcgaccaccgcggtgttttatagcattagcaactgccaagatggcctgcgcggcattagctttggcaacttt

[0112] ctgattaaacaagtggtggatgatctgcgccgcgattttccgggcctgaaaaactttgtgaccctgagcccggtgccgggttttgcgcgttggttaagccgcgt

[0113] gcgcggcgtgggcgcggatggcgcgctgggcgatgaagaactggaaaccctgcgcctgctggatgatccggattgggcggcgaacgaagatgcggcga

[0114] ccgaagtggaacgcattctgctgccgctggcggcgcgctattttctggtggagcgtaccccggaaggccgtccgttagatccggtggcgcgctttcatctggg

[0115] caacggcgcgcgcctggaacgcctgaactttcgcggcgatcgcagcagcaaagcgatgcatcaagcgcatggcctgatggtgaactatctgtatgaactgg

[0116] aagatattattgcgaaccatgaagcgctggcgcagcgcggcgaagtgattgcgagcaccggcgtgcgcagcctgctgaaaaagaaagatgaaagccgcg

[0117] gccatcgcccggatggcagccgcaaatttggtcagattattagcagtacgctgggcggtggccgtaaa

[0118] In one embodiment of the present invention, the enzyme reaction system is any one of the following (a) to (c):

[0119] (a) mixing the obtained pantothenate kinase, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme, phosphopanthene adenylate transferase, dephospho-CoA kinase, acyl-CoA synthetase and malonyl-CoA decarboxylase;

[0120] (b) using recombinant Escherichia coli to overexpress pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase and dephospho-CoA kinase to obtain a genetically engineered bacterium, and using recombinant Escherichia coli to overexpress acyl-CoA synthetase and malonyl-CoA decarboxylase to obtain a genetically engineered bacterium; after culturing, collecting the bacterial cells respectively, crushing the bacterial cells to obtain a crude enzyme solution or purifying the bacterial cells to obtain a pure enzyme solution, and mixing the obtained products;

[0121] (c) using recombinant Escherichia coli to overexpress any one or more of pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase, dephospho-CoA kinase, acyl-CoA synthetase, and malonyl-CoA decarboxylase to obtain a genetically engineered bacterium; after culturing, collecting the bacterium, crushing the bacterium to obtain a crude enzyme solution or purifying the bacterium to obtain a pure enzyme solution, that is, an enzyme reaction system.

[0122] In one embodiment of the present invention, the NCBI number of the pantothenate kinase is: WP_000862728.1, the NCBI number of the phosphopanthenoylcysteine ​​synthetase-phosphopanthenoylcysteine ​​decarboxylase bifunctional enzyme is: WP_000050139.1, the NCBI number of the phosphopanthenoylthiothioate adenylate transferase is: WP_040356089.1, and the NCBI number of the dephospho-CoA kinase is: WP_001269520.1; the GenBank number of the acyl-CoA synthetase is: MBW8796441.1, and the GenBank number of the malonyl-CoA decarboxylase is: CAH0342705.1.

[0123] In one embodiment of the present invention, the added amount of calcium pantothenate is 2.5-10 mM, the added amount of cysteine ​​is 15-60 mM, the added amount of malonic acid is 10-40 mM; the added amount of CTP is 20-80 mM; and the added amount of ATP is 35-140 mM.

[0124] In one embodiment of the present invention, the reaction system further contains metal ions magnesium ions or manganese ions, and the added amount of the metal ions is: 0.5-10mM.

[0125] In one embodiment of the present invention, the reaction conditions are: temperature: 25-50° C., pH: 5.0-8.5.

[0126] In one embodiment of the present invention, the culture method of the genetically engineered bacteria is: adding the genetically engineered bacteria to a seed culture medium to prepare a seed solution; inoculating the prepared seed solution into a fermentation medium, and obtaining a culture solution after induction under the conditions of 17-37°C and 0.1-0.5mM IPTG; and obtaining bacterial cells after centrifuging the prepared culture solution.

[0127] In one embodiment of the present invention, the amount of pantothenate kinase added is 0.1 to 100 μM; the amount of phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme added is 0.1 to 100 μM; the amount of phosphopantetheine adenylate transferase added is 0.1 to 100 μM; the amount of dephospho-CoA kinase added is 0.1 to 100 μM; the amount of acyl-CoA synthetase added is 0.1 to 100 μM; the amount of malonyl-CoA decarboxylase added is 0.1 to 100 μM.

[0128] In one embodiment of the present invention, the added amount of the genetically engineered bacteria is 5 to 25 g / L.

[0129] In one embodiment of the present invention, the genetically engineered bacteria uses E. coli BL21 (DE3), B. subtilis 168 or Pichia sp. GS115 as host cells and pET28a, pCDF-Duet-1, pRSF-Duet-1, pET21a or pGEX-6p-1 as expression vectors.

[0130] In one embodiment of the present invention, the genetically engineered bacteria is a bacteria that uses pET-28(a) and pET-21(a) as vectors to co-express the malonyl-CoA synthetase and malonyl-CoA decarboxylase in E. coli BL21(DE3); and uses pET-28(a) as a vector to express the pantothenate kinase, phosphopanthenoylcysteine ​​synthetase-phosphopanthenoylcysteine ​​decarboxylase bifunctional enzyme, phosphopanthenoylthiothioate adenylate transferase or dephospho-CoA kinase in E. coli BL21(DE3).

[0131] In one embodiment of the present invention, the genetically engineered bacteria uses pCDF-Duet-1 as a vector to co-express the pantothenate kinase, phosphopanthocyanate synthetase-phosphopanthocyanate decarboxylase bifunctional enzyme, phosphopanthocyanate adenylate transferase, and dephospho-CoA kinase in E. coli BL21 (DE3).

[0132] In one embodiment of the present invention, the culture method of the genetically engineered bacteria is: inoculating the genetically engineered bacteria into a culture medium, inducing culture at 0.5 mM IPTG and 17° C. for 16 to 20 hours.

