Bio-based nylon precursors with reduced organic and inorganic impurities

Abstract

Described herein is an improved method for producing bio-based nylon precursors with reduced organic and inorganic impurities. The method generally involves fermenting a microorganism engineered to produce lysine in a modified medium with low or reduced inorganic ion content, preferably by employing a medium with an ammonium dicarboxylate buffer system lacking non-essential inorganic ions, and directly crystallizing lysine from the spent lysine fermentation supernatant by adding a sufficient amount of dicarboxylic acid. Such a strategy aims to produce lysine dicarboxylate crystals that can be employed in downstream bioconversion processes for the production of cadaverine dicarboxylate with reduced organic and inorganic impurities, thereby improving downstream performance in polymerization reactions, for example, for polyamide synthesis.

Description

[Technical Field]

[0001] This disclosure relates to bio-based nylon precursors. More particularly, described herein are improved processes for the production of bio-based nylon precursors, such as lysine, cadaverine, and their dicarboxylates, that have reduced organic and inorganic impurities suitable for use in the synthesis of nylon. [Background technology]

[0002] Growing concerns about the global environment have led to a growing demand for polyamides derived from biological precursors, commonly known as biobased nylons. Most nylons are polymers made from the reaction of diamines and dicarboxylic acids. Biobased nylons, such as biobased nylon 6,6, are becoming increasingly important industrial materials. Biobased nylon 5,6 has been gaining increasing attention, but one challenge facing biobased nylon 5,6 is purity requirements, as impurities in nylon 5,6 products can cause yellowing, high-temperature intolerance, and other undesirable properties in subsequent applications. A key component of biobased nylon 5,6 is the five-carbon diamine cadaverine (pentamethylenediamine, PDA), which can be produced industrially, for example, by fermentation of microorganisms specially engineered for this purpose or by bioconversion involving enzymatic decarboxylation of L-lysine (Tsuge et al., 2016). The former has the disadvantage of low productivity due to the harmful effects of cadaverine on the host microorganism, while the latter is difficult to practically implement on an industrial scale due to the high cost and low purity of the substrate, L-lysine. It has been known that the fermentative production of cadaverine or lysine inevitably produces large amounts of inorganic salts due to the substrate, medium, pH adjustment process, etc., and that these inorganic salts require complex processing steps to remove. Furthermore, prior art studies have focused on reducing the amount of inorganic salts formed in the fermentative production of cadaverine or lysine. However, when producing cadaverine by fermentation, the field of biological fermentation traditionally favors a "one-pot" continuous process, which minimizes intermediate purification steps (e.g., intermediate purification steps) to improve overall efficiency and reduce operating costs, and the costly purification steps are usually postponed until the final product is produced. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] EP 1182261 [Patent Document 2] US 2019 / 0040429 [Patent Document 3] U.S. Patent No. 7,189,543 Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there remains a need for technological advances that will better enable bio-based nylon to compete with petroleum-based nylon on an industrial scale, and in particular, there remains a need for technological advances to produce bio-based nylon and its precursors in highly efficient, high-purity processes. [Means for solving the problem]

[0005] The inventors have unexpectedly discovered that, by using the methods disclosed herein, cadaverine dicarboxylate can be obtained in high yield and purity, and that nylon 5,X polymers prepared from such cadaverine dicarboxylate are of high quality and do not, for example, yellow or tolerate high temperatures during use.

[0006] In one aspect, described herein is a method for producing cadaverine dicarboxylate having reduced inorganic ion content, comprising the steps of: (a) providing a fermentation broth comprising a microorganism submerged in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) adding less than one equivalent or an excess of dicarboxylic acid to obtain lysine dicarboxylate crystals from the fermentation broth. (d) subjecting lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system to said solution; and (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of an organic solvent to the solution.

[0007] In a further aspect, described herein is a method for producing cadaverine dicarboxylate having reduced inorganic ion content, comprising the steps of: (a) providing a fermentation broth comprising a microorganism submerged in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) obtaining a lysine dicarboxylate stream from the fermentation broth, wherein the inorganic ion content of the lysine dicarboxylate stream is reduced compared to a corresponding lysine mineral stream obtained by a method that uses inorganic anions instead of dicarboxylate anions in the buffer system. (d) subjecting lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the lysine dicarboxylate stream at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system to said stream, thereby producing a solution comprising cadaverine dicarboxylate; and (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol, and particularly preferably isopropanol.

[0008] In a further aspect, described herein is a method for producing cadaverine dicarboxylate having reduced inorganic ion content, comprising the steps of: (a) providing a fermentation broth comprising a microorganism submerged in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) obtaining a lysine dicarboxylate stream from the fermentation broth, wherein inorganic anions are substituted for dicarboxylate anions in the buffer system. (d) subjecting lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the lysine dicarboxylate stream at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system; adding a highly active lysine decarboxylase described herein at a lysine decarboxylase addition rate that is significantly reduced compared to the prior art, thereby producing a solution comprising cadaverine dicarboxylate; and (e) adding a sufficient volume of an organic solvent to the solution to crystallize the cadaverine dicarboxylate.

[0009] The highly active lysine decarboxylase described herein has relatively high activity, allowing it to be used in significantly reduced amounts during fermentation. Furthermore, the inventors have discovered that the use of the highly active lysine decarboxylase described herein can also improve the yield and purity of the target product, cadaverine dicarboxylate. The highly active lysine decarboxylase described herein is a lysine decarboxylase produced by a method comprising the following steps: (1) a plasmid containing the kdc gene of the lysine decarboxylase represented by SEQ ID NO: 1 was transformed into Escherichia coli (E. coli) cells; (2) a positive single transformed colony was selected and inoculated into LB (10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride) test tube medium, which was then inoculated overnight at 30°C and 180 RPM; (3) a flask containing TB medium (12 g / L peptone, 24 g / L yeast extract, and 4 g / L glycerol) was inoculated from the overnight culture at a 5% inoculum volume; and (4) the flask was placed in a shaker and incubated. Incubation conditions: 30°C, 250 RPM, and approximately 2 hours. (5) Protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Induction conditions: 30°C, 250 RPM, and approximately 4 hours. (6) The fermentation broth was centrifuged to harvest the cells, and the wet cells after centrifugation were stored at -80°C.

