Improved variants of lysine decarboxylase active at higher temperatures and higher ph
E. coli lysine decarboxylase variants with specific amino acid substitutions address the challenge of high yield and purity in PMDA production by enhancing enzyme activity at higher temperatures and pH, improving separation efficiency.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ARCHER DANIELS MIDLAND CO
- Filing Date
- 2025-11-07
- Publication Date
- 2026-05-15
AI Technical Summary
Existing methods for producing 1,5 diaminopentane (PMDA) from lysine face challenges in achieving high yield and purity, particularly due to the complex mixture of amino and carboxylic acids in the culture medium, and the need for improved lysine decarboxylase enzymes with higher specific activity at pH values closer to the pH of PMDA as a free base.
Development of E. coli lysine decarboxylase variants with specific amino acid substitutions, such as I303V, D501N, and K595R, to enhance the enzyme's activity in converting lysine to pentamethadiamine (PMDA), optimized for higher temperatures and pH conditions.
The variants exhibit greater specific activity in converting lysine to pentamethadiamine, reducing the need for acidic pH adjustment and improving the separation process efficiency.
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Abstract
Description
[0001] IMPROVED VARIANTS OF LYSINE DECARBOXYLASE ACTIVE AT HIGHER TEMPERATURES AND HIGHER PH
[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing, which has been submitted electronically in .xml format. The contents of the electronic sequence listing (IND0240W001 xml; Size 105,008 bytes, and Date of Creation: November 5, 2025) are incorporated by reference herein in its entirety.
[0004] TECHNICAL FIELD
[0005] This disclosure relates generally to amino acid substitutions that improve the activity of lysine decarboxylase useful for the enzymatic production of 1,5 diaminopentane, (a.k.a., cadaverine in common vernacular or pentamethadi amine (PMDA) in the polymer industry) from lysine.
[0006] Four to six carbon diamines are used for the synthesis of polyamides and polyurethanes. For example, 1 ,4-diaminobutane and 1,6-diaminohexane are used for the synthesis of nylon-4, 6 and nylon-6, 6 while 1,5 diaminopentane ( a.k.a. pentamethaldiamine (PMDA) in the polymer industry or cadaverine in common vernacular) is used for the production of nylon- 1,5. The diamines may be produced by biochemical or chemical methods. Chemical methods rely on classical synthesis of monomers such as 1,4-diaminobutane or PMDA from nonrenewable hydrocarbon resources. Biochemical methods rely on production of ornithine or lysine from a biological source by fermentation fed with renewable resources such as dextrose followed by use of an appropriate decarboxylase enzyme to decarboxylate the amino acid to form the diamine. In the case of PMDA lysine decarboxylase is the appropriate enzyme in some practices the lysine is made in a fermentation step separate from a subsequent decarboxylation step. In other practices the microorganism engineered for lysine production is also engineered to express the decarboxylase and export it from the cell, so the decarboxylation reaction occurs simultaneously in the broth as lysine is being exported. The best studied of this enzyme is the lysine decarboxylase from E. coli which has the amino acid sequence according to SEQ ID NO: 1. The biochemical method utilizes culturing a transformed microorganism engineered to secrete either a diamine to the culture medium or an amino acid decarboxylase to a culture medium containing the amino acid (see for example U. S. patents 11,124,812; 11,053,524, 11,053,525:10,626,425; 10,870,871; 10,711,289; 10,640,798; 10,472,636; 10,150,977; 9,745,608; 9,644,220; 9,365,876; 9,115,362; 8.741,623; each of which is incorporated herein by reference in its entirety). In the case of microorganisms secreting amino acid decarboxylase (see for example 8,871,477, incorporated herein by reference in its entirety), an amino acid such as lysine or ornithine is added to the culture medium clarified of the biomass to produce the diamine by action of the decarboxylase. Some methods utilize diammonium salts of carboxylic acids to produce the diamines (see for example U. S. 8,742.060, incorporated herein by reference in its entirety). In any of the above-mentioned methods, the crude product is a complex mixture containing amino and / or carboxylic acid(s) from the amino and / or carboxylic acid used to produce the diamine and the culture media usually contain one or more carboxylic acid metabolites such as acetic acid, oxalic acid, succinic acid, tartaric acid, lactic acid and / or salts thereof, as well as the conjugate ions of amine or or carboxylate functional groups such as phosphate, sulfate, HCL, chloride, ammonium, sodium, potassium and the like, which may also be present due the their addition as mineral nutrients in a culture medium. Thus, the separation of the diamine efficiently high yield and purity as well as low cost from such a complex mixture continues to be a challenging process.