[0133] In one embodiment of the present invention, the culture medium formula is 10 g / L peptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0134] The present invention also provides an enzyme preparation for catalyzing the synthesis of acetyl-CoA and a method for preparing acetyl-CoA by using the enzyme preparation. The enzyme preparation contains one or more of the above-mentioned pantothenate kinase, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme, phosphopanthene adenylate transferase, dephospho-CoA kinase, malonyl-CoA synthetase and malonyl-CoA decarboxylase.

[0135] In one embodiment of the present invention, the enzyme preparation is a liquid preparation, which contains one or more of the pantothenate kinase, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme, phosphopantothenylthioethylamine adenylate transferase, dephospho-CoA kinase, malonyl-CoA synthetase, malonyl-CoA decarboxylase and auxiliary materials.

[0136] Beneficial Effects

[0137] (1) The present invention expresses a series of recombinant enzymes with high enzyme activity in Escherichia coli, realizes the synthesis of high-value product acetyl-CoA from calcium pantothenate as a starting substrate, and is applied to the production of acetyl-CoA.

[0138] (2) The enzyme catalysis method was applied to the production of acetyl-CoA. D-pantothenate calcium, L-cysteine ​​hydrochloride and malonic acid were used as substrates, and the catalytic reaction was mediated by 10 g / L E.coli BL21(DE3) / pET28a-ScACS / pET21a-RsMCD and 10 g / L E.coliBL21(DE3) / pCDF-Duet-CoABCDE crude enzyme solutions. When 15 mM pantothenate was added as a substrate, the maximum yield reached 8.3 g / L in 90 minutes of reaction. Compared with chemical or biological methods for preparing acetyl-CoA, this method produces acetyl-CoA more efficiently and has a simple reaction system, which is green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1:SDS-PAGE analysis of pure enzyme, M,turecolor two-color pre-strained protein marker (wide range); 1, SaCoaA; 2, CoaBC; 3, CoaD; 4, CoaE; 5, CaCoaA.

[0140] Figure 2 : Liquid phase diagram of products catalyzed by SaCoaA, CoaBC, CoaD and CoaE.

[0141] Figure 3 :The effect of different reaction conditions on the conversion rate of pantothenic acid to coenzyme A or acetyl-CoA; A is the effect of temperature on the conversion rate of coenzyme A, B is the effect of pH on the conversion rate of coenzyme A, C is the effect of cysteine ​​concentration on the conversion rate of coenzyme A, D is the effect of ATP concentration on the conversion rate of coenzyme A, E is the effect of CTP concentration on the conversion rate of coenzyme A, F is Mg 2+ The effect of ATP concentration on the conversion rate of acetyl-CoA, H is the effect of Mg 2+ I is the effect of concentration on the conversion rate of acetyl-CoA, and I is the effect of pH on the conversion rate of acetyl-CoA.

[0142] Figure 4 : Liquid phase diagram of products catalyzed by SaCoaA, CoaBC, CoaD, CoaE, SsACS and RsMCD.

[0143] Figure 5 :The conversion rate and yield of crude enzyme solution catalyzing 5-20mM pantothenic acid to synthesize acetyl-CoA. DETAILED DESCRIPTION

[0144] Technical terms:

[0145] Expression: The term "expression" includes any step involved in the production of the enzyme including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0146] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding an enzyme described in the present invention and operably linked to control sequences that provide for its expression.

[0147] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The host cell can be any cell useful in the recombinant production of the enzyme, such as a prokaryotic cell or a eukaryotic cell. Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silvae, Neisseria, Pseudomonas, Salmonella, and Ureaplasma. The host cell can also be a eukaryotic organism, such as a mammal, insect, plant, or fungal cell.

[0148] Acetyl CoA: an organic substance with the chemical formula C 23 H 38 N 7 O 17 P 3 S is a derivative of coenzyme A.

[0149] Enzyme preparations: refers to biological products with catalytic functions after the enzymes are purified and processed. They are mainly used to catalyze various chemical reactions in the production process. They have the characteristics of high catalytic efficiency, high specificity, mild action conditions, reduced energy consumption, and reduced chemical pollution. Their application areas are in food (bread baking industry, flour deep processing, fruit processing industry, etc.), textiles, feed, detergents, papermaking, leather, medicine, energy development, environmental protection, etc. Enzyme preparations are derived from organisms and are generally safe. They can be used in appropriate amounts according to production needs.

[0150] The culture medium involved in the following examples is as follows:

[0151] Luria-Bertani (LB) medium: tryptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L.

[0152] The detection methods involved in the following embodiments are as follows:

[0153] Detection of Coenzyme A and Acetyl Coenzyme A Concentration:

[0154] Since coenzyme A and acetyl-coenzyme A have better response values ​​at UV 254nm, and calcium pantothenate has better response values ​​at 210nm, high performance liquid chromatography was used to detect coenzyme A and acetyl-coenzyme A;

[0155] Coenzyme A and acetyl-CoA were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column. Mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid, and mobile phase B was methanol containing 0.1% trifluoroacetic acid. The detection conditions were: injection of 10 μL, gradient elution of 10% to 67% B solution, time of 20 min, flow rate of 1 mL / min, column temperature of 30 ° C, and detection at UV 254 nm and 210 nm. Coenzyme A and acetyl-CoA standards were used to draw a standard curve, and the content was calculated by peak area and standard curve energy.

[0156] Calculation method of the yield of Coenzyme A: The peak area of ​​Coenzyme A is X (mAU×s) determined by the above-mentioned HPLC method, the amount of pantothenic acid input is Y (mM), and the Coenzyme A yield = X×1000 / (8254.64×767×Y), wherein 8254.64 is the slope of the Coenzyme A standard curve, and 767 is the molecular weight of Coenzyme A.

[0157] The 2.5 mM D-calcium pantothenate (5 mM pantothenic acid) mentioned in the following examples means that the concentration of calcium pantothenate in the reaction system is 2.5 mM. At this time, since calcium pantothenate is dissolved in water to obtain pantothenic acid, the concentration of pantothenic acid is 5 mM.