[0010] In a further aspect, described herein is a method for producing cadaverine dicarboxylate having a reduced inorganic ion content, comprising the steps of: (a) providing a fermentation broth comprising a microorganism submerged in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) adding less than one equivalent or an excess of a dicarboxylic acid to obtain lysine dicarboxylate crystals, and dissolving the crystals in an aqueous solution to obtain a lysine dicarboxylate stream from the fermentation broth, wherein the lysine dicarboxylate stream has a reduced inorganic ion content compared to a lysine inorganic salt stream obtained by a corresponding method that uses an inorganic anion instead of a dicarboxylate anion in the buffer system. (d) subjecting lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system; and adding a high activity lysine decarboxylase described herein at a lysine decarboxylase addition rate that is significantly reduced compared to the prior art, thereby producing a solution containing cadaverine dicarboxylate. (e) crystallizing cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol, and particularly preferably isopropanol.

[0011] In further aspects, described herein are cadaverine dicarboxylates produced by the methods described herein and the use of cadaverine dicarboxylates produced by the methods described herein for making nylon.

[0012] In a further aspect, described herein is a fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, and the medium is modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, wherein the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth that employs inorganic anions instead of dicarboxylate anions in the buffer system.

[0013] In a further aspect, described herein is a lysine dicarboxylate stream obtained from a lysine fermentation, the lysine dicarboxylate stream comprising lysine cations, dicarboxylate anions, and a medium modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, the lysine dicarboxylate stream having a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0014] In a further aspect, described herein is a method for producing lysine dicarboxylate crystals with reduced inorganic impurities and improved performance in downstream enzymatic bioconversion reactions to produce cadaverine dicarboxylate. The method generally comprises, or consists essentially of, the steps of: (a) providing a fermentation broth containing a microorganism immersed in a modified medium, the microorganism engineered to produce lysine from a carbon source, the medium modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by adding ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) adding a sufficient amount of dicarboxylic acid to the spent fermentation broth to induce the formation of lysine dicarboxylate crystals. The produced lysine dicarboxylate crystals have a reduced inorganic ion content compared to lysine inorganic salt obtained by a corresponding method that uses inorganic anions instead of dicarboxylate anions in the buffer system.

[0015] In a further aspect, described herein is a method for producing cadaverine dicarboxylate having reduced organic and inorganic impurities, comprising or consisting essentially of steps (a)-(c) as defined above, and further comprising the steps of: (d) dissolving the lysine dicarboxylate crystals isolated from step (c) in an aqueous solution and subjecting the lysine to an enzymatic decarboxylation reaction while adding a dicarboxylic acid to the solution to maintain the pH of the solution at a level sufficient for the reaction to occur, thereby producing a solution containing cadaverine dicarboxylate; and (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol.

[0016] In further aspects, described herein are lysine dicarboxylate or cadaverine dicarboxylate produced by the methods described herein, and the use of lysine dicarboxylate or cadaverine dicarboxylate produced by the methods described herein for making nylon.

[0017] In a further aspect, described herein is a fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, and the medium is modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, wherein the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth that employs inorganic anions instead of dicarboxylate anions in the buffer system.

[0018] In a further aspect, described herein is a lysine dicarboxylate stream obtained from a lysine fermentation, the lysine dicarboxylate stream comprising lysine cations, dicarboxylate anions, and a medium modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, the lysine dicarboxylate stream having a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0019] General definition Headings and other identifiers, such as (a), (b), (i), (ii), etc., are provided solely to facilitate the readability of the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements to be performed in alphabetical or numerical order or in the order in which they are presented.

[0020] When used in conjunction with the term "comprising" in the claims and / or specification, the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0021] The term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. Generally, the term "about" is meant to indicate a possibility of up to a 10% variation. Thus, variations in values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10% are encompassed by the term "about." Unless otherwise indicated, the use of the term "about" before a range applies to both ends of the range.

[0022] As used herein, the terms "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include") or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or process / method steps.

[0023] As used herein, the phrase "consisting essentially of" refers to elements necessary and non-essential for a given embodiment, excluding elements that would materially alter the invention. For example, the methods described herein generally relate to reducing or minimizing the amount of inorganic ions (or non-dicarboxylate ions) and / or increasing the purity of the nylon precursors discussed herein. Thus, the phrase "consisting essentially of" in this context refers to the excluding steps and elements that introduce undesirable additional inorganic ions (or non-dicarboxylate ions) and / or decrease the purity of the nylon precursor, thereby contradicting the teachings of the present description.

[0024] As used herein, the term "inorganic" in the context of the ions / counterions described herein refers to ions without C-H bonds (e.g., negative ions or anions), such as phosphate, sulfate, and / or chloride. Such inorganic ions are commonly used in the art of reaction solutions and / or acids / bases in fermentation buffers and / or buffering agents utilized to adjust or control pH.

[0025] As used herein, the term "lysine" generally refers to L-lysine unless otherwise specified. Generally, the fermentation broth in the methods described herein contains lysine in its monocationic form (Lys+), primarily as a dicarboxylate, depending on the pH range of the fermentation (e.g., pH 3-8). [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a graph showing the time course of a conventional lysine fermentation process in ammonium sulfate-containing medium, carried out as described in Example 4A. DETAILED DESCRIPTION OF THE INVENTION

[0027] As an important precursor in biobased nylon production, cadaverine (also known as pentamethylenediamine or 1,5-pentanediamine [PDA]) can be produced by enzymatically converting L-lysine with lysine decarboxylase. While the free base form of L-lysine is commercially available in relatively small quantities, the amino acid is expensive to purify as a free base, so it is currently produced and sold in industrial quantities only as relatively impure inorganic lysine hydrochloride or lysine sulfate. Because the majority of lysine produced on an industrial scale by fermentation is intended for use in animal feed mixtures, the lack of purity of commercially available lysine hydrochloride or lysine sulfate is not significant for this purpose. However, the presence of chloride or sulfate ions (and other organic and / or inorganic impurities) in commercially available lysine hydrochloride or lysine sulfate reduces the purity of the cadaverine and / or cadaverine dicarboxylate ultimately produced therefrom, thereby reducing their performance in nylon synthesis. Therefore, costly purification steps are traditionally employed to remove organic and inorganic impurities from biobased nylon precursors. Therefore, an improved method for producing high purity bio-based nylon precursors with reduced organic and inorganic impurities without sacrificing yield is highly desirable.