[0007] To further improve the capacity to produce PMDA from renewable resources, it is desirable to have improved lysine decarboxylase enzymes that have higher specific activity particularly at a pH value that is as close as possible to the pH of the PMDA as a free base, which becomes more basic as more lysine is exported into the broth The present invention provides for such improved lysine decarboxylase enzymes. SUMMARY OF THE INVENTION
[0008] In one aspect, disclosed herein are variants of an E. coll lysine carboxylase enzy me having a protein sequence according to SEQ ID NO: 1 except comprising at least three amino acid substitutions selected from the group consisting of a) I303V. D501N, and K595R; b) L166I, H286R, and N426H; c) N76Y, P293L, and D577A; d) N76Y, 1359F, and A622T; e) R28A, D501N, and P293A; and f) R28A, K320N, and E711A, wherein the variant enzyme exhibits greater specific activity' in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1.
[0009] In another aspect disclosed herein are variants of the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1, except comprising two amino acid substitution of D501N, K595R, and at least one other amino acid substitution wherein the variant enzyme exhibits greater specific activity7in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1. In certain embodiments of this aspect the at least one other amino acid substitution comprises I303V. A further embodiment includes wherein the at least one other amino acid substitution further comprises an amino acid substitution selected from the group consisting of N76Y, K320N, D506E, L637I, and S713N. In other embodiments of this aspect the at least one other amino acid substitution further comprises each of K320N and D506E. In other embodiments the at least one other amino acid substitution further comprises each of K320N and D506E and at least one additional amino acid substitution selected from the group consisting of R28A, T444S. L637I, I644V, D577N, and S713N. In other embodiments, the at least one additional amino acid substitution further comprises each of K320N, D506E, L637I, I644V, and S713N.
[0010] In other aspect, disclosed herein are variants of an E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1 except comprising each amino acid substitution of K320N, D501N, D506E, K595R, and L637I, , wherein the variant enzyme exhibits greater specific activity' in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1. Some embodiments of this aspect the sequence further comprises at least one substitution selected from the group consisting of R28A, N76Y, I303V, T461D, A700E, and S713N. Some embodiments further include at least one additional substitution selected from the group consisting of Y80H, I200L, T444S, and S469A. Other embodiments of this aspect include at least two substitutions of R28A, N76Y, I303V, T461D, and S713N. Some embodiments include at least three substitutions of R28A, N76Y. I303V. T461D, and S713N. Still other embodiments include at least four substitutions of R28A. N76Y, I303V, T461D, and S713N.
[0011] In another aspect disclosed herein are variants of an E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1 except comprising each amino acid substitution of K320N, T416D, D501N, D506E, K595R, and L637I wherein the variant enzyme exhibits greater specific activity in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1. Some embodiments of this aspect further include at least one amino acid substitution selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. Some embodiments of this aspect further include at least two amino acid substitutions selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. Some embodiments of this aspect further include at least three amino acid substitutions selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. Some embodiments of this aspect include a substitution of 1303V. Some refines of this last aspect include at least one additional amino acid substitution selected from the group consisting of substitution of R28A, N76Y, A702E, and S713N.
[0012] Exemplary embodiments of the variants include any amino acid substitution indicated in any row of the table shown in Figure 3.
[0013] BRIEF DESCRIPTION OF THE FIGURES
[0014] Figure 1 is a chart that shows the relative lysine decarboxylase specific activity measured as a result of screening 96 candidate lysine decarboxylase genes identified by internal ID number. E.c. CadA is the lysine decarboxylase gene from E.coli which has the amino acid sequence according to SEQ ID NO: 1. Specific activity is expressed relative to E.coli enzyme.
[0015] Figure 2 are charts that show the relative specific activity of six selected candidate lysine decarboxylase enzymes measured at different time points at pH 8.4 and 9.0 and at 55°C or 60°C. Results are expressed relative to the specific activity of the E.c. CadA protein at a given time point, which has internal ID number 396212. Left to right ID number order for each panel is 79322, 396212, 396241, 396243, 396304, and 369305.
[0016] Figure 3 is a table showing specific amino acid substitutions made in the E. coli CadA protein according to SEQ ID NO: 1 and improvement in activity over the immediate parent.
