Engineered Lysine Decarboxylase for the Preparation of 1,5-Diaminopentane

Abstract

The present invention provides an engineered lysine decarboxylase that can be used to synthesize 1,5-diaminopentane under industrially relevant conditions. The present invention also provides a polynucleotide encoding the engineered lysine decarboxylase, a host cell capable of expressing the engineered lysine decarboxylase, and a method for preparing 1,5-diaminopentane using the engineered lysine decarboxylase. The engineered lysine decarboxylase of the present invention was developed from a wild-type lysine decarboxylase through a creative process of directed evolution, and the engineered lysine decarboxylase of the present invention has better activity and / or stability and can tolerate higher substrate concentrations than other lysine decarboxylases in the preparation of 1,5-diaminopentane, and therefore has good prospects for industrial application.

Description

[Technical Field]

[0001] The present invention relates to the field of bioengineering technology, and in particular to a genetically engineered lysine decarboxylase and a method for preparing 1,5-diaminopentane therefrom. [Background technology]

[0002] 1,5-Diaminopentane, also known as cadaverine, is a natural five-carbon nitrogen-containing base with wide applications in agriculture, medicine, and chemistry. In agriculture, 1,5-diaminopentane participates in various physiological processes in crops, promoting amphidromic development and regulating plant senescence. In medicine, 1,5-diaminopentane is a precursor for the synthesis of quinolizine, an effective drug for the treatment of dysentery. In chemical engineering, polymer compounds polymerized from 1,5-diaminopentane monomers can be used as chelating agents and curing agents in a wide range of industrial applications. Furthermore, the annual global demand for high-performance polyamides exceeds 6 million tons, and their prices are high. In this context, it has been found that 1,5-diaminopentane can be polymerized with sebacic acid to form polyamide PA510, which has superior performance compared to polyamides derived from conventional petrochemical synthesis. Therefore, market demand for 1,5-diaminopentane is rapidly growing due to its wide range of applications and excellent properties.

[0003] In previous work, Chong Li and colleagues at Northwestern University produced 1,5-diaminopentane by chemical hydrogenation of 1,5-pentaninitrile in the presence of an amorphous nickel catalyst at 3 MPa and 70°C, yielding a 66.8% pure product. Chemical synthesis of 1,5-diaminopentane has low product selectivity, harsh preparation conditions, high costs, and pollution. Furthermore, the chemical method requires non-renewable petroleum as a raw material, posing the dilemma of scarcity and rising petroleum prices. Therefore, the development of a bioenzymatic method for the synthesis of 1,5-diaminopentane is crucial and an inevitable trend in the pursuit of carbon neutrality.

[0004] Currently, biosynthetic methods for 1,5-diaminopentane mainly involve microbial fermentation and whole-cell conversion. Microbial fermentation typically suffers from long fermentation periods and low yields. Furthermore, the microbial fermentation system is complex and contains many impurities, making product isolation and purification difficult and resulting in increased production costs. Instead, whole-cell conversion, unlike microbial fermentation, concentrates strains through high-density fermentation culture, increasing the amount of enzymes used as catalysts to convert the substrate lysine into 1,5-diaminopentane. This system has the advantage of fewer impurities and easier purification.

[0005] However, the drawback of the whole-cell conversion method is the narrow profit margin between the substrate L-lysine and the product 1,5-diaminopentane. Therefore, an economically effective enzyme-catalyzed process suitable for industrialization requires high concentrations of L-lysine and the ability to completely convert L-lysine to 1,5-diaminopentane; otherwise, production costs will rise and profits will decrease. Most studies on the whole-cell conversion of 1,5-diaminopentane are based on the lysine decarboxylases CadA or LdcC from Escherichia coli. Patent US7189543B2 discloses a method for producing 1,5-diaminopentane by adjusting the pH with dibasic acid and converting lysine through cellular overexpression of a wild-type lysine decarboxylase peptide. However, the drawback of this method is the very low substrate concentration of only 50 g / L and the substrate supply to enzyme dosage ratio (substrate-to-enzyme ratio) of less than 7.5. Initial bacterial OD 600A study reported using genetically engineered E. coli XL1-Blue bacteria overexpressing LdcC, increasing the initial lysine concentration to 200 g / L at 50°C. However, the drawbacks were the long reaction time of 120 hours of catalytic reaction required to obtain 133.75 g / L of 1,5-diaminopentane, and the low product concentration. Patent CN107164352B also disclosed several CadA mutants, including a genetically engineered bacterium (M176V / Y230H / K44R) with a dry weight of 0.2 g / L, capable of converting 84% of 25 g / L of substrate after 4 hours at 37°C. The drawback of this method is that the product concentration is still too low, making it unsuitable for large-scale industrialization. Patents CN102851307A and CN104762336A disclose a genetically engineered lysine decarboxylase derived from the bacterium Hafnia alvei, which is used to catalyze the decarboxylation of L-lysine to produce 1,5-diaminopentane. The input of lysine hydrochloride was increased to 450 g / kg, and the substrate-to-enzyme ratio was approximately 10. After 48 hours of catalytic reaction, 251 g / kg of 1,5-diaminopentane was obtained, resulting in significant improvements in overall catalytic efficiency and temporal and spatial yield. However, the drawback is that the activity of the lysine decarboxylase is insufficient, resulting in long catalytic reaction times and inefficient production, leading to high production costs. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a genetically engineered lysine decarboxylase polypeptide. Under conditions where the feed dose of the substrate, L-lysine hydrochloride, reached 650 g / L, a very small amount of wet cells expressing the genetically engineered lysine decarboxylase polypeptide (enzyme to substrate ratio of 100:1) was used, and the substrate was completely converted to 1,5-diaminopentane after only 6 hours of catalytic reaction. The present invention also provides gene and protein sequences of the genetically engineered lysine decarboxylase polypeptide, recombinant expression vectors containing the genes, genetically engineered bacterial strains, and efficient methods for their preparation, as well as a reaction process for preparing 1,5-diaminopentane using the genetically engineered polypeptide.