[0028] In some aspects, described herein are methods for producing lysine dicarboxylate crystals with reduced inorganic impurities, which can be directly employed in downstream bioconversion processes for the production of cadaverine dicarboxylate as a precursor for bio-based nylon synthesis. The methods generally involve fermenting a microorganism engineered to produce lysine from a carbon source in a modified medium with a low or reduced inorganic ion content compared to standard media traditionally employed in the fermentation of such engineered microorganisms (e.g., media utilizing inorganic counteranions such as ammonium sulfate or ammonium chloride). More generally, the methods described herein relate, in part, to strategies for reducing or minimizing the overall inorganic anion content (and / or the content of anions other than the desired dicarboxylate in the lysine salt product), thereby reducing or minimizing the amount of counteranion impurities present in the final intermediates and / or products (e.g., lysine dicarboxylate, cadaverine dicarboxylate, and nylons produced therefrom). More specifically, the methods described herein replace the inorganic ammonium salts (e.g., ammonium sulfate or ammonium chloride) employed in traditional methods with an ammonium dicarboxylate buffer system. Such a strategy may reduce production costs by reducing or avoiding downstream purification steps to remove undesirable inorganic ions (and / or non-dicarboxylate ions), such as by ion exchange and / or distillation, and may provide a higher purity product with improved performance in nylon synthesis reactions.

[0029] In some embodiments, the lysine fermentation methods described herein further comprise the step of crystallizing lysine as lysine dicarboxylate salt by adding a sufficient amount of the corresponding dicarboxylic acid directly to the spent fermentation broth / supernatant after fermentation is complete. Surprisingly, the yield / purity of lysine salt crystals obtained using dicarboxylic acids was comparable to or higher than that obtained using inorganic anions / inorganic acids (e.g., sulfate ions / sulfuric acid or chloride ions / hydrochloric acid), even though the latter method includes an additional desalting step by passage through an ion exchange column. In some embodiments, the amount of dicarboxylic acid added to crystallize the lysine dicarboxylate salt may be at least 1 equivalent relative to the moles of lysine in the spent fermentation broth. In some embodiments, the amount of dicarboxylic acid added to crystallize the lysine dicarboxylate salt may be in excess of the moles of lysine in the spent fermentation broth. Interestingly, it has been found that inducing crystallization of the lysine salt using an excess of dicarboxylic acid promotes crystal formation more than using an equal equivalent of dicarboxylic acid, resulting in increased yield. In particular, crystallization of lysine dicarboxylate with excess adipic acid was found to offer the highest combination of both yield and purity among the various conditions tested herein.

[0030] As used herein, the expressions "lysine dicarboxylate," "lysine dicarboxylate crystals," and "lysine dicarboxylate stream," when used in the context of the methods described herein, refer to a downstream product of the lysine fermentation method described herein, which has a reduced inorganic ion content compared to a lysine salt stream obtained from a corresponding lysine fermentation method that uses inorganic anions (e.g., sulfate, phosphate, or chloride) instead of dicarboxylate anions in the dicarboxylate buffer system. For clarity, the lysine dicarboxylate and lysine dicarboxylate stream described herein include compositions obtained downstream of lysine fermentation that may be subjected to centrifugation and / or filtration steps to remove cells and / or cell debris, and / or may be subjected to a crystallization step to obtain lysine dicarboxylate crystals. However, the lysine dicarboxylate stream described herein preferably excludes additional purification steps aimed at removing inorganic ions, such as ion exchange and / or other desalting methods conventionally employed for this purpose.

[0031] In some aspects, described herein are methods for producing lysine dicarboxylate, lysine dicarboxylate crystals, and lysine dicarboxylate streams having reduced inorganic ion content (and / or reduced non-dicarboxylate ion content), comprising or consisting essentially of providing a fermentation broth comprising a microorganism submerged in a modified medium, wherein the microorganism has been engineered to produce lysine from a carbon source, the medium being modified to include a dicarboxylate buffer system (e.g., an ammonium dicarboxylate buffer system such as an ammonium adipate buffer system), e.g., in place of a medium or buffer system employing an inorganic counteranion (e.g., phosphate, sulfate, and / or chloride) or a non-dicarboxylate counteranion (e.g., carbonate / bicarbonate). The method further comprises fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production, while controlling the pH of the fermentation broth by addition of a nitrogen source (e.g., selected from ammonium hydroxide and ammonium dicarboxylate) to maintain the pH in a range that promotes lysine production. The method further comprises obtaining lysine dicarboxylate crystals and / or a lysine dicarboxylate stream from the fermentation broth, wherein the lysine dicarboxylate stream has a reduced inorganic ion content compared to a lysine inorganic salt stream obtained by a corresponding method that uses inorganic anions instead of dicarboxylate anions in the buffer system.