[0017] DETAILED DESCRIPTION
[0018] This disclosure provides improved lysine decarboxylase enzymes useful for conversion of lysine to pentamethyldiamine (PMDA), a.k.a., 1,5 diaminopentane, a.k.a., cadaverine. The improvements were developed by synthesizing codon optimized nucleic acids encoding candidate lysine decarboxylase genes or mutations thereof in an E. coli expression system, expressing the candidates in E. coli, forming crude extracts of the E. coli cells and measuring the lysine decarboxylase in the crude extracts using equal amounts of protein.
[0019] Expression of Lysine Decarboxylase Variants in E. coli
[0020] Genes for lysine decarboxylases were designed for high expression in E. coli and cloned into the rhamnose inducible expression vector pD861. The resulting plasmid was used to transform E. coli strain BL21 by standard procedures. Transformants were selected by growth on LB agar plates containing antibiotics. Selected colonies were used to inoculate 0.5 ml of LB media containing antibiotics in a deep-well microplate and cultures were incubated overnight with shaking at 37°C. Overnight culture was used to inoculate 0.5 ml of fresh LB media containing antibiotics and the resulting culture was incubated, induced for expression and Harvested after 3 hours of growth. Cell pellets were lysed and of the lysate was recovered from supernatant after centrifugation at 6100 x g for 30 minutes. Soluble decarboxylase level was analyzed by polyacrylamide gel electrophoresis and densitometry.
[0021] Candidate transformants were grown to mid log phase then the cells w ere lysed, and the cell debris was removed forming crude extracts that were used as the samples for the expressed lysine decarboxylase genes. Equal amounts of protein from the crude extracts were added to assay wells containing a clarified lysine fermentation broth with a desirable amount of lysine that was adjusted to a desirable pH (i.e., 7.5, 8.4, or 9.0) using phosphoric acid in a final assay volume of 100 pl. Lysine decarboxylase from E. coll uses pyridoxal phosphate (PLP) as a cofactor so 0.05 to 1.0 mM PLP was included in all reactions. The reactions w ere incubated at a desired temperature (37°C, 55°C, or 60°C) for various times from as short as 10 minutes to as long as twenty hours and stopped by addition of 10% volume 2% HC1 and chilling to 4°C. Reactions were filtered through a PES membrane at a 10 kDa molecular weight cutoff (Pall) prior to HPLC analysis.
[0022] Sequence Panning for Candidate Lysine Decarboxylase Genes
[0023] Several sequence libraries were initially probed to identify sequences that showed > 20%-30% or higher amino acid homology to the E.c. CadA gene or had notations indicating an actual or predicted amino acid decarboxylase activity. Synthetic DNA constructs encoding the candidates with codons optimized for expression in E. coll were evaluated via the above E. coll expression system protocol. The assay conditions for the initial screening were conducted at pH 8.4 with a reaction time of 20 hours at a temperature of 37 °C or 55 °C with and without 0.5 mM PLP present in the reaction. Figure 1 shows the relative activity of 96 initial candidate sequences relative to the E. coli lysine decarboxylase where the amount of PMDA produced by E. coli at each pH was assigned a value of 1.0 and the amount produced by the other candidate at each temperature after the 20 h reaction is expressed relative to the amount produced by the E. coli enzyme. A small cache of candidate sequences showed significant or higher activity than the E. coli enzyme at 55 °C, two of which, designated with intern ID reference numbers 396304 and 396305 also displayed high activity in the absence of PLP, Five of the higher performing candidate sequences and the E. coll enzyme (which has internal ID reference number 396212) were selected for further screening at higher temperatures and at a higher pH. Activity at higher pH is desirable because a liquid lysine product derived after purification of fermentation derived lysine is basic, having a pH typically in the range of 9.5 -10.0 therefore enzy mes with activity' at higher pHs reduces the amount of an acidic conjugate acid such as phosphoric acid needed to lower the pH. The selected candidates were evaluated relative to the E.coli enzyme at 55 °C at pH 8.4 and 9.0, and the activity at pH 8.4 was also evaluated at 60 °C. All were assayed in the presence of 0.5 mM PLP. Figure 2 are bar graphs that show the relative activity of each selected candidate at each time point relative to the activity of the E. coll enzyme. The candidate ID number for each bar for each time point in each graph are from left to right - 49322, 396212, 396241, 393243, 396304, then 396305. No candidate enzy me showed higher activity than the E.coli enzyme at any of the temperatures or pHs tested, so the E. coli enzy me was selected for further improvement.