[0007] The first aspect of the present invention provides an improved genetically engineered lysine decarboxylase polypeptide. This genetically engineered polypeptide is derived from a wild-type lysine decarboxylase modified by an artificially directed evolutionary process involving a specific number of mutations, such as amino acid substitutions, insertions, or deletions. The wild-type lysine decarboxylase is derived from Plesiomonas shigelloides, and its sequence is set forth in SEQ ID NO: 2. This wild-type lysine decarboxylase has low activity, low thermostability, and product inhibition toward 1,5-diaminopentane. Tests conducted by the inventors have shown that when the lysine decarboxylase of SEQ ID NO: 2 was used in the following reaction, after 2 hours of reaction (reaction temperature: 35°C-40°C) and a loading level of 450 g / L of L-lysine hydrochloride, no further 1,5-diaminopentane was produced, and the conversion rate did not exceed 20%. [ka]

[0008] The present invention provides improved engineered lysine decarboxylase polypeptides that have increased activity and / or stability, overcome product inhibition, and can more efficiently catalyze the production of 1,5-diaminopentane from L-lysine compared to the wild-type lysine decarboxylase corresponding to SEQ ID NO:2. These improved lysine decarboxylase polypeptides can comprise an amino acid sequence that differs by one or more residues from SEQ ID NO:2 at residue positions corresponding to X5, X11, X12, X13, X24, X48, X85, X95, X99, X108, X111, X116, X119, X143, X144, X145, X316, X334, X368, X378, X383, X422, X440, X441, X445, X516, X521, X551, X561, X591, X595, X693, X694, X701, X710. The improved lysine decarboxylase polypeptides include A5V, G11A, G11C, G11P, G11R, G11T, G11S, A12E, A12V, A12R, A12L, A12T, A12Q, A12K, F13I, F13W, F13V, F13P, F13Y, A24K, N48S, T85D, D95G, N99D, N99A, N99G, N99Y, N99T, N99S, N99Q, D108Q, S111I, K116Q, Q119P, Q119D, Q119R, G143P, K144L, K144F, K144Q, K144R, K144E, Y145 and an amino acid sequence comprising at least one of the following features: D, Y145V, Y145W, Y145L, D316E, V334I, L368C, I378V, D383E, K422Q, K440R, K441Q, K441R, E445Y, E445L, E445A, K516R, R521L, R521M, R551L, R551Q, R551N, D561E, Q591G, Q591S, K595R, I693V, H694Q, D701G, K710Q, or an insertion or deletion of 1, 2, 3, 4, 5 or more amino acid residues based on these differences.

[0009] More specifically, in some embodiments, the engineered lysine decarboxylase polypeptides that improve SEQ ID NO:2 are selected from the group consisting of SEQ ID NOs:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 , 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170.

[0010] In some embodiments, the improved engineered lysine decarboxylase polypeptide is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114 , 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, and the like.

[0011] The identity of two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the technical field, and can be calculated using NCBI Blastp and Blastn software based on default parameters, or by using the Clustal W algorithm (Nucleic Acid Research, 22(22):4673-4680, 1994). For example, when the Clustal W algorithm is used, the identity of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 68 is 99.3%.

[0012] In another aspect, the present invention provides polynucleotide sequences encoding engineered lysine decarboxylase polypeptides. In some embodiments, the polynucleotide can be part of an expression vector having one or more regulatory sequences for expressing the engineered lysine decarboxylase polypeptide. In some embodiments, the polynucleotide can be selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 138 7, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169.

[0013] As those skilled in the art know, due to degeneration of nucleotide codons, the sequences of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 8, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, The polynucleotide sequences encoding the 170 amino acid sequences are set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169 The nucleic acid sequences encoding the lysine decarboxylase of the present invention are SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 , 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, or any other nucleic acid sequence encoding the amino acid sequence shown in

[0014] In another aspect, the disclosure provides expression vectors and host cells comprising polynucleotides encoding or capable of expressing engineered lysine decarboxylases. In some embodiments, the host cells can be bacterial host cells, such as E. coli. The host cells can be used to express and isolate the engineered lysine decarboxylases described herein, or, optionally, can be used directly to reactively convert substrates to products.

[0015] In some embodiments, the engineered lysine decarboxylase in the form of a whole cell, a crude extract, an isolated polypeptide, or a purified polypeptide may be used alone or in immobilized form (e.g., immobilized on a resin).

[0016] The present disclosure also provides methods for converting an L-lysine compound represented by structural formula (II) to a 1,5-diaminopentane compound represented by structural formula (I) using the engineered lysine decarboxylase polypeptides disclosed herein. [ka] [ka]

[0017] Specific embodiments of engineered lysine decarboxylase polypeptides for use in the methods are provided further in the detailed description. Improved engineered lysine decarboxylase polypeptides that can be used in the above methods include those set forth in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, , 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170.