[0032] As used herein, the phrases "non-dicarboxylate ions" or "non-dicarboxylate anions" refer to anions of dicarboxylic acids other than those employed in the ammonium dicarboxylate buffer systems described herein or other than those present in the lysine dicarboxylate or cadaverine dicarboxylate produced by the methods described herein. Preferably, a single type of non-essential dicarboxylate anion (e.g., ammonium adipate) and corresponding dicarboxylic acid (e.g., adipic acid) are employed throughout the lysine fermentation and cadaverine dicarboxylate production methods described herein, thereby providing a greater degree of uniformity in the dicarboxylates produced therefrom. For clarity, unless otherwise specified, the phrases "dicarboxylate" and "dicarboxylic acid" as used herein refer to the species of dicarboxylate anion and corresponding dicarboxylic acid intended to be incorporated into the dicarboxylate (e.g., lysine dicarboxylate and / or cadaverine dicarboxylate) produced by the methods described herein.

[0033] In some embodiments, modified media employed in the methods described herein are preferably formulated to lack non-essential inorganic counterions (and / or non-essential non-dicarboxylate anions). As used herein, the term "non-essential" in the context of inorganic and / or non-dicarboxylate counter anions refers to reducing the amount of such anions (e.g., phosphate, sulfate, chloride, and / or carbonate / bicarbonate) in a given medium or fermentation broth so that the cultured / fermented engineered microorganism can fully perform its desired function in the fermentation process, while removing excess inorganic and / or non-dicarboxylate counter anions. Such modified media may be referred to as "minimal inorganic anion media" or "minimal non-dicarboxylate anion media," and may be adapted for various microorganisms and / or fermentation conditions and fermentation objectives (i.e., cell growth versus lysine production). For example, in the case of microorganisms fermented to produce lysine (e.g., stationary-phase microorganisms), non-essential inorganic and / or non-dicarboxylic acid counteranions may refer to such anions present in the culture medium or fermentation broth that are not required for this purpose or that are present in an amount greater than the minimum required for lysine production. For clarity, the minimum essential inorganic and / or non-dicarboxylic acid counteranions in the context of lysine production (e.g., the production fermentation phase) may not be the same as those essential for microbial growth (e.g., the growth fermentation phase). Thus, in some embodiments, a first modified medium lacking non-essential inorganic and / or non-dicarboxylic acid counteranions for cell growth can be employed in the growth phase of fermentation, and the microorganisms are cultured until a desired level of cell biomass is reached for cell growth. In some embodiments, a second modified medium lacking non-essential inorganic and / or non-dicarboxylic acid counteranions for lysine production can be employed in the production phase of fermentation, and the microorganisms are cultured for lysine production instead of cell growth. It is understood that the type and exact concentration of inorganic anions and / or non-dicarboxylic acid anions present in the minimal medium will vary depending on the microorganism selected for lysine fermentation and may be determined empirically as a function of the fermentation process conditions employed.

[0034] In some embodiments, described herein is a modified medium for use in an industrial process for the fermentative production of a lysine dicarboxylate stream using an engineered microorganism, the modified medium comprising a dicarboxylate buffer system (e.g., an ammonium dicarboxylate buffer system such as an ammonium adipate buffer system) and preferably lacking non-essential inorganic and / or non-dicarboxylic acid counter anions. In some embodiments, described herein is a modified fermentation broth for use in an industrial process for the fermentative production of a lysine dicarboxylate stream, the modified fermentation broth comprising a microorganism engineered to produce lysine from a carbon source immersed in a modified medium comprising a dicarboxylate buffer system (e.g., an ammonium dicarboxylate buffer system such as an ammonium adipate buffer system), and preferably lacking non-essential inorganic and / or non-dicarboxylic acid counter anions. In some embodiments, the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth employing inorganic anions instead of dicarboxylate anions in the buffer system.

[0035] In some embodiments, described herein is a fermentation broth comprising a microorganism immersed in a modified medium, the microorganism engineered to produce lysine from a carbon source, the medium modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions. In some embodiments, the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth employing inorganic anions instead of dicarboxylate anions in the buffer system. In some embodiments, the fermentation broth may be supplemented with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production. In some embodiments, the fermentation broth may have a dicarboxylate salt ratio (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0036] In some embodiments, described herein is a lysine dicarboxylate stream obtained from lysine fermentation, the lysine dicarboxylate stream comprising lysine cations, dicarboxylate anions, and a medium modified to include an ammonium dicarboxylate buffer system and preferably modified to lack non-essential inorganic ions, the lysine dicarboxylate stream having a dicarboxylate ratio (DSR), as calculated above, of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%.

[0037] In some embodiments, the methods described herein further comprise, during the lysine fermentation step, supplementing the fermentation broth with a nitrogen source sufficient for lysine production by the microorganism engineered to produce lysine. In some embodiments, the methods described herein comprise supplementing the fermentation broth with a nitrogen source selected from ammonium hydroxide and ammonium dicarboxylate. In some embodiments, the only nitrogen source supplemented to the fermentation broth after the start of fermentation is selected from ammonium hydroxide and ammonium dicarboxylate. The choice of whether to supplement with ammonium hydroxide or ammonium dicarboxylate can be made based on the level of pH to be maintained, as the former is more basic than the latter. In some embodiments, the methods described herein comprise supplementing the fermentation broth with ammonium hydroxide and / or ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production. In some embodiments, the nitrogen source is replenished when the total ammonium level drops to about 0.15% (wt / vol) and / or to maintain the total ammonium concentration at a level of about 0.05%-0.5%, 0.05%-0.45%, 0.05%-0.4%, 0.05%-0.35%, 0.05%-0.3%, 0.05%-0.25%, 0.05%-0.2%, 0.1%-0.4%, 0.1%-0.45%, 0.1%-0.4%, 0.1%-0.35%, 0.1%-0.3%, 0.1%-0.25%, 0.1%-0.2%, or about 0.15% (wt / vol).

[0038] In some embodiments, the methods described herein comprise obtaining lysine dicarboxylate crystals and / or a lysine dicarboxylate stream from a fermentation broth via a crystallization process comprising adding an equivalent or excess amount of dicarboxylic acid to obtain lysine dicarboxylate crystals, which can then be filtered, dried, and dissolved in an aqueous solution. Advantageously, the lysine dicarboxylate crystals can be dissolved in an aqueous solution and then directly employed in a subsequent bioconversion process to enzymatically convert lysine to cadaverine, without additional lysine purification steps, such as ion exchange or desalting methods conventionally used in the art.