[0158] Example 1: Construction of recombinant Escherichia coli

[0159] The specific steps are as follows:

[0160] (1) Construction of recombinant plasmid

[0161] The pantothenate kinase SaCoaA (WP_000862728.1) from Staphylococcus aureus, the pantothenate kinase CaCoaA (GenBank: EFG81976.1) from Corynebacterium ammoniagenes, the bifunctional enzyme phosphopanthenylcysteine ​​synthetase-phosphopanthenylcysteine ​​decarboxylase CoaBC (WP_000050139.1) from Escherichia coli, the phosphopanthenylthiothioate adenylate transferase CoaD (WP_040356089.1) from Brevibacterium ammoniagenes, and the dephospho-CoA kinase CoaE (WP_001269520.1) from Escherichia coli were sent to Shanghai Biotechnology Co., Ltd. for codon optimization and then cloned into the vector pET-28a (+) with the cloning site of BamH I / Xho I. The vector resistance is kanamycin resistance, and the recombinant plasmids pET28a-SaCoaA, pET28a-CaCoaA, pET28a-CoaBC, pET28a-CoaD, and pET28a-CoaE were obtained and stored at -20°C.

[0162] (2) Construction of recombinant strains

[0163] Take 1 μL of plasmid obtained in step (1) and add it to 100 μL of E. coli BL21 (DE3) competent cell suspension. After ice bathing for 30 minutes, place it in a 42°C metal bath for heat shock for 90 seconds. After heat shock, quickly place it on ice for 3 to 5 minutes. Add 700 μL of LB liquid culture medium to the centrifuge tube and shake and culture it at 37°C, 200 rpm for 1 hour. Take 50 μL of bacterial liquid and spread it on an LB solid culture medium plate containing 50 μg / mL kanamycin sulfate. Invert it and culture it overnight in a 37°C constant temperature incubator (about 10 hours).

[0164] The recombinant strains E. coli BL21 (DE3) / pET28a-SaCoaA, E. coli BL21 (DE3) / pET28a-CaCoaA, E. coli BL21 (DE3) / pET28a-CoaBC, E. coli BL21 (DE3) / pET28a-CoaD, and E. coli BL21 (DE3) / pET28a-CoaE were prepared respectively.

[0165] At the same time, the recombinant strains expressing acyl-CoA synthetase SsACS (GenBank No. MBW8796441.1): E. coli BL21 (DE3) / pET28a-SsACS and malonyl-CoA decarboxylase RsMCD (GenBank: CAH0342705.1): E. coli BL21 (DE3) / pET28a-RsMCD were prepared according to the above method.

[0166] Example 2: Expression and purification of enzymes related to coenzyme A synthesis

[0167] The specific steps are as follows:

[0168] (1) Pick up the positive clones prepared in Example 1 and place them in 5 mL LB liquid culture medium test tubes containing 50 μg / mL kanamycin sulfate, and culture them at 37° C., 200 rpm, with shaking for about 8 h; prepare seed solutions respectively.

[0169] (2) The above seed solution was inoculated into 250 mL LB liquid medium containing 50 μg / mL kanamycin sulfate at a 1% (v / v) inoculum, and cultured at 37°C, 200 rpm, with shaking until OD 600 After reaching 0.6-0.8 (about 2 hours), the shake flask was placed in an ice-water mixture to cool for 10 minutes, and 0.5 mM IPTG was added to induce the reaction at 17°C. After about 20 hours of induction culture, the bacteria were collected by centrifugation at 8,000×g for 10 minutes and washed twice with saline.

[0170] The cells were resuspended in protein purification buffer A (100 mM HEPSE, 500 mM NaCl, 10% (w / v) glycerol, pH 7.5), and the cells were disrupted with an ultrasonic disruptor in an ice bath. The disrupted liquid was centrifuged at 10,000 × g, 4°C for 30 min to remove cell debris, and the supernatant was filtered with a 0.22 μm water filter to prepare crude enzyme solutions.

[0171] (3) The crude enzyme solution was purified using a HisTrap HP column (GE Healthcare). First, 5 column volumes were balanced with protein purification buffer A. After loading, the impurities were washed with 5% protein purification buffer B (100 mM HEPSE, 500 mM NaCl, 10% (w / v) glycerol, 1 M imidazole, pH 7.5), and then the target protein was eluted with 50% protein purification buffer B and collected.

[0172] The enzyme solution purified by the nickel column was desalted using a Sephadex G-25 desalting column to remove imidazole in the enzyme solution. First, three column volumes were washed and balanced with ultrapure water and protein purification buffer A, and then the sample was loaded. The protein was eluted with protein purification buffer A. The pure enzyme solution after desalting was subjected to SDS-PAGE electrophoresis. The band results were observed to determine whether a single purified protein of interest was obtained. The following pure enzymes were prepared: SaCoaA, CoaBC, CoaD, CoaE, and CaCoaA. The results are shown in the figure. Figure 1 shown.

[0173] The results showed that SaCoaA, CoaBC, CoaD, CoaE, and CaCoaA could be expressed soluble in Escherichia coli, and pure enzymes could be obtained after purification for subsequent experiments.

[0174] SsACS pure enzyme solution and RsMCD pure enzyme solution were prepared according to the above methods respectively.

[0175] Example 3: Synthesis of Coenzyme A

[0176] 1. Using D-calcium pantothenate as a substrate and the pure enzyme solution obtained in Example 2 as a catalyst, coenzyme A was prepared in different reaction systems;

[0177] (1) Using ATP as a coenzyme, adding different metal ions to prepare coenzyme A

[0178] Reaction system (200 μL): Add 5 mM MgCl to a reaction system containing 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM ATP, and 100 mM HEPES. 2 、MnCl 2 、FeCl 2 、CoCl 2 or MgCl 2 / MnCl 2 (MgCl 2 、MnCl 2 Added at a ratio of 1:1), and no metal ions were added as a control;

[0179] Reaction conditions: under pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. The production and yield of coenzyme A were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column; the results are shown in Table 1:

[0180] Table 1: Yields of Coenzyme A prepared with different metal ions using ATP as coenzyme

[0181]

[0182] It can be seen that the above reaction system cannot produce coenzyme A.

[0183] (2) Using GTP as a coenzyme, adding different metal ions to prepare coenzyme A

[0184] Reaction system (200 μL): Add 5 mM MgCl to a reaction system containing 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM GTP, and 100 mM HEPES. 2 、MnCl 2 、FeCl 2 、CoCl 2 or MgCl 2 / MnCl 2 (MgCl 2 、MnCl 2 Added at a ratio of 1:1), and no metal ions were added as a control;

[0185] Reaction conditions: under pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. The production and yield of coenzyme A were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column; the results are shown in Table 2:

[0186] Table 2: Yields of Coenzyme A prepared with different metal ions using GTP as coenzyme

[0187]

[0188] It can be seen that the above reaction system cannot produce coenzyme A.