[0018] Any of the methods using engineered peptides to prepare compounds of Formula (I) disclosed herein can be carried out within a range of suitable reaction conditions, including, but not limited to, pH, temperature, substrate dosage, peptide dosage, cofactor dosage, and reaction time. For example, in some embodiments, preparation of compounds of Formula (I) can be carried out under suitable reaction conditions including: (a) a substrate compound (II) concentration of about 10 g / L to about 650 g / L; (b) an engineered polypeptide concentration of about 0.1 g / L to about 50 g / L; (c) a PLP cofactor concentration of about 0.1 mM to about 5 mM; (d) a pH of about 5.5 to about 8.5; (e) a temperature of about 10°C to 60°C; and (f) a reaction time of about 1 hour to about 12 hours. DETAILED DESCRIPTION OF THE INVENTION

[0019] Improved engineered lysine decarboxylase The engineered lysine decarboxylase polypeptides developed according to the present invention are set forth below in Table 1. Each row provides the polynucleotide and amino acid sequence numbers for a particular engineered lysine decarboxylase polypeptide, as well as the residue difference from SEQ ID NO: 2. The level of catalytic performance (overall performance in the reaction, activity, stability, and ability to overcome product inhibition) of each exemplary engineered lysine decarboxylase polypeptide is indicated by a "+", the specific meaning of which is provided in Table 2. JPEG2026507552000004.jpg27170JPEG2026507552000005.jpg255164JPEG2026507552000006.jpg255170JPEG2026507552000007.jpg100170

[0020] The amino acid sequences shown in Table 1 (i.e., including sequence identifiers SEQ ID NOS:2-170) all contain 711 amino acid residues. The wet cells set forth in Table 2 contain approximately the same amount of expression of the engineered lysine decarboxylase polypeptide protein.

[0021] The wild-type enzyme SEQ ID NO: 2 has low activity, low thermal stability, and is subject to product inhibition. The improved engineered lysine decarboxylase polypeptides provided by the present invention have higher activity, higher stability, overcome product inhibition, and catalyze a higher conversion of L-lysine to 1,5-diaminopentane than the wild-type lysine decarboxylase corresponding to SEQ ID NO: 2.

[0022] Polynucleotides, control sequences, expression vectors and host cells that can be used to produce engineered lysine decarboxylase polypeptides In another aspect, the present disclosure provides polynucleotides encoding the engineered polypeptides having lysine decarboxylase activity described herein. The polynucleotides can be operatively linked to one or more heterologous regulatory sequences that control gene expression to produce a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered lysine decarboxylase can be introduced into a suitable host cell to express the corresponding engineered lysine decarboxylase polypeptide.

[0023] As will be apparent to those skilled in the art, the availability and knowledge of protein sequences with respect to the codons corresponding to various amino acids provides examples of all possible polynucleotides encoding the target protein sequence. The redundancy of the genetic code allows for the generation of numerous polynucleotides encoding the improved lysine decarboxylase polypeptides disclosed herein, since the same amino acid may be encoded by alternative or synonymous codons. Thus, once a particular amino acid sequence is determined, one skilled in the art can generate a variety of polynucleotides simply by altering the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates all possible variations of polynucleotides that can be made by selecting combinations based on possible codon choices, any polypeptide disclosed herein, including the amino acid sequences of the exemplary engineered polypeptides shown in Table 1, and the sequence identifiers of any polypeptide disclosed in the sequence listing as even-numbered sequences of SEQ ID NOS: 4-170, incorporated herein by reference, and all such variations are considered specifically disclosed.

[0024] In various embodiments, codons are preferred and selected according to the host cell in which the recombinant protein will be produced, for example, bacterial-preferred codons are used for gene expression in bacteria, yeast-preferred codons are used for gene expression in yeast, and mammalian-preferred codons are used for gene expression in mammalian cells.

[0025] In some embodiments, the polynucleotide encodes a lysine decarboxylase polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence that is an even-numbered sequence identifier of SEQ ID NO:4-170, and the polypeptide has lysine decarboxylase activity, one or more of the improved properties described herein, e.g., the ability to convert lysine to the product 1,5-diaminopentane with increased activity, compared to the polypeptide of SEQ ID NO:2.

[0026] In some embodiments, the polynucleotide encodes an engineered lysine decarboxylase polypeptide, wherein the engineered lysine decarboxylase polypeptide comprises an amino acid sequence having a percent identity as described above compared to SEQ ID NO:2, and has one or more selected amino acid residue differences. In some embodiments, the disclosure provides engineered polypeptides with lysine decarboxylase activity, wherein the engineered polypeptides comprise a combination of residue differences at a position selected from X5, X11, X12, X13, X24, X48, X85, X95, X99, X108, X111, X116, X119, X143, X144, X145, X316, X334, X368, X378, X383, X422, X440, X441, X445, X516, X521, X551, X561, X591, X595, X693, X694, X701, and X710, from a reference sequence having at least 99.3% sequence identity to SEQ ID NO:2.

[0027] In some embodiments, the polynucleotide encoding the engineered lysine decarboxylase polypeptide comprises a sequence having an odd-numbered sequence identifier of SEQ ID NO: 3-169.

[0028] In some embodiments, the polynucleotide encodes a polypeptide as described herein, but at the nucleotide level, the polynucleotide has about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a reference polynucleotide encoding an engineered lysine decarboxylase. In some embodiments, the reference polynucleotide sequence is selected from sequences having odd-numbered sequence identifiers of SEQ ID NOs: 3-169.

[0029] An isolated polynucleotide encoding an engineered lysine decarboxylase polypeptide can be manipulated in a variety of ways to allow for expression of the polypeptide, including further modification of the sequence by codon optimization to improve expression, insertion into appropriate expression elements with or without additional regulatory sequences, and transformation into a host cell suitable for expression and production of the engineered polypeptide.

[0030] Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to its insertion into the vector. The techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, edited by Ausubel, F., Greene Pub. Associates, 1998, updated 2010.