[0039] In some embodiments, the methods described herein do not include the addition of any further sources of non-essential inorganic ions, thereby minimizing the amount of inorganic ions present in the lysine dicarboxylate stream and thus in downstream intermediates or products derived therefrom. In some embodiments, the methods described herein do not include a purification step to remove inorganic and / or non-dicarboxylate anions from the fermentation broth and / or lysine dicarboxylate stream.

[0040] In some embodiments, the inorganic anions referred to in the methods described herein are or include phosphate, sulfate, chloride, and / or other non-essential inorganic anions conventionally used in microbial fermentation. This contrasts with conventional fermentation methods for producing lysine, which typically employ sulfate or chloride anions added to the medium as lysine counteranions to maintain electroneutrality, with ammonium sulfate being the primary source of sulfate. Furthermore, conventional lysine fermentation methods require the purification of lysine from the fermentation broth, most commonly by ion exchange. For example, in the case of lysine, the fermentation broth is made weakly acidic, and the lysine is typically adsorbed onto an ion exchange resin and then desorbed from the resin with ammonium ions. The desorbed lysine is then used "as is" as a lysine base or crystallized as lysine hydrochloride with hydrochloric acid. On the other hand, if a lysine purification step is not employed in conventional methods, residual sulfate and chloride ions remain, reducing the purity and quality of the product. While such low-purity products are not commercially important for lysine use in animal feed mixtures, etc., they are prohibited for use as a precursor in bio-based nylon synthesis.

[0041] Methods such as those described in EP 1182261 and US 2019 / 0040429 employ carbonate / bicarbonate counterions to attempt to reduce the presence of chloride or sulfate ions in the fermentation broth. However, such approaches require an additional heating step to distill off the carbonate / bicarbonate ions at the end of fermentation, thereby increasing the complexity and expense of the lysine production process. In contrast, in some embodiments, the methods described herein do not include the use of a carbonate buffer system (e.g., ammonium carbonate and / or ammonium bicarbonate) and / or do not include carbonate or carbonate ions as lysine counter anions. Furthermore, in some embodiments, the methods described herein do not include or require a distillation step to purify lysine from the lysine dicarboxylate stream, thereby streamlining the process.

[0042] In some embodiments, any suitable microorganism engineered to produce lysine from a carbon source may be employed. Such engineered microorganisms are widely used in the field of commercial lysine production, and any such engineered microorganism may be suitable for the methods described herein. In some embodiments, the microorganism engineered to produce lysine described herein may belong to the genus Corynebacterium (e.g., Corynebacterium glutamicum) or Brevibacterium (e.g., Brevibacterium flavum or Brevibacterium lactofermentum). In some embodiments, the microorganism engineered to produce lysine may be engineered Escherichia coli. In some embodiments, the microorganism engineered to produce lysine may be a fungus or yeast. In some embodiments, the yeast or fungus may belong to the genus Saccharomyces (e.g., Saccharomyces cerevisiae), Pichia (e.g., Pichia kudriavzevii), or Candida (e.g., Candida albicans), or other industrially suitable yeast or fungal species.

[0043] In some embodiments, the methods described herein involve fermenting a microorganism in the presence of a suitable carbon source (e.g., sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose; oils and fats, such as soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids, such as palmitic acid, stearic acid, linoleic acid; alcohols, such as glycerol and ethanol; and organic acids, such as acetic acid). In some embodiments, these substances may be used individually or as a mixture.

[0044] In some embodiments, the methods described herein include fermenting the microorganism under culture conditions that allow for lysine production while maintaining the pH of the fermentation broth at a level that promotes lysine production by the microorganism by adding a suitable base and / or acid. In some embodiments, controlling the pH during fermentation may include adding a suitable base, such as ammonium hydroxide, to the fermentation broth in an amount sufficient to prevent a decrease in the pH of the fermentation broth. In some embodiments, the addition of ammonium hydroxide also provides a continuous nitrogen source to the microorganism for lysine production, thereby serving a dual purpose (pH control and nitrogen source) during fermentation. If necessary, the pH of the fermentation broth in some embodiments may be lowered / adjusted by adding organic acids, such as dicarboxylic acids (e.g., adipic acid) and / or ammonium dicarboxylates (e.g., ammonium adipate). The dicarboxylic acid and / or ammonium dicarboxylate may be selected from succinic acid / ammonium succinate, glutaric acid / ammonium glutarate, adipic acid / ammonium adipate, pimelic acid / ammonium pimelate, suberic acid / ammonium suberate, azelaic acid / ammonium azelaate, sebacic acid / ammonium sebacate, undecanedicarboxylic acid / ammonium undecanedicarboxylate, dodecanedicarboxylic acid / ammonium dodecanedicarboxylate, tridecanedicarboxylic acid / ammonium tridecanedicarboxylate, tetradecanedicarboxylic acid / ammonium tetradecanedicarboxylate, pentadecanedicarboxylic acid / ammonium pentadecanedicarboxylate, hexadecanedicarboxylic acid / ammonium hexadecanedicarboxylate, heptadecanedicarboxylic acid / ammonium heptadecanedicarboxylate, octadecanedicarboxylic acid / ammonium octadecanedicarboxylate. Preferably, the dicarboxylic acid and / or ammonium dicarboxylate is selected from adipic acid / ammonium adipate, pimelic acid / ammonium pimelate, suberic acid / ammonium suberate, azelaic acid / ammonium azelate.