[0024] Generation of Variants of the SEQ ID NO: 1
[0025] Based on methods described in US 8,635,029, nucleotide changes encoding amino acid variations were introduced into the E.c. CadA gene to generate specific amino acid substitutions that were expressed and assayed using the E. coli expression system. E. coli CAD A were subjected to multiple rounds of diversification and machine learning (US 8,635,029) where substitutions having positive impacts on activity and stability are enriched
[0026] Figure 3 is table showing the specific amino acid substitutions introduced in sequential rounds of variant production. The substitutions are expressed in the form XNZ, where X is the one letter code for the original amino acid present in SEQ ID NO: 1, N is the amino acid position counting from the N terminus of the E.coli protein, and Z is the one letter code for the specific amino acid substitution made at that position. Those amino acid substitutions most preserved in multiple rounds of variant production are written across the top of the table. Those amino acid substitutions not preserved more than twice are listed in the penultimate column as “other substitutions.” The relative activity shown in the last column is the best measured activity relative to the immediate parent under any condition. For example, some substitutions showed better activity at pH 9.0 than pH 8.4, or better activity in a 1- hour reaction than a 3 hour reaction than its parent, but only the activity of the best performing condition relative the parent under the same condition is shown. The relative activity may be lower at a different pH or for a different time. Each reaction was done in triplicate and the relative activity reported is the average thereof.
[0027] Round 1 variants produced the highest step improvement of over the parent (E.c., CadA) than any other round of variant production. Figure 3 shows that six round 1 run variants had improved activity ranging from 1.3 to 2. 1 -fold over the parent. Those round 1 variants had the following amino substitutions: I303V, D501N and K595R; L166I, H286R and N426H; N76Y, P293L and D577A; N76Y, I359F and A622T; R28A, D501N and P293A; or R28A, K320N, and E711A. Because the variant containing the I303V. D501N and K595R substitutions showed the highest step improvement over the original enzyme, it was used as the parent for round 2 variant production.
[0028] The variants produced in round 2 all retained at least two to the three substitutions of their parent. Figure 3 shows that 14 round 2 variants had improved activity over their immediate parent. All of these variants showing an improvement retained the substitutions D501N, K595R, and contained at least one other amino acid substitution. Most of these variants retained the substitution 1303V. Several also contained substitutions at least one other amino acid substitution of N76Y, K320N, D506E, L637I. and S713. Of these, the substitutions K320N and D506E appeared most frequently. These two additional substitutions included at least one additional amino acid substitution selected from the group consisting of R28A, T444S, L637I, I644V, D577N, and S713N. Many of these round 2 variants contained each substitution of K320N, D506E, L637I, I644V, and S713N. Many of the round two variants contained the substitutions R28A, N76Y, I303V, T461D, A700E, and / or S713N. Some round 2 variants contained at least one additional substitution selected from the group consisting of Y80H, I200L, T444S, and S469A. Many of the round two variants contained at least one additional substitution selected from the group consisting ofY80H, I200L, T444S, and S469A, several round 2 variants contained at least two substitutions of R28A, N76Y, I303V, T461D. and S713N. Several round 2 variants contained at least three substitutions of R28A, N76Y, I303V, T461D, and S713N. Some round 2 variants contained at least four substitutions of R28A, N76Y, I303V, T461D, and S713N. Based on these observations a parent for round 3 substitutions was selected that contained substitutions at I303V, K320N, D501N, D506E, K595R, and L637I.
[0029] In the first round 3 of variant production retaining each substitution of K320N, T416D, D501N, D506E, K595R, and L637I from the immediate parent, only 5 variants were produced with similar or slightly improved activity over the round 3 patent. Therefore, a second round of round 3 variants were produced designated as round 3B. Figure 3 shows that the sum of round 3 and 3B variants exhibited similar, slightly improved or significantly improved activity over the round 3 patent. Collectively, these variants contained amino acid substitution of K320N, T416D, D501N, D506E, K595R, and L637I. All of these variants contained at least one other amino acid substitution selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. Many variants contain data at least two amino acid substitution selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. Some of these variants contained at least three amino acid substitutions selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N. A great majority of these variants comprising a substitution of 1303V. Many of those containing the variant 1303V also contained at least one additional amino acid substitution selected from the group consisting of substitution of R28A, N76Y, A702E, and S713N.