[0031] In another aspect, the present disclosure also relates to recombinant expression vectors, appropriate for the type of host into which they will be introduced, that include a polynucleotide encoding an engineered lysine decarboxylase polypeptide or variant thereof, and one or more expression control elements, such as a promoter, terminator, or origin of replication. Alternatively, nucleic acid sequences of the present disclosure can be expressed by inserting the nucleic acid sequence, or a nucleic acid construct containing the sequence, into an appropriate expression vector. To generate an expression vector, a coding sequence is placed into the vector such that the coding sequence is linked to appropriate control sequences for expression.

[0032] A recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently used in recombinant DNA techniques and can result in expression of a polynucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. The expression vector can be an autonomously replicating vector, i.e., a vector that exists as a superchromosomal entity that replicates independently of chromosomal replication, such as a plasmid, superchromosomal element, minichromosome, or artificial chromosome. The vector can contain any tool to ensure self-replication. Alternatively, the vector can be a vector that, upon introduction into a host cell, integrates into the genome and replicates along with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid containing the total DNA to be introduced into the genome of the host cell, or two or more vectors or plasmids, can be used.

[0033] Many expression vectors useful for embodiments of the present disclosure are commercially available. An exemplary expression vector can be prepared by inserting a polynucleotide encoding an improved lysine decarboxylase polypeptide into the plasmid pACYC-Duet-1 (Novagen).

[0034] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an engineered lysine decarboxylase polypeptide of the present disclosure, the polynucleotide being linked to one or more regulatory sequences for expression of the lysine decarboxylase in the host cell. Host cells for expression of the polypeptide encoded by the expression vector of the present disclosure are well known in the art and include, but are not limited to, bacterial cells such as Escherichia coli, Arthrobacter sp. KNK168, actinomycetes, and Salmonella typhimurium cells, fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris), insect cells such as Drosophila melanogaster S2 and Subtilis subtilis Sf9 cells, animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells, and plant cells. An exemplary host cell is E. coli BL21(DE3). The host cells may be wild-type or genetically engineered through genome editing, such as knockout of the wild-type lysine decarboxylase gene retained in the host cell's genome. Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0035] The polynucleotides used to express the engineered lysine decarboxylase can be introduced into cells by a variety of methods known in the art, including electroporation, bioparticle bombardment, liposome-mediated transformation, calcium chloride transformation, and protoplast fusion. The various methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0036] Process for preparing genetically engineered lysine decarboxylase polypeptides A polynucleotide encoding a lysine decarboxylase can be subjected to mutagenesis and / or directed evolution techniques to obtain an engineered lysine decarboxylase. Exemplary directed evolution techniques can be found in Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing (2009 John Wiley & Sons Asia (Pte) Ltd. ISBN. 978-0-470-82314-9).

[0037] If the sequence of the engineered polypeptide is known, the encoding polynucleotide can be prepared by standard solid-phase synthesis methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then linked (e.g., by enzymatic or chemical ligation or polymerase-mediated methods) to form any contiguous desired sequence. For example, polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis, for example, using the classical phosphoramgide method described by Beaucage et al. (1981, Tet Lett 22:1859-69) or the method described by Matthes et al. (1984, EMBO J. 3:801-05), as commonly performed in automated synthesis methods. According to the phosphoramgide method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned into an appropriate vector, for example, in an automated DNA synthesizer. Furthermore, essentially any nucleic acid is available from a variety of commercial sources.

[0038] In some embodiments, the present disclosure also provides a process for preparing or manufacturing an engineered lysine decarboxylase polypeptide, the process comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered polypeptide under culture conditions suitable for expression of the polypeptide. In some embodiments, the process for preparing the polypeptide further comprises isolating the polypeptide. The engineered polypeptide is expressed in a suitable cell and can be isolated (or recovered) from the host cell and / or culture medium using any one or more techniques known for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, chromatography, etc.

[0039] Use of genetically engineered lysine decarboxylase and compounds prepared therefrom In another aspect, the improved engineered L-lysine decarboxylase polypeptides provided herein convert L-lysine to 1,5-diaminopentane. In some embodiments, the engineered L-lysine decarboxylase polypeptides can be used in a process for preparing a compound of structural formula (I): [ka] The method includes contacting L-lysine with an improved engineered L-lysine decarboxylase polypeptide disclosed herein.

[0040] In some embodiments of the above process, the compound of formula (I) is produced at a conversion of at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0041] Specific embodiments of engineered L-lysine decarboxylase polypeptides for use in the above processes are further provided in the detailed description. Engineered lysine decarboxylase polypeptides that can be used in the above processes are those represented by SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 2, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

[0042] As described herein and as shown in the Examples, the present disclosure contemplates a range of suitable reaction conditions that may be used in the processes herein, including, but not limited to, pH, temperature, substrate dosage, polypeptide dosage, and reaction time. Additional suitable reaction conditions for carrying out the methods described herein for biocatalytically converting substrate compounds to product compounds using engineered lysine decarboxylase polypeptides can be readily optimized by routine experimentation, including, but not limited to, contacting the engineered lysine decarboxylase polypeptide with substrate compounds under experimental reaction conditions that vary the dosage, pH, and temperature conditions of the individual reaction components, and detecting the product compounds. For example, utilizing the methods described in the embodiments provided herein.

[0043] As noted above, the engineered polypeptides with lysine decarboxylase activity used in the processes of the present disclosure generally comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference amino acid sequence selected from any even numbered sequence of SEQ ID NOs: 4-170.