[0045] In some embodiments, the methods described herein include obtaining a lysine dicarboxylate stream at the end of fermentation. In some embodiments, the methods described herein may not include or require a lysine purification step to remove inorganic and / or non-dicarboxylate ions (e.g., phosphate, sulfate, chloride, carbonate / bicarbonate, or other non-dicarboxylate anions). In some embodiments, the fermentation broth may be subjected to a centrifugation and / or filtration step to remove cells and / or cell debris, and / or a crystallization step to obtain lysine dicarboxylate crystals, which may be subsequently redissolved in an aqueous solution. In particularly preferred embodiments, the fermentation broth may be subjected to a crystallization step to obtain lysine dicarboxylate crystals, which may be subsequently redissolved in an aqueous solution. In some embodiments, the lysine dicarboxylate stream thus obtained may advantageously be used directly in a subsequent decarboxylation reaction to produce cadaverine and / or cadaverine dicarboxylate.

[0046] The present inventors have discovered that, compared to the production method of cadaverine by one-pot fermentation, purification by crystallization of an intermediate lysine salt can increase the yield and purity of the product cadaverine (significantly reducing, and in some cases even reducing to zero, the amount of impurities such as acetic acid, protein, lactic acid, and pyruvic acid), as well as increase light transmittance and avoid yellowing, without requiring an additional purification step of lysine, such as an ion exchange method or desalting method conventionally used in the art. In some embodiments, compared to cadaverine dicarboxylate (preferably adipate) produced by one-pot fermentation, cadaverine dicarboxylate produced by crystallizing an intermediate lysine dicarboxylate (preferably adipate) can have one or more of the following characteristics: (1) increased yield; (2) absence of impurities such as acetic acid, lactic acid, and pyruvic acid; (3) significant reduction in the amount of protein; (4) increased light transmittance; and (5) absence of yellowing. In another embodiment, it has been found that crystallization using an excess amount of dicarboxylic acid can further increase the yield and purity of the product cadaverine salt compared to crystallization using an equal equivalent amount of dicarboxylic acid.

[0047] In some embodiments, the cadaverine dicarboxylate produced using the crystalline lysine dicarboxylate stream has a higher yield and purity compared to the yield and purity of the corresponding inorganic salts of cadaverine (e.g., sulfate and chloride salts) produced from crystalline lysine sulfate or lysine chloride. In another embodiment, when an excess amount of dicarboxylic acid is used to form the lysine dicarboxylate, the final cadaverine dicarboxylate can be produced in a higher yield compared to lysine dicarboxylate formed using an equal equivalent amount of dicarboxylic acid.

[0048] In yet a further embodiment, when forming the lysine dicarboxylate salt described herein, the dicarboxylic acid used may be selected from succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedicarboxylic acid, dodecanedicarboxylic acid, tridecanedicarboxylic acid, tetradecanedicarboxylic acid, pentadecanedicarboxylic acid, hexadecanedicarboxylic acid, heptadecanedicarboxylic acid, and octadecanedicarboxylic acid. In particular, adipic acid, suberic acid, and azelaic acid can be used. Furthermore, it has surprisingly been found that if the carbon chain length of the dicarboxylic acid used is too long (e.g., dodecanedicarboxylic acid), the yield and purity of the final cadaverine dicarboxylate salt product decrease.

[0049] In some embodiments, the methods described herein include obtaining a lysine dicarboxylate stream from a fermentation broth that has a reduced inorganic ion content compared to a fermentation broth produced by a corresponding method that substitutes inorganic ions for dicarboxylate ions (i.e., in the buffer system employed in the culture medium).

[0050] In some embodiments, the methods described herein may include a growth phase prior to the lysine production phase that comprises culturing the microorganism in a growth medium (e.g., formulated to include an excess of inorganic ions considered non-essential for the lysine production phase) to optimize or maximize cell growth until a desired level of cell mass is reached, whereafter the growth medium is replaced with a modified medium lacking the non-essential inorganic ions described herein. Alternatively, in some embodiments, the methods described herein may include a growth phase prior to the lysine production phase that comprises culturing the microorganism in a minimal growth medium (e.g., formulated to include an ammonium dicarboxylate buffer system and lacking non-essential inorganic salts) to optimize or maximize cell growth until a desired level of cell mass is reached, where the minimal growth medium is a modified medium or is replaced with a modified medium described herein.

[0051] In some embodiments, the microorganisms may be immobilized to facilitate medium exchange and / or downstream lysine purification. Examples of suitable cell immobilization methods are discussed and reviewed in Zhu 2007.

[0052] In some aspects, described herein are methods for producing cadaverine dicarboxylate with reduced inorganic salt content. The methods may comprise or consist essentially of the method steps described herein for the fermentative production of lysine with reduced inorganic ion content, and further comprise the step of subjecting a lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for the reaction to occur by adding a dicarboxylic acid to the solution, thereby producing a solution containing cadaverine dicarboxylate. In various embodiments, any suitable lysine decarboxylase enzyme can be employed in the decarboxylation reaction. In some embodiments, the lysine decarboxylase enzyme employed can be encoded by a nucleic acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1. In some embodiments, the lysine decarboxylase enzyme employed can comprise an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:2.

[0053] The lysine decarboxylase addition ratio can be defined as the ratio of the mass of lysine decarboxylase added (calculated on a dry basis of lysine decarboxylase cells) to the mass of lysine in the lysine fermentation broth (based on the molecular weight of lysine dicarboxylate). In the art, it is common for lysine decarboxylase addition ratios to be relatively high, for example, 1:80 to 1:295. However, when using the highly active lysine decarboxylases described herein, the lysine decarboxylase addition ratio during fermentation can be dramatically reduced due to their high activity. For example, the lysine decarboxylase addition ratio for the highly active lysine decarboxylases described herein can be 1:600 ​​to 1:1000.