[0030] The process of producing the variants shown in Figure 3 indicates that so long as any of the set key substitutions shown in any round of variant production are retained, it is possible to generate further variants having similar or improved activity starting with any of the set of variants shown in Figure 3.
[0031] The amino acid sequence for the parent E.c., Cad A is provided herewith in the sequence listing as SEQ ID NO: 1 and exemplary variants having the features described herein are provided as SEQ ID NOs: 2-66.
Claims
CLAIMS1. A variant of an E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1 except comprising at least three amino acid substitutions selected from the group consisting of a) I303V, D501N, and K595R; b) L166I, H286R, and N426H; c) N76Y. P293L. and D577A; d) N76Y, I359F, and A622T; e) R28A, D501N, and P293A; and f) R28A, K320N, and E711A wherein the variant enzyme exhibits greater specific activity in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1.
2. A variant of an E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1, except comprising two amino acid substitution of D501N, K595R, and at least one other amino acid substitution wherein the variant enzyme exhibits greater specific activity in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1.
3. The variant E. coli lysine decarboxylase enz me of claim 2, wherein the at least one other amino acid substitution comprises I303V.
4. The variant lysine decarboxylase enzyme of claim 3 wherein the at least one other amino acid substitution further comprises an amino acid substitution selected from the group consisting of N76Y, K320N, D506E, L637I, and S713N.
5. The variant E. coli lysine decarboxylase enz me of claim 2, wherein the at least one other amino acid substitution further comprises each of K320N and D506E.
6. The variant E. coli lysine decarboxylase enzy me of claim 2, wherein the at least one other amino acid substitution further comprises each of K320N and D506E and at least one additional ammo acid substitution selected from the group consisting of R28A, T444S, L637I, I644V, D577N, and S713N.
7. The variant E. coll lysine decarboxylase enzyme of claim 2, wherein the at least one additional amino acid substitution further comprises each of K320N, D506E, L637I, I644V, and S713N.
8. A variant of an E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1 except comprising each amino acid substitution of K320N, D501N, D506E, K595R, and L637I, wherein the variant enzyme exhibits greater specific activity7in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1.
9. The variant of an E. coli lysine carboxylase enzy me of claim 8 further comprising at least one substitution selected from the group consisting of R28A, N76Y, I303V. T461D, A700E, and S713N.
10. The variant of an E. coli lysine carboxylase enzy me of claim 9 further comprising at least one additional substitution selected from the group consisting of Y80H, I200L, T444S, and S469A.
11. The variant of an E. coli lysine carboxylase enzy me of claim 8 comprising at least two substitutions of R28A, N76Y, I303V, T461D, and S713N.
12. The variant of an E. coli lysine carboxylase enzyme of claim 8 comprising at least three substitutions of R28A, N76Y, I303V, T461D, and S713N.
13. The variant of an E. coli lysine carboxylase enzyme of claim 8 comprising at least four substitutions of R28A, N76Y, I303V, T461D, and S713N.
14. A variant of an A. coli lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1 except comprising each amino acid substitution of K.320N, T416D, D501N, D506E, K595R, and L637I wherein the variantenzy me exhibits greater specific activity in converting lysine to pentamethadiamine (PMDA) than the E. coll lysine carboxylase enzyme having a protein sequence according to SEQ ID NO: 1.
15. The variant s, coli lysine carboxylase enzyme of claim 14 further comprising at least one amino acid substitution selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N.
16. The variant s, coli lysine carboxylase enzyme of claim 14 further comprising at least two amino acid substitutions selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N.
17. The variant s, coli lysine carboxylase enzyme of claim 14 further comprising at least three amino acid substitutions selected from the group consisting of R28A, N76Y, 1303V, A702E, and S713N.
18. The variant E. coli lysine carboxylase enzy me of claim 14 further comprising a substitution of 1303V.
19. The variant E. coli lysine carboxylase enzy me of claim 18 further comprising at least one additional amino acid substitution selected from the group consisting ofR.28A, N76Y, A702E. and S713N.
20. A variant E. coli lysine carboxylase enzy me having a protein sequence according to SEQ ID NO: 1 except comprising each amino acid substitution indicated in any row of the table shown in Figure 3 wherein the variant enzyme exhibits greater specific activity in converting lysine to pentamethadiamine (PMDA) than the E. coli lysine carboxylase enzy me having a protein sequence according to SEQ ID NO: 1.