[0189] (3) Using UTP as a coenzyme and adding different metal ions to prepare coenzyme A

[0190] Reaction system (200 μL): Add 5 mM MgCl to a reaction system containing 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM UTP, and 100 mM HEPES. 2 、MnCl 2 、FeCl 2 、CoCl 2 or MgCl 2 / MnCl 2 (MgCl 2 、MnCl 2 Added at a ratio of 1:1), and no metal ions were added as a control;

[0191] Reaction conditions: under pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. The production and yield of coenzyme A were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column; the results are shown in Table 3:

[0192] Table 3: Yields of Coenzyme A prepared with different metal ions using UTP as coenzyme

[0193]

[0194] It can be seen that the above reaction system cannot produce coenzyme A.

[0195] (4) Using CTP as a coenzyme and adding different metal ions to prepare coenzyme A

[0196] Reaction system (200 μL): Add 5 mM MgCl to a reaction system containing 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM CTP, and 100 mM HEPES. 2 、MnCl 2 、FeCl 2 、CoCl 2 or MgCl 2 / MnCl 2 (MgCl 2 、MnCl 2Added at a ratio of 1:1), and no metal ions were added as a control;

[0197] Reaction conditions: under pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left standing at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. The production and yield of coenzyme A were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column; the results are shown in Table 4:

[0198] Table 4: Yields of Coenzyme A prepared with different metal ions using CTP as coenzyme

[0199]

[0200] It can be seen that the reaction system with CTP as the coenzyme can detect the generation of coenzyme A, and when MnCl is added 2 The conversion rate of coenzyme A prepared by metal ions is the highest.

[0201] (5) ATP and CTP are present simultaneously as coenzymes, and different metal ions are added to prepare coenzyme A

[0202] Reaction system (200 μL): Add 5 mM MgCl to a reaction system containing 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM ATP / CTP (ATP and CTP were added at a concentration ratio of 1:1), and 100 mM HEPES. 2 、MnCl 2 、FeCl 2 、CoCl 2 or MgCl 2 / MnCl 2 (MgCl 2 、MnCl 2 Added at a concentration ratio of 1:1), and no metal ions were added as a control;

[0203] Reaction conditions: under pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left standing at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. The production and yield of coenzyme A were detected using an Agilent 1260 liquid chromatograph and a Waters C18 column; the results are shown in Table 5:

[0204] Table 5: Yields of Coenzyme A prepared with different metal ions using ATP and CTP as coenzymes

[0205]

[0206] It can be seen that the generation of coenzyme A can be detected with ATP and CTP as coenzymes, and when MgCl is added 2 or MnCl 2 When metal ions are present, the conversion rate of the prepared coenzyme A is very high.

[0207] 2. Effects of pantothenate kinase from different sources

[0208] The specific steps are as follows:

[0209] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution or 10 μM SaCoaA pure enzyme solution (prepared according to the method of Examples 1-2), 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM calcium D-pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 20 mM ATP, 20 mM CTP, 100 mM HEPES;

[0210] Reaction conditions: pH 8.0, placed in a 1.5 mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, 200 μL of methanol was added to terminate the reaction, left to stand at room temperature for 30 minutes, centrifuged at 12,000 × g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as the sample. Coenzyme A was detected using an Agilent 1260 liquid chromatograph and a Waters C18 column.

[0211] The results showed that the catalytic effect of SaCoaA (the yield of Coenzyme A was 58.3%) was better than that of CaCoaA (the yield of Coenzyme A was 41.9%), and the yield was higher.

[0212] 3. Verification of Coenzyme A

[0213] The specific steps are as follows:

[0214] The reaction system is: 200 μL, 10 μM SaCoaA, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM calcium D-pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 10 mM ATP, 10 mM CTP, 100 mM HEPES;

[0215] Reaction conditions: pH 8.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 30°C for 30 minutes, added 200μL methanol to terminate the reaction, left at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as a sample. Coenzyme A was detected using liquid chromatography-mass spectrometry. The results are as follows Figure 2 shown.

[0216] The results showed that the molecular weight of the product catalyzed by recombinant SaCoaA, CoaBC, CoaD and CoaE was the same as that of the Coenzyme A standard, and was Coenzyme A. Coenzyme A can be formed by recombinant SaCoaA, CoaBC, CoaD and CoaE.

[0217] Example 4: Optimization of conditions for coenzyme A synthesis

[0218] The reaction system was as follows (200 μL): 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM calcium D-pantothenate (5 mM pantothenic acid), 10 mM L-cysteine ​​hydrochloride, 40 mM ATP, 40 mM CTP, 100 mM HEPES;

[0219] Reaction conditions: pH 8.0, placed in a 1.5 mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, 200 μL of methanol was added to terminate the reaction, left to stand at room temperature for 30 minutes, centrifuged at 12,000 × g for 10 minutes to remove the denatured protein precipitate, and the supernatant was taken as the sample. Coenzyme A was detected using an Agilent 1260 liquid chromatograph and a Waters C18 column.

[0220] 1. Optimization of optimal temperature

[0221] The optimal reaction temperature for the synthesis of coenzyme A was determined under different temperature conditions. The specific reaction system was the same as above, except that the reaction temperature was adjusted to 25°C, 30°C, 35°C, 40°C, and 50°C, respectively, and the pH was 8.0. The conversion rate of coenzyme A measured under different temperature reactions is shown in the figure below. Figure 3 As shown in A.

[0222] The results showed that the conversion rate of pantothenic acid to coenzyme A was the highest at 35°C (58.7%).

[0223] 2. Optimization of the optimal pH

[0224] The optimal reaction temperature for the synthesis of coenzyme A was determined under different pH conditions. The specific reaction system was the same as above, except that the pH of the reaction was adjusted to 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, and 10.5, respectively, and the reaction was carried out at 35°C. The conversion rates of coenzyme A measured under different pH reactions are shown in Figure 2. Figure 3 As shown in B.