[0044] The dosage of the substrate compound in the reaction mixture can be varied, taking into account, for example, the amount of the desired product compound, the effect of substrate concentration on enzyme activity, the stability of the enzyme under reaction conditions, and the conversion of substrate to product. In some embodiments of the process, suitable reaction conditions include a substrate dosage of at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L, at least about 150 g / L, at least about 200 g / L, at least about 250 g / L, at least about 300 g / L, at least about 350 g / L, at least about 400 g / L, at least about 450 g / L, at least about 500 g / L, and at least about 650 g / L of L-lysine hydrochloride. The values ​​for the substrate dosage provided herein are based on molecular weight, but it is contemplated that equivalent molar amounts of various hydrates and salts of Compound (II) can also be used in the process.

[0045] In the processes described herein, an engineered lysine decarboxylase polypeptide catalyzes the decarboxylation of L-lysine to form 1,5-diaminopentane. In some embodiments, the lysine in the reaction is L-lysine, which in embodiments includes the option of being applied in the form of a salt (e.g., lysine hydrochloride, lysine sulfate, etc.).

[0046] In some embodiments, suitable reaction conditions include a solution pH of about 5.5 to about 8.5. In some embodiments, reaction conditions include a solution pH of about 5, 5.5, 6, 6.5, 7, 7.5, 8, or 8.5.

[0047] In embodiments of the processes herein, suitable temperatures can be used as reaction conditions, for example, taking into account increased reaction rates at higher temperatures and enzyme activity over the reaction duration. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 60°C, about 25°C to about 50°C, about 25°C to about 45°C, or about 30°C to about 40°C. In some embodiments, suitable reaction temperatures include temperatures of about 25°C, 30°C, 35°C, 40°C, or 45°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the reaction.

[0048] The process using genetically engineered lysine decarboxylase is usually carried out in water or a solvent. Carbon dioxide produced during the decarboxylation reaction can cause foam formation, so a defoaming agent can be added if necessary.

[0049] Suitable reaction conditions can include a combination of reaction parameters that allow for the biocatalytic conversion of a substrate compound to a corresponding product compound. Thus, in some embodiments of the process, the combination of reaction parameters includes: (a) a dosage of substrate L-lysine hydrochloride of about 10 g / L to about 650 g / L, (b) a dosage of engineered polypeptide of about 0.1 g / L to about 10 g / L, (c) a concentration of PLP cofactor of about 0.1 mM to about 5 mM, (d) a pH of about 5.5 to about 8.5, (e) a temperature of about 10°C to about 60°C, and (f) a time period of about 1 hour to about 24 hours.

[0050] In some embodiments, the method comprises contacting ≥ 10 g / L of L-lysine hydrochloride substrate with ≥ 0.1 g / L of wet cells of an engineered lysine decarboxylase polypeptide described herein at a temperature of about 10°C to about 60°C, a pH of about 5.5 to about 8.5, and a reaction time of about 1 hour to about 24 hours, wherein at least 70%, 80%, 90% or more of the L-lysine hydrochloride substrate is converted to 1,5-diaminopentane product. In some embodiments, the lysine decarboxylase polypeptide capable of carrying out the above reaction is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 15 , 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, and 170.

[0051] Exemplary reaction conditions include the assay conditions shown in Table 2 and Examples 7 and 8.

[0052] When carrying out the decarboxylation reactions described herein, the engineered lysine decarboxylase polypeptide may be added to the reaction mixture in a partially purified or purified form, in whole cells harboring a gene encoding the engineered lysine decarboxylase polypeptide, and / or in the form of a cell extract and / or lysate of such cells. Whole cells or cell extracts harboring a gene encoding the engineered lysine decarboxylase, lysates therefrom, and isolated enzymes can be used in a variety of different forms, including solid (e.g., lyophilized, spray-dried, etc.) or semi-solid (e.g., a coarse paste such as wet cells). Cell extracts or cell lysates may be partially purified using precipitation (e.g., ammonium sulfate, polyethyleneamine, heat treatment, etc.), followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) before lyophilization. Any of the enzyme preparations can be stabilized by cross-linking using known cross-linking agents, such as glutaraldehyde, or by immobilization on a solid-phase material (e.g., a resin).

[0053] In some embodiments of the decarboxylation reaction described herein, the reaction is carried out under suitable reaction conditions, as described herein, and the engineered lysine decarboxylase polypeptide is immobilized on a solid support. Solid supports useful for immobilizing the engineered lysine decarboxylase to carry out the reaction include, but are not limited to, beads or resins such as epoxy-functionalized polymethacrylate, aminoepoxy-functionalized polymethacrylate, polymethacrylate, styrene / DVB copolymer, or octadecyl-functionalized polymethacrylate. Exemplary solid supports include, but are not limited to, chitosan beads, SEPABEAD (Mitsubishi), including different types of SEPABEAD, including Eupergit C, EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0054] In some embodiments, the engineered polypeptide may be expressed in the form of a secreted polypeptide, and the culture medium containing the secreted polypeptide may be used in the processes herein.

[0055] In some embodiments, solid reactants (e.g., enzymes, salts, etc.) can be supplied to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray-dried, etc.), solutions, emulsions, and suspensions. Reactants can be readily lyophilized or spray-dried using methods and equipment known to those skilled in the art. For example, a protein solution can be frozen in small aliquots at -80°C and added to a pre-chilled lyophilization chamber, after which a vacuum is applied.

[0056] In some embodiments, the order of addition of the reactants is not important: the reactants may be added together to the solvent at the same time (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or some reactants may be added separately and some reactants may be added together at different times. [Example]

[0057] The following examples further illustrate the present invention without limiting it thereto, in which experimental methods for which conditions are not specified were carried out according to commonly used conditions or supplier suggestions.