[0054] Furthermore, the present inventors have discovered that the use of the highly active lysine decarboxylase described herein can also improve the yield and purity of the target product, cadaverine dicarboxylate. The highly active lysine decarboxylase described herein is a lysine decarboxylase produced by a method comprising the following steps: (1) A plasmid containing the kdc gene of the lysine decarboxylase represented by SEQ ID NO: 1 was transformed into E. coli cells. (2) A single positive transformed colony was selected and inoculated into LB (10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride) test tube medium, which was then inoculated overnight at 30°C and 180 RPM. (3) A flask containing TB medium (12 g / L peptone, 24 g / L yeast extract, and 4 g / L glycerol) was inoculated from the overnight culture at a 5% inoculum size. (4) The flask was placed in a shaker and incubated. Incubation conditions: 30°C, 250 RPM, and approximately 2 hours. (5) Protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Induction conditions: 30°C, 250 RPM, and approximately 4 hours. (6) The fermentation broth was centrifuged to harvest the cells, and the wet cells after centrifugation were stored at -80°C. In a preferred embodiment, the E. coli is BL21(DE3) E. coli. In a more preferred embodiment, the incubation conditions in step (2) were 30°C, 180 RPM, and incubation with shaking for 16 hours. In a more preferred embodiment, a flask containing TB medium was inoculated with a 5% inoculum volume from 40 mL of overnight-cultured LB medium fermentation broth. In a more preferred embodiment, the incubation time in step (4) was 2 hours. In a more preferred embodiment, the IPTG concentration was 0.2 mM. In a more preferred embodiment, in step (5), incubation with shaking was performed at 30°C for an additional 4 hours.

[0055] U.S. Patent No. 7,189,543 describes a method for producing cadaverine dicarboxylate, which involves subjecting a lysine solution to an enzymatic decarboxylation reaction while maintaining the pH within a target range by adding a dicarboxylic acid to the solution, and isolating the resulting cadaverine dicarboxylate, for example, by crystallization. Attempts to reproduce the separation / crystallization method described in U.S. Patent No. 7,189,543 have not resulted in commercially viable yields of cadaverine dicarboxylate (see, e.g., Example 18, where only a 45% yield was obtained). Accordingly, various crystallization methods have been explored and tested, some of which are described herein. Surprisingly, it has been found that crystallization can be significantly improved by adding a sufficient amount of organic solvent to the solution, preferably a cooled organic solvent, e.g., at a temperature below 25°C, 20°C, or 15°C (e.g., in the range of 5°C to 20°C or 10°C to 15°C). Thus, in some embodiments, the methods for producing cadaverine described herein include crystallizing cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution, where the addition of the organic solvent increases the amount of cadaverine dicarboxylate crystals recovered (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, and the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol.

[0056] Furthermore, the present inventors have surprisingly discovered that when cadaverine dicarboxylate is crystallized using an alcoholic solvent, both the yield and purity of the product obtained using isopropanol are higher than those obtained using common lower aliphatic alcohols (e.g., methanol, ethanol, and n-propanol). In one embodiment, the use of isopropanol to crystallize cadaverine adipate results in an increase in product yield of at least 10%, e.g., at least 11%, 12%, 13%, 14%, or 15%, compared to the yield obtained by crystallizing cadaverine adipate from n-propanol.

[0057] In some aspects, described herein are methods for producing cadaverine dicarboxylate having a reduced content of inorganic salts, comprising, or consisting essentially of, providing an aqueous solution comprising lysine dicarboxylate; subjecting the lysine dicarboxylate to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for the reaction to occur by adding a dicarboxylic acid to the solution, thereby producing a solution comprising cadaverine dicarboxylate; and adding a sufficient volume of organic solvent to the solution to crystallize the cadaverine dicarboxylate, wherein the addition of the organic solvent increases the amount of cadaverine dicarboxylate crystals recovered (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent.

[0058] In some embodiments, the enzymatic decarboxylation reactions described herein may be carried out by subjecting the lysine product or lysine dicarboxylate to viable or intact cells of a microorganism expressing lysine decarboxylase. In some embodiments, the viable or intact cells may be immobilized, for example, as described in Zhu 2007. In some embodiments, the enzymatic decarboxylation reactions described herein may be carried out by subjecting the lysine product or lysine dicarboxylate to a cell lysate of a microorganism expressing lysine decarboxylase. The use of viable or intact cells instead of a cell lysate may facilitate downstream purification of cellular components from the reaction solution.

[0059] In some embodiments, the terms "dicarboxylate" and "dicarboxylic acid," as used herein, refer to dicarboxylates and / or dicarboxylic acids useful as precursors or intermediates for the production of nylon salts, nylon, industrially relevant lysine salts, and / or industrially relevant cadaverine salts. In some embodiments, the dicarboxylates and / or dicarboxylic acids may be dicarboxylates and / or dicarboxylic acids having 4 to 10 carbons (e.g., succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, or sebacic acid). In particular embodiments, the dicarboxylates and / or dicarboxylic acids employed in the methods and products described herein are adipate and / or adipic acid.

[0060] In some aspects, described herein are cadaverine dicarboxylate salts produced by the methods described herein. In some aspects, described herein are uses of cadaverine dicarboxylate salts produced by the methods described herein for making nylon (e.g., nylon 5,6).

[0061] item In various aspects, described herein are one or more of the following:

[0062] 1. A method for producing lysine dicarboxylate crystals having reduced inorganic impurities, comprising, or consisting essentially of, the steps of: (a) providing a fermentation broth containing a microorganism submerged in a modified medium, wherein the microorganism has been engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system, preferably modified to lack non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production, while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; and (c) adding a sufficient amount of a dicarboxylic acid to the spent fermentation broth to induce the formation of lysine dicarboxylate crystals, wherein the inorganic ion content of the lysine dicarboxylate crystals is reduced compared to a corresponding lysine inorganic salt obtained by a method that substitutes inorganic anions for dicarboxylate anions in the buffer system.

[0063] 2. The method according to item 1, wherein the sufficient amount of dicarboxylic acid added in step (c) corresponds to at least 1 equivalent per mole of lysine in the spent fermentation broth.

[0064] 3. The method according to item 1, wherein the sufficient amount of dicarboxylic acid added in step (c) corresponds to an excess equivalent relative to the moles of lysine in the spent fermentation broth.

[0065] 4. The method according to any one of items 1 to 3, wherein step (b) further comprises supplementing the fermentation broth with an ammonium dicarboxylate solution so as to maintain the total ammonium concentration at a level that promotes lysine production, preferably so as to maintain the total ammonium concentration at 0.05% w / v to 0.5% w / v.