[0225] The results showed that the conversion rate of pantothenic acid to coenzyme A was the highest (75.6%) at pH 7.0.

[0226] 3. Optimization of substrate concentration

[0227] (1) Cysteine ​​concentration

[0228] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 0-25 mM L-cysteine ​​hydrochloride, 40 mM ATP, 40 mM CTP, 100 mM HEPES, 35°C, pH 7.0; the conversion rate of coenzyme A measured at different cysteine ​​concentrations is shown in Figure 3 As shown in C.

[0229] The results showed that the conversion rate of coenzyme A was relatively high when the cysteine ​​concentration was greater than 5 mM, and the conversion rate of coenzyme A was the highest (76.3%) when the cysteine ​​concentration was 15 mM.

[0230] (2) ATP concentration

[0231] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 0-80 mM ATP, 40 mM CTP, 100 mM HEPES, 35 °C, pH 7.0; the conversion rate of coenzyme A measured at different ATP concentrations is shown in Figure 3 As shown in D.

[0232] The results showed that the conversion rate of coenzyme A was relatively high when the ATP concentration was 20-40 mM, and the conversion rate of coenzyme A was the highest (78.6%) when the ATP concentration was 20 mM.

[0233] (3) CTP concentration

[0234] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 5 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 20 mM ATP, 0-80 mM CTP, 100 mM HEPES, 35 °C, pH 7.0; the conversion rate of coenzyme A measured at different CTP concentrations is shown in Figure 3 As shown in E.

[0235] The results showed that the conversion rate of coenzyme A was relatively high when the CTP concentration was 10-30 mM, and the conversion rate of coenzyme A was the highest (91.4%) when the CTP concentration was 20 mM.

[0236] (4)MgCl 2 concentration

[0237] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 0-10 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 20 mM ATP, 20 mM CTP, 100 mM HEPES, 35 °C, pH 7.0; the conversion rate of coenzyme A measured at different CTP concentrations is shown in Figure 3 As shown in F.

[0238] The results showed that in MgCl 2 The conversion rate of coenzyme A is relatively high when the concentration is 3-4 mM. 2 The conversion rate of coenzyme A was the highest (93.3%) when the concentration was 4 mM.

[0239] Example 5: Synthesis of acetyl-CoA by multi-enzyme cascade catalysis

[0240] Using calcium pantothenate and cysteine ​​as starting substrates, the products obtained after catalysis by cascade CoA synthesis-related enzymes SaCoaA, CoaBC, CoaD, CoaE, as well as acyl-CoA synthetase SsACS and malonyl-CoA decarboxylase RsMCD were verified and identified. The specific steps are as follows:

[0241] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 10 μM SsACS pure enzyme solution, 10 μM RsMCD pure enzyme solution, 3 mM MgCl2 , 2.5 mM calcium D-pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 20 mM ATP, 20 mM CTP, 10 mM malonic acid, 100 mM HEPES;

[0242] Reaction conditions: pH 7.0, placed in a 1.5mL centrifuge tube, reacted in a constant temperature metal bath at 35°C for 30 minutes, added 200μL methanol to terminate the reaction, left at room temperature for 30 minutes, centrifuged at 12,000×g for 10 minutes to remove the denatured protein precipitate, and took the supernatant as a sample. Acetyl CoA was detected by high performance liquid chromatography. The results are as follows Figure 4 shown.

[0243] The results showed that acetyl-CoA can be synthesized from pantothenic acid through the joint catalysis of pantothenate kinase, phosphopantothenylcysteine ​​synthetase, phosphopantothenylcysteine ​​decarboxylase-phosphopantothenylthioethylamine adenylate transferase bifunctional enzyme, dephospho-CoA kinase, malonyl-CoA synthetase, and malonyl-CoA decarboxylase.

[0244] Example 6: Optimization of conditions for acetyl-CoA synthesis

[0245] (1) ATP concentration

[0246] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 10 μM SsACS pure enzyme solution, 10 μM RsMCD pure enzyme solution, 3 mM MgCl 2 , 2.5mM D-calcium pantothenate (5mM pantothenic acid), 15mM L-cysteine ​​hydrochloride, 20-70mM ATP, 20mM CTP, 10mM malonic acid, 100mM HEPES; the conversion rate of acetyl-CoA measured at different ATP concentrations is as follows Figure 3 As shown in G.

[0247] The results showed that the conversion rate of acetyl-CoA was higher when the ATP concentration was 35-70 mM, and the conversion rate of acetyl-CoA was the highest (78.2%) when the ATP concentration was 35 mM.

[0248] (2)MgCl 2 concentration

[0249] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 10 μM SsACS pure enzyme solution, 10 μM RsMCD pure enzyme solution, 0.5-10 mM MgCl 2, 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 35 mM ATP, 20 mM CTP, 10 mM malonic acid, 100 mM HEPES, 35 °C, pH 7.0; the conversion rate of acetyl-CoA measured at different ATP concentrations is shown in Figure 3 As shown in H.

[0250] The results showed that in MgCl 2 The conversion rate of acetyl-CoA is higher when the concentration is 2-5 mM, and the conversion rate of acetyl-CoA is higher when the concentration is 2-5 mM. 2 The conversion rate of acetyl-CoA was the highest (78.9%) when the concentration was 3 mM.

[0251] (3) pH value

[0252] The reaction system is: 200 μL, 10 μM SaCoaA pure enzyme solution, 10 μM CoaBC pure enzyme solution, 10 μM CoaD pure enzyme solution, 10 μM CoaE pure enzyme solution, 10 μM SsACS pure enzyme solution, 10 μM RsMCD pure enzyme solution, 3 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 35 mM ATP, 20 mM CTP, 10 mM malonic acid, 100 mM HEPES, 35 ° C, pH 5.5-8.5; the conversion rate of acetyl-CoA measured at different ATP concentrations is as follows Figure 3 As shown in I.

[0253] The results showed that the conversion rate of acetyl-CoA was higher when the pH was 6-7.5, and the conversion rate of acetyl-CoA was the highest (90.0%) when the pH was 6.6.