[0058] Example 1: Gene cloning and expression vector construction The amino acid sequence of wild-type lysine decarboxylase from Plesiomonas shigelloides was available from NCBI, and the corresponding nucleic acid was synthesized by a vendor using standard techniques and cloned into the expression vector pACYC-Duet-1. The recombinant expression plasmid was transformed into competent E. coli BL21(DE3) cells by heat shock at 42°C for 90 seconds. The transformation solution was plated on LB agar plates containing chloramphenicol and incubated overnight at 37°C to obtain recombinant transformants.

[0059] Example 2: Expression of lysine decarboxylase polypeptides The recombinant E. coli BL21(DE3) obtained in Example 1 was inoculated into 50 mL of LB medium (peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH 7.0±0.2, 25°C) containing chloramphenicol in a 250 mL Erlenmeyer flask, and then cultured overnight in a shaking incubator at 30°C and 250 rpm. The OD of the overnight culture was 600 When the OD reached 2, it was divided into 1000 mL flasks containing 250 mL of TB medium (tryptone 12 g / L, yeast extract 24 g / L, disodium hydrogen phosphate 9.4 g / L, dipotassium hydrogen phosphate 2.2 g / L, pH 7.2 ± 0.2, 30 °C) at a 5% (v / v) inoculum. Lactose was added at a final concentration of 6 g / L as an inducer, and the culture was placed in a shaking incubator at 30 °C and 250 rpm overnight. After 20 h of induction at 30 °C, the culture was centrifuged, the cells were collected, and the supernatant was discarded to obtain wet cells. The resulting wet cells were then placed in a -20 °C refrigerator for future use. If an enzyme solution was required, the resulting wet cells were resuspended in 50 mL of PBS buffer (pH 7.4) and sonicated in an ice bath for 5 minutes (5 s / s, 50% power). The resulting cell lysate was clarified by centrifugation at 8000 rpm for 10 min at 4°C using a Thermo Multifuge X3R centrifuge, and the clarified supernatant was frozen at -20°C.

[0060] Example 3: Construction of a lysine decarboxylase mutant library All reagents used herein were commercially available, preferably the Quikchange kit (supplier: Agilent). The mutagenesis primer sequences were designed according to the kit's instructions. The PCR reaction consisted of 10 μL of 5x buffer, 1 μL of 10 mM dNTPs, 1 μL of plasmid DNA template (50 ng / μL), 0.75 μL each of upstream and downstream primers (10 μM), 0.5 μL of high-fidelity enzyme, and 36 μL of ddHO. The PCR primers contained an NNK codon at the mutation site.

[0061] The PCR amplification steps were: (1) pre-denaturation at 98°C for 3 min, (2) denaturation at 98°C for 10 s, (3) annealing and extension at 72°C for 3 min, (4) repeating steps (2)–(3) 25 times, (5) extension at 72°C for 10 min, followed by cooling to 4°C. 2 μL of DpnI was added to the PCR product, and the plasmid template was removed by overnight digestion at 37°C. The digested PCR product was transformed into competent E. coli BL21(DE3) cells and plated on LB agar plates containing chloramphenicol to obtain the mutagenesis library.

[0062] Example 4: High-throughput screening of a lysine decarboxylase mutation library For expression of the lysine decarboxylase mutant library, the shake flask preparation protocol was scaled down to a 96-well plate. Mutant colonies were selected from LB agar plates and inoculated into LB medium containing chloramphenicol in a 96-well shallow plate with 200 μL of LB medium per well. The culture was grown overnight (18-20 hours) in a shaking incubator at 180 rpm, 80% humidity, and 30°C. The OD of this overnight culture was 0.05. 600 When the OD of the deep well culture reached approximately 2.0, 20 μL was used to inoculate 400 μL / well of TB medium (containing chloramphenicol) into a 96-well deep well plate. The deep well plate was placed in a shaking incubator at 250 rpm, 30°C, and 80% humidity to continue culturing. The OD of the deep well culture was 600 When the Λ / Λ ratio reached 0.6-0.8, IPTG was added at a final concentration of 1 mM as an inducer to induce expression, and the expression was carried out overnight at 30°C. Finally, the deep-well plate was removed and centrifuged at 4000 rpm for 10 minutes, the culture medium was removed, and the wet cell pellet in the deep-well plate was placed in a -20°C freezer.

[0063] The assay protocol for screening the mutant library for catalytic conversion of L-lysine to 1,5-diaminopentane is as follows.

[0064] Using a pipette, 300 μL of cell lysis solution (containing 0.2 mM PLP and 0.1 M phosphate buffer, pH 6.0) was added to a 96-well plate containing wet cells. The plate was sealed with a membrane and placed on a plate oscillator at 700 rpm for 1 hour. A 500 g / L L-lysine hydrochloride solution (containing 0.2 mM PLP) was prepared, and 180 μL was added to each deep-well plate to achieve a final L-lysine hydrochloride concentration of 450 g / L. Twenty μL of cell lysis solution was added to each 96-deep-well plate, for a total reaction volume of 200 μL. The 96-well plate was heat-sealed with aluminum foil and placed in a shaking incubator at 35°C for 20 hours at 200 rpm. At the end of the reaction, 20 μL of the reaction solution was removed, quenched with 280 μL of acetonitrile, and shaken at 700 rpm for 1 hour. The quenched reactions were centrifuged and the supernatants were diluted for HPLC analysis to detect conversion.