[0254] Example 7: Construction of a coenzyme A synthetase recombinant co-expression strain

[0255] 1. Construction of genetically engineered bacteria co-expressing SaCoaA, CoaBC, CoaD, and CoaE

[0256] (1) Construction of recombinant plasmid pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE

[0257] Table 6: Primers used in the construction of pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE

[0258]

[0259] The reaction system for PCR amplification is: 2× PrimeSTAR 25 μL, plasmid 1 μL, ddH 2O 10μL, primers 2μL each. PCR program: 98℃5min; 98℃30s, 55℃30s, 72℃30s, 30 cycles; 72℃10min, 16℃10min.

[0260] (2) After the PCR is completed, add 5.5 μL of 10× Quickcut Buffer to each PCR tube and mix well. Use 1% agarose gel electrophoresis to verify whether the band size is correct, and use a kit to recover the gel to obtain the DNA fragment and linearized vector.

[0261] (3) The obtained DNA fragment and linearized vector were homologously recombined using the Vazyme single fragment homologous recombination kit according to the instructions. The homologous recombination system was added to 100 μL E. coli BL21 (DE3) competent cells. After ice bathing for 30 minutes, it was placed in a 42°C metal bath for heat shock for 90 seconds. After heat shock, it was quickly placed on ice for 3 to 5 minutes. 700 μL LB liquid culture medium was added to the centrifuge tube and cultured at 37°C, 200 rpm, and shaken for 1 hour. Centrifuge at 6,000×g and 4°C for 5 minutes, collect the cells, pour off the culture medium, and resuspend the cells after about 50 to 100 μL is left.

[0262] Take 50 μL of bacterial solution and spread it on a LB solid culture medium plate containing 50 μg / mL streptomycin. Invert it in a 37°C constant temperature incubator and culture overnight (about 10 hours). Pick a single colony in a 5mL LB liquid culture medium test tube containing 50 μg / mL streptomycin, 37°C, 200rpm, shake culture for 10-12 hours, extract the plasmid for sequencing. Obtain the recombinant plasmid pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE, and further obtain the recombinant strain E.coli BL21(DE3) / pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE.

[0263] 2. Preparation of recombinant bacteria co-expressing acyl-CoA synthetase SsACS and malonyl-CoA decarboxylase RsMCD

[0264] (1) Take 1 μL of pET21a-RsMCD and pET28a-SsACS plasmids (preparation method is the same as the above embodiment), which are preserved in this laboratory, and add them to 100 μL of E. coli BL21 (DE3) competent cell suspension. After ice bathing for 30 minutes, place them in a 42°C metal bath for heat shock for 90 seconds. After heat shock, quickly place them on ice for 3-5 minutes. Add 700 μL of LB liquid culture medium to the centrifuge tube, and shake and culture at 37°C, 200 rpm for 1 hour. Take 50 μL of bacterial solution and spread it on an LB solid culture medium plate containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin sodium. Invert and culture overnight in a 37°C constant temperature incubator (about 10 hours) to prepare the recombinant bacteria E. coli BL21 (DE3) / pET28a-ScACS / pET21a-RsMCD.

[0265] (2) Pick single colonies from the solid culture medium plates and place them in 5 mL LB liquid culture tubes containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin sodium. Incubate at 37°C, 200 rpm, with shaking for about 8 to 10 h to obtain seed solution.

[0266] (3) The above seed solution was inoculated into 250 mL LB liquid medium containing 50 μg / mL kanamycin sulfate and 100 μg / mL ampicillin sodium at a 1% (v / v) inoculum volume, and cultured at 37°C, 200 rpm, with shaking until OD 600 After reaching 0.6-0.8 (about 2 hours), the shake flask was placed in an ice-water mixture to cool for 10 minutes, 0.5 mM IPTG was induced at 17°C, and after about 20 hours of induction culture, the bacteria were collected by centrifugation at 6,000×g for 10 minutes and washed twice with saline.

[0267] 3. Construction of recombinant strains co-expressing SaCoaA, CoaBC, CoaD, CoaE, acyl-CoA synthetase SsACS and malonyl-CoA decarboxylase RsMCD

[0268] (1) Construction of recombinant plasmid pRSF-Duet-SsACS-RsMCD

[0269] Table 7: Primers used in the construction of pRSF-Duet-SsACS-RsMCD

[0270]

[0271] The reaction system for PCR amplification is: 2× PrimeSTAR 25 μL, plasmid 1 μL, ddH 2O 10μL, primers 2μL each. PCR program: 98℃5min; 98℃30s, 55℃30s, 72℃30s, 30 cycles; 72℃10min, 16℃10min.

[0272] (2) After the PCR is completed, add 5.5 μL of 10× Quickcut Buffer to each PCR tube and mix well. Use 1% agarose gel electrophoresis to verify whether the band size is correct, and use a kit to recover the gel to obtain the DNA fragment and linearized vector.

[0273] (3) The obtained DNA fragment and linearized vector were homologously recombined using the Vazyme single fragment homologous recombination kit according to the instructions. The homologous recombination system was added to 100 μL E. coli BL21 (DE3) competent cells. After ice bathing for 30 minutes, it was placed in a 42°C metal bath for heat shock for 90 seconds. After heat shock, it was quickly placed on ice for 3 to 5 minutes. 700 μL LB liquid culture medium was added to the centrifuge tube and cultured at 37°C, 200 rpm, and shaken for 1 hour. Centrifuge at 5,000×g and 4°C for 5 minutes, collect the cells, pour off the culture medium, and resuspend the cells after about 50 to 100 μL is left.

[0274] Take 50 μL of bacterial solution and spread it on a LB solid medium plate containing 50 μg / mL kanamycin sulfate. Invert it in a 37°C constant temperature incubator and culture it overnight (about 10 hours). Pick a single colony in a 5mL LB liquid medium test tube containing 50 μg / mL kanamycin sulfate, shake and culture it at 37°C, 200 rpm for 10-12 hours, extract the plasmid and sequence it. Get the recombinant plasmid pRSF-Duet-SsACS-RsMCD.