[0065] Example 5: HPLC analysis High-throughput screening analytical method: analytical column Chirex3126(D)-penicillamine 30*4.6mM, mobile phase 1mM copper sulfate, flow rate: 1.2mL / min, column temperature: 45°C, detection wavelength: 280nm, injection volume: 10μL.

[0066] Example 6: Fermentation and downstream processing A single microbial colony of E. coli BL21(DE3) harboring a plasmid containing the target lysine decarboxylase gene was inoculated into 50 mL of LB broth containing 30 μg / mL chloramphenicol (yeast extract 5.0 g / L, tryptone 10 g / L, NaCl 10 g / L). The cells were incubated overnight at 30 °C in a shaker at 250 rpm for 16 h. The OD of the culture was measured. 600 When the reached 3.5–4.5, the culture was immediately inoculated into the fermenter medium.

[0067] A 1.0 L fermenter containing 0.4 L of medium was sterilized in an autoclave at 121°C for 30 minutes. The fermenter was inoculated with the aforementioned culture. The temperature of the fermenter was maintained at 37°C. The medium in the fermenter was stirred at 200–1000 rpm, and air was supplied to the fermentation vessel at 0.4–0.8 L / min to maintain the dissolved oxygen level above 60%. The culture was maintained at pH 6.5–7.0 by adding 25–28% v / v ammonium hydroxide. Cell growth was maintained by feeding a feed solution containing 500 g / L dextrose monohydrate, 12 g / L ammonium chloride, and 5 g / L magnesium sulfate heptahydrate. The OD of the culture was 0.01. 600 After the pH reached 25 ± 5, the fermenter temperature was lowered and maintained at 30°C, and target polypeptide expression was induced by adding α-lactose to a final concentration of 15 g / L. The fermentation process continued for an additional 16–22 hours. After the fermentation process was completed, the cells were harvested by centrifugation at 8000 rpm for 10 minutes at 4°C using a Thermo Multifuge X3R centrifuge. The harvested cells were either used directly for downstream recovery or stored frozen at -20°C.

[0068] Six grams of cell pellet was resuspended in 30 mL of 100 mM potassium phosphate buffer containing 250 μM pyridoxal 5'-phosphate (PLP) at pH 7.5 at 4°C. The cells were then homogenized twice at 800 bar using a homogenizer to release the lysate. The lysate was clarified using a Thermo Multifuge X3R centrifuge at 8000 rpm for 10 min at 4°C. The clarified supernatant was frozen at -20°C.

[0069] Example 7: Reaction process for catalytic production of 1,5-diaminopentane by engineered lysine decarboxylase polypeptides This embodiment provides a method for the enzymatic preparation of 1,5-diaminopentane, comprising the following steps:

[0070] S1: 50 g of L-lysine hydrochloride was dissolved in 60 mL of water to obtain a substrate solution, and the substrate solution was placed in a reaction flask.

[0071] S2: 0.5 g of wet cells containing the engineered lysine decarboxylase prepared in Example 6 was added to a 2 mL centrifuge tube, and 1.5 mL of pure water was added to thoroughly resuspend the wet cells. The resuspended cells were added to a reaction flask, and the centrifuge tube was washed with 1 mL of water, which was also added to the reaction flask. Next, 2 mL of 10 mM pyridoxal phosphate solution was added to the reaction flask to initiate the decarboxylation reaction, and the temperature of the decarboxylation reaction was controlled at 30°C. The reaction was stirred at 300 rpm for 6 hours, and the conversion rate was detected using the analytical method described in Example 5 and is shown in the table below.

[0072] S3: 100 mL of methanol was added to the above reaction solution and heated to inactivate the decarboxylase. The water-cooled reaction was cooled to room temperature, then the pH was adjusted to 2 using hydrochloric acid, followed by filtration to obtain the filtrate. The filtrate was then filtered under reduced pressure until crystals precipitated from the liquid surface. The filtration was stopped, and the liquid was cooled to room temperature. 250 mL of ethanol was then added, shaken, and kept at 4 °C overnight. Finally, filtration was performed to remove the ethanol and water. The solid was then washed with 100 mL of ethanol and dried in an oven at 50 °C for 12 hours to obtain 1,5-diaminopentane hydrochloride. To obtain 1,5-diaminopentane, aqueous sodium hydroxide solution was added, and 1,5-diaminopentane was separated by distillation. JPEG2026507552000009.jpg38170

[0073] Example 8: High-scale reaction process S1: 5g of wet cells containing the genetically engineered lysine decarboxylase sequence number 170 produced in Example 6 above was added to 630mL of pure water, 0.055g of pyridoxal phosphate was weighed and added to the system, the reaction temperature was controlled at 35°C or 40°C, and the system was stirred at 300 rpm. 500g of L-lysine hydrochloride was slowly added to the system, and the reaction was carried out for 6 hours to obtain a reaction solution, which was detected using the analytical method described in Example 5, and the conversion rate was greater than 99.99%.

[0074] S2: The pH of the reaction solution was adjusted to 1 using hydrochloric acid, and the solution was filtered to obtain a filtrate, which was then subjected to electrodialysis. The aqueous solution of 1,5-diaminopentane obtained by electrodialysis was distilled to obtain 1,5-diaminopentane.

[0075] Example 9: Reaction process for catalytic production of 1,5-diaminopentane by engineered lysine decarboxylase polypeptide (SEQ ID NO: 144) This embodiment provides a method for the enzymatic preparation of 1,5-diaminopentane at different substrate dosages, comprising the following steps:

[0076] S1, 50 g, 60 g, 65 g of L-lysine hydrochloride were added to 60 mL, 53 mL, 49 mL of water, respectively, to obtain substrate stock solutions, and then the substrate solutions were added to the reaction flask.