[0275] (4) Take 1 μL of pRSF-Duet-SsACS-RsMCD and pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE plasmids respectively, add them to 100 μL of E. coli BL21 (DE3) competent cell suspension, place them in an ice bath for 30 minutes, heat shock them in a 42°C metal bath for 90 seconds, and quickly place them on ice for 3-5 minutes. Add 700 μL of LB liquid culture medium to the centrifuge tube, shake and culture at 37°C, 200 rpm for 1 hour. Take 50 μL of bacterial solution and spread it on an LB solid culture medium plate containing 50 μg / mL kanamycin sulfate and 50 μg / mL streptomycin. Invert and culture in a 37°C constant temperature incubator overnight (about 10 hours) to prepare the recombinant bacteria E. coli BL21 (DE3) / pRSF-Duet-SsACS-RsMCD / pCDF-Duet-SaCoaA-CoaBC-CoaD-CoaE.

[0276] Example 8: Synthesis of Acetyl-CoA Catalyzed by Crude Enzyme Solution

[0277] In order to reduce industrial production costs, the crude enzyme solution after ultrasonic disruption of the recombinant strain cells was used for catalytic reaction.

[0278] (1) Preparation of crude enzyme solution

[0279] The cells of the recombinant strain (Table 8) were resuspended in protein purification buffer A (100 mM HEPSE, 500 mM NaCl, 10% (w / v) glycerol, pH 7.5) (the final concentration of the cells was 50 g / L), and the cells were disrupted with an ultrasonic disruptor under ice bath conditions. The disrupted liquid was centrifuged at 10,000 × g and 4°C for 30 min to remove cell debris, and the supernatant was filtered with a 0.22 μm water filter to obtain crude enzyme solutions.

[0280] (2) Synthesis of acetyl-CoA catalyzed by crude enzyme solution

[0281] The reaction system is: 3mM MgCl 2 , 2.5mM D-calcium pantothenate (5mM pantothenic acid), 15mM L-cysteine ​​hydrochloride, 20mM CTP, 35mM ATP, 10mM malonic acid, 100mM HEPES, pH 6.6, 35℃; the amount of crude enzyme solution added is 20g / L corresponding to the amount of wet bacteria; 100rpm, reaction for 20min.

[0282] Different recombinant strains (as shown in Table 8, the construction method of the strains is the same as the above embodiment) were selected to prepare crude enzyme solution for addition, and the addition amount of crude enzyme solution was the corresponding wet bacterial amount, and the corresponding bacterial amount was 20 g / L. The addition amount of crude enzyme solution prepared by different strains in the same reaction system was the same.

[0283] Table 8: Effect of different protein addition methods on low yield

[0284]

[0285] The results showed that when the crude enzyme solution of the recombinant strain E.coli / pCDF-DueT-CoaA-CoaD-CoaBC-CoaE / pRSFDueT-ScACS-RsMCD co-expressing SaCoaA, CoaBC, CoaD, CoaE, acyl-CoA synthetase SsACS and malonyl-CoA decarboxylase RsMCD was added, the conversion rate was low, only 23.2%. When the crude enzyme solutions of E.coli / pCDF-DueT-SaCoaA-CoaD-CoaBC-CoaE and E.coli / pET28a-ScACS / pET21a-RsMCD were added at the same time for catalysis, the conversion rate was 90.7%, which was comparable to the effect of other addition methods.

[0286] Example 9: Synthesis of acetyl-CoA from high-concentration substrates by multi-enzyme cascade catalysis

[0287] Effects of different substrate concentrations on the preparation of acetyl-CoA using E. coli / pCDF-DueT-SaCoaA-CoaD-CoaBC-CoaE and E. coli / pET28a-ScACS / pET21a-RsMCD; the specific steps are as follows:

[0288] 1. Preparation of crude enzyme solution

[0289] The cells of the recombinant strain were resuspended in protein purification buffer A (100 mM HEPSE, 500 mM NaCl, 10% (w / v) glycerol, pH 7.5) (final cell concentration was 50 g / L), the cells were disrupted with an ultrasonic disruptor in an ice bath, the disrupted liquid was centrifuged at 10,000 × g and 4°C for 30 min to remove cell debris, and the supernatant was filtered with a 0.22 μm water filter to obtain crude enzyme solutions.

[0290] 2. The reaction system is as follows:

[0291] (1) 3 mM MgCl 2 , 2.5 mM D-calcium pantothenate (5 mM pantothenic acid), 15 mM L-cysteine ​​hydrochloride, 35 mM ATP, 20 mM CTP, 10 mM malonic acid, 100 mM HEPES, crude enzyme solution, pH 6.6, 35 °C;

[0292] (2) 3 mM MgCl 2 , 5 mM D-calcium pantothenate (10 mM pantothenic acid), 30 mM L-cysteine ​​hydrochloride, 70 mM ATP, 40 mM CTP, 20 mM malonic acid, 100 mM HEPES, crude enzyme solution, pH 6.6, 35 °C;

[0293] (3) 3 mM MgCl 2 , 7.5 mM D-calcium pantothenate (15 mM pantothenic acid), 45 mM L-cysteine ​​hydrochloride, 105 mM ATP, 60 mM CTP, 30 mM malonic acid, 100 mM HEPES, crude enzyme solution, pH 6.6, 35 °C;

[0294] (4) 3 mM MgCl 2 , 10 mM D-calcium pantothenate (20 mM pantothenic acid), 60 mM L-cysteine ​​hydrochloride, 140 mM ATP, 20-80 mM CTP, 40 mM malonic acid, 100 mM HEPES, crude enzyme solution, pH 6.6, 35°C;

[0295] The amount of crude enzyme solution added was 10 g / L E. coli BL21 (DE3) / pET28a-ScACS / pET21a-RsMCD and 10 g / L E. coli BL21 (DE3) / pCDF-Duet-CoABCDE corresponding to the wet bacterial volume; 100 rpm, reaction time 0-120 min, the test results were as follows Figure 5 The results showed that as the substrate concentration increased, the conversion rate decreased. When 15 mM pantothenic acid was added as the substrate, the maximum yield reached 8.3 g / L after 90 min of reaction.

[0296] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for synthesizing coenzyme A, characterized in that: The method comprises the following steps: adding calcium pantothenate, cysteine, CTP or a mixture of CTP / ATP to an enzyme reaction system containing pantothenate kinase derived from Staphylococcus aureus, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme derived from Escherichia coli, phosphopantothenylthiothioate adenylate transferase derived from Corynebacterium ammoniagenes and dephospho-CoA kinase derived from Escherichia coli, and then catalytically preparing CoA.