[0077] S2: 0.5 g, 0.6 g, and 0.65 g of wet cells using the genetically engineered lysine decarboxylase prepared in Example 6 were added to 2 mL centrifuge tubes, respectively. 1.5 mL of purified water was added to completely resuspend the wet cells. The resuspended cells were added to the reaction flask, and the centrifuge tube was washed with 1 mL of water, which was then added to the reaction flask. Next, 2 mL of 10 mM pyridoxal phosphate solution was added to the reaction flask to initiate the decarboxylation reaction. The temperature of the decarboxylation reaction was controlled at 35°C, and the reaction was stirred at 300 rpm for 6 hours. The conversion rates were then detected using the analytical method mentioned in Example 5, and were 99.99%, 97.52%, and 96.94%, respectively.

[0078] In S3, 100 mL of methanol was added to the above reaction solution, and the solution was heated to inactivate the decarboxylase. The water-cooled reaction was cooled to room temperature, then the pH was adjusted to 2 using hydrochloric acid, followed by filtration to obtain the filtrate. The filtrate was then filtered under reduced pressure until crystals precipitated from the liquid surface. The filtration was stopped, and the liquid was cooled to room temperature. 250 mL of ethanol was then added, shaken, and kept at 4 °C overnight. Finally, filtration was performed to remove the ethanol and water. The solid was then washed with 100 mL of ethanol and dried in an oven at 50 °C for 12 hours to obtain 1,5-diaminopentane hydrochloride. To obtain 1,5-diaminopentane, aqueous sodium hydroxide solution was added, and 1,5-diaminopentane was separated by distillation.

Claims

1. 1. A genetically engineered lysine decarboxylase polypeptide capable of converting L-lysine to 1,5-diaminopentane and carbon dioxide, said polypeptide comprising any one of the following amino acids selected from the group consisting of X5, X11, X12, X13, X24, X48, X85, X95, X99, X108, X111, X116, X119, X143, X144, X145, X316, X334, X368, X378, X383, X422, X423, X424, X425, X426, X428, X429, X430, X431, X432, X433, X434, X435, X436, X437, X438, X439, X440, X441, X442, X443, X445, X446, X447, X448, X449, X450, X451, X452, X453, X454, X455, X456, X457, X458, X459, X460, X461, X462, X463, X464, X465, X466, X467, X468, X469, X470, X471, X472, X473, X474, X475, X476, X477, X478, X479, X480, X481, X482, X483, X484, X485, X486, X487, X488, X489, X490, X491, X492, X493, X494, X495, , X440, X441, X445, X516, X521, X551, X561, X591, X595, X693, X694, X701, X710, wherein the polypeptide has lysine decarboxylase activity and an amino acid sequence having 99.3% or greater sequence identity to the reference sequence of SEQ ID NO:

2.

2. The amino acid sequence of the lysine decarboxylase polypeptide further comprises one or more of the following amino acid residues: X5 is V, X11 is A, C, P, R, T or S; X12 is E, V, R, L, T, Q or K; X13 is I, W, V, P or Y; X24 is K, X48 is S, X85 is D, X95 is G, X99 is D, A, G, Y, T, S or Q; X is Q, X is I, X116 is Q, X119 is P, D or R; X143 is P, X144 is L, F, Q, R or E; X145 is D, V, W or L; X316 is E, X334 is I, X368 is C, X378 is V, X383 is E, X422 is Q, X440 is R, X441 is Q or R, X445 is Y, L or A; X516 is R, X521 is L or M; X551 is L, Q or N; X561 is E, X591 is G or S, X595 is R, X693 is V, X694 is Q, X701 is G, X710 is Q, The lysine decarboxylase polypeptide of claim 1, wherein the number refers to SEQ ID NO:

2.

3. The engineered polypeptides are selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 160, 162, 164, 166, 168, 170, or an amino acid sequence having at least 95% sequence identity to the above reference sequences.

4. 2. The genetically engineered lysine decarboxylase polypeptide of claim 1, wherein the reaction conditions comprise about 10 g / L to 650 g / L of L-lysine hydrochloride, about 0.1 mM to 1 mM of PLP, and a temperature of 10 to 60°C.

5. A polynucleotide encoding the polypeptide of claim 3.

6. The polynucleotide sequences are SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, or 169.

7. An expression vector comprising the polynucleotide of claim 5 or 6.

8. A host cell comprising the expression vector of claim 7.

9. 10. A method for preparing a lysine decarboxylase polypeptide, comprising culturing the host cell of claim 8 and obtaining the lysine decarboxylase polypeptide from the culture.

10. 10. A lysine decarboxylase catalyst comprising the culture obtained in claim 9, cells or culture medium containing the lysine decarboxylase polypeptide obtained from the culture, or a product processed therefrom, wherein the product is an extract obtained from the cells, an isolated product obtained by separation or purification of the lysine decarboxylase in the extract, an isolated product obtained by immobilizing the cells and their extract, or an immobilized product obtained by immobilizing the extract or an isolated product of the extract.

11. A process for preparing a compound of formula (I) comprising: 【Chemistry 1】 10. The process comprising the step of contacting L-lysine, a compound of formula (II), with the engineered polypeptide of any of claims 1-4 in a suitable solvent and under reaction conditions suitable for converting said compound of formula (II) to 1,5-diaminopentane of formula (I). 【Chemistry 2】

12. 12. The process of claim 11, wherein the reaction conditions comprise a temperature of 10°C to 60°C, preferably 30°C to 40°C, 10 g / L to 650 g / L of L-lysine hydrochloride, and about 0.1 mM to 1 mM of PLP.

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