2. The method according to claim 1, characterized in that The enzyme reaction system is: The mixture is obtained by mixing pantothenate kinase, phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme, phosphopanthene adenylate transferase, and dephospho-CoA kinase; Or the genetically engineered bacteria obtained by overexpressing one or more enzymes selected from pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase, and dephospho-CoA kinase are cultured, and the crude enzyme solution is collected or the purified enzyme solution is mixed to obtain the obtained product.

3. The method according to claim 1 or 2, characterized in that: The amino acid sequence of the pantothenate kinase is shown in SEQ ID NO.1, the amino acid sequence of the bifunctional enzyme phosphopantetheine synthetase-phosphopantetheine decarboxylase is shown in SEQ ID NO.3, the amino acid sequence of the phosphopantetheine adenylate transferase is shown in SEQ ID NO.5, the amino acid sequence of the dephospho-CoA kinase is shown in SEQ ID NO.7, the amino acid sequence of the malonyl-CoA synthetase is shown in SEQ ID NO.9, and the amino acid sequence of the malonyl-CoA decarboxylase is shown in SEQ ID NO.11; Preferably, the amount of calcium pantothenate added is 2.5-10 mM, the amount of cysteine ​​added is 15-60 mM; the amount of CTP added is 20-80 mM; the amount of ATP added is 35-140 mM; Preferably, the reaction system further contains metal ions magnesium ions or manganese ions, and the amount of the metal ions added is: 0-10mM; Preferably, the reaction conditions are: temperature: 25-50°C, pH: 5.0-10.

5.

4. The method according to any one of claims 1 to 3, characterized in that: The amount of pantothenate kinase added is 0.1-100 μM; the amount of phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme added is 0.1-100 μM; the amount of phosphopantotheine adenylate transferase added is 0.1-100 μM; the amount of dephospho-CoA kinase added is 0.1-100 μM; Preferably, the added amount of the genetically engineered bacteria is 5 to 25 g / L.

5. A method for synthesizing acetyl-CoA, characterized in that: The method comprises the following steps: adding calcium pantothenate, cysteine, malonate, CTP or a mixture of CTP / ATP to an enzyme reaction system containing pantothenate kinase derived from Staphylococcus aureus, phosphopantothenylcysteine ​​synthetase-phosphopantothenylcysteine ​​decarboxylase bifunctional enzyme derived from Escherichia coli, phosphopantothenylthioethylamine adenylate transferase derived from Corynebacterium ammoniagenes, dephospho-CoA kinase derived from Escherichia coli, acyl-CoA synthetase derived from Streptomyces sp. and malonyl-CoA decarboxylase derived from Rhizobium sp., and then catalytically preparing acetyl-CoA.

6. The method according to claim 5, characterized in that The enzyme reaction system is any one of the following (a) to (c): (a) a mixture of pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase, dephospho-CoA kinase, acyl-CoA synthetase and malonyl-CoA decarboxylase; (b) overexpressing pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase, and dephospho-CoA kinase, and overexpressing acyl-CoA synthetase and malonyl-CoA decarboxylase; culturing and collecting crude enzyme solutions or purifying and mixing the purified enzyme solutions; (c) Genetically engineered bacteria are obtained by overexpressing one or more enzymes of pantothenate kinase, phosphopantetheine synthetase-phosphopantetheine decarboxylase bifunctional enzyme, phosphopantetheine adenylate transferase, dephospho-CoA kinase, acyl-CoA synthetase, and malonyl-CoA decarboxylase; after culturing, crude enzyme solution is collected or purified to obtain pure enzyme solution, which is then mixed to obtain an enzyme reaction system.

7. The method according to claim 5 or 6, characterized in that: The amino acid sequence of the pantothenate kinase is shown in SEQ ID NO.1, the amino acid sequence of the bifunctional enzyme phosphopantetheine synthetase-phosphopantetheine decarboxylase is shown in SEQ ID NO.3, the amino acid sequence of the phosphopantetheine adenylate transferase is shown in SEQ ID NO.5, the amino acid sequence of the dephospho-CoA kinase is shown in SEQ ID NO.7, the amino acid sequence of the malonyl-CoA synthetase is shown in SEQ ID NO.9, and the amino acid sequence of the malonyl-CoA decarboxylase is shown in SEQ ID NO.11; Preferably, the amount of calcium pantothenate added is 2.5-10 mM, the amount of cysteine ​​added is 15-60 mM, the amount of malonic acid added is 10-40 mM; the amount of CTP added is 20-80 mM; the amount of ATP added is 35-140 mM; Preferably, the reaction system further contains metal ions magnesium ions or manganese ions, and the amount of the metal ions added is: 0.5-10mM; Preferably, the reaction conditions are: temperature: 25-50°C, pH: 5.0-8.

5.

8. The method according to any one of claims 1 to 7, characterized in that: The method for culturing the genetically engineered bacteria comprises: adding the genetically engineered bacteria to a seed culture medium to prepare a seed solution; inoculating the prepared seed solution into a fermentation culture medium, and obtaining a culture solution after culturing and induction at 17-37° C. and 0.1-0.5 mM IPTG.

9. The method according to claim 8, characterized in that The amount of pantothenate kinase added is 0.1-100 μM; the amount of phosphopantotheine synthetase-phosphopantotheine decarboxylase bifunctional enzyme added is 0.1-100 μM; the amount of phosphopanthene adenylate transferase added is 0.1-100 μM; the amount of dephospho-CoA kinase added is 0.1-100 μM; the amount of acyl-CoA synthetase added is 0.1-100 μM; the amount of malonyl-CoA decarboxylase added is 0.1-100 μM; Preferably, the added amount of the genetically engineered bacteria is 5 to 25 g / L.

10. The method according to claim 9, characterized in that The genetic engineering bacteria use E.coliBL21 (DE3), B.subtilis 168 or Pichia sp.GS115 as host cells and pET28a, pCDF-Duet-1, pRSF-Duet-1, pET21a or pGEX-6p-1 as expression vectors.

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