[0066] 5. The method of any one of items 1 to 4, wherein prior to crystallization, the spent fermentation broth has a dicarboxylate ratio (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0067] 6. The method according to any one of items 1 to 5, wherein the method (i) does not include the addition of any further source of non-essential inorganic anions, thereby minimizing the amount of inorganic anions present in the lysine dicarboxylate stream, and / or (ii) does not include a purification step (e.g., desalting and / or ion exchange) to remove inorganic ions from the fermentation broth and / or the lysine dicarboxylate stream.

[0068] 7. The method according to any one of items 1 to 6, wherein the inorganic ions are or include phosphate anions, sulfate anions, and / or chloride anions.

[0069] 8. The method according to any one of items 1 to 7, which does not include the use of a carbonate buffer system (e.g., ammonium carbonate and / or ammonium bicarbonate) and / or does not include carbonate or carbonate anions as lysine counter anions.

[0070] 9. The method according to any one of items 1 to 8, which does not include a distillation step for purifying the lysine.

[0071] 10. The method of any one of items 1 to 9, wherein prior to step (a), the microorganism is cultured in (i) a growth medium formulated to include inorganic salts or (ii) a growth medium formulated to lack non-essential inorganic salts until a desired cell mass is reached, and then the growth medium is replaced with the modified medium comprising an ammonium dicarboxylate buffer system and lacking non-essential inorganic ions.

[0072] 11. The method according to any one of items 1 to 10, wherein the microorganisms engineered to produce lysine are immobilized to facilitate medium exchange and / or lysine dicarboxylate stream processing.

[0073] 12. The method according to any one of items 1 to 11, wherein the microorganism engineered to produce lysine is a bacterium, preferably belonging to the genus Corynebacterium (e.g., Corynebacterium glutamicum) or Brevibacterium (e.g., Brevibacterium flavum or Brevibacterium lactofermentum).

[0074] 13. A method for producing cadaverine dicarboxylate salt having reduced organic and inorganic impurities, comprising or consisting essentially of steps (a) to (c) as defined in any one of items 1 to 12, and further comprising: (d) dissolving the lysine dicarboxylate crystals separated from step (c) in an aqueous solution, and subjecting the lysine to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for the reaction to occur by adding a dicarboxylic acid to the solution, thereby producing a solution containing cadaverine dicarboxylate; and (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol.

[0075] 14. A method for producing cadaverine dicarboxylate having reduced organic and inorganic impurities, comprising, or consisting essentially of, the steps of: (i) providing an aqueous solution containing lysine dicarboxylate; (ii) subjecting the lysine dicarboxylate to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for the reaction to occur by adding a dicarboxylic acid to the solution, thereby producing a solution containing cadaverine dicarboxylate; and (iii) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method not involving the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol.

[0076] 15. The method according to item 13 or 14, wherein the enzymatic decarboxylation reaction is carried out by providing lysine to living or intact cells of a microorganism expressing lysine decarboxylase.

[0077] 16. The method according to item 15, wherein viable or intact cells of a microorganism expressing lysine decarboxylase are immobilized.

[0078] 17. The method according to item 15 or 16, wherein the enzymatic decarboxylation reaction is carried out by providing lysine to a cell lysate of a microorganism expressing lysine decarboxylase.

[0079] 18. The method according to any one of items 15 to 17, wherein the lysine decarboxylase is the lysine decarboxylase of SEQ ID NO: 2 or a variant thereof having lysine decarboxylase activity comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

[0080] 19. The method according to any one of items 1 to 18, wherein the dicarboxylate and / or dicarboxylic acid contains 4 to 18 carbons, 4 to 16 carbons, 4 to 14 carbons, 4 to 12 carbons, 4 to 10 carbons, 4 to 9 carbons, 6 to 10 carbons, or 6 to 9 carbons.

[0081] 20. - The dicarboxylate is succinate and / or the dicarboxylic acid is succinic acid, thereby producing lysine dicarboxylate, which is lysine succinate; - the dicarboxylate is glutarate and / or the dicarboxylic acid is glutaric acid, thereby producing a lysine dicarboxylate that is lysine glutarate; - the dicarboxylate is adipate and / or the dicarboxylic acid is adipic acid, thereby producing a lysine dicarboxylate that is lysine adipate; - the dicarboxylate is pimelate and / or the dicarboxylic acid is pimelic acid, thereby producing a lysine dicarboxylate that is lysine pimelate; - the dicarboxylic acid salt is suberate and / or the dicarboxylic acid is suberic acid, thereby producing a lysine dicarboxylate which is lysine suberate; - the dicarboxylic acid salt is azelaic acid salt and / or the dicarboxylic acid salt is azelaic acid, thereby producing a lysine dicarboxylate salt that is lysine azelaate; - the dicarboxylate is sebacate and / or the dicarboxylic acid is sebacic acid, thereby producing a lysine dicarboxylate that is lysine sebacate; - the dicarboxylate is undecanedicarboxylate and / or the dicarboxylic acid is undecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine undecanedicarboxylate; - the dicarboxylate is dodecanedicarboxylate and / or the dicarboxylic acid is dodecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine dodecanedicarboxylate; - the dicarboxylate is tridecanedicarboxylate and / or the dicarboxylic acid is tridecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine tridecanedicarboxylate; - the dicarboxylate is tetradecanedicarboxylate and / or the dicarboxylic acid is tetradecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine tetradecanedicarboxylate; - the dicarboxylate is pentadecanedicarboxylate and / or the dicarboxylic acid is pentadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine pentadecanedicarboxylate; - the dicarboxylate is hexadecanedicarboxylate and / or the dicarboxylic acid is hexadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine hexadecanedicarboxylate; - the dicarboxylate is heptadecanedicarboxylate and / or the dicarboxylic acid is heptadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine heptadecanedicarboxylate; 20. The method according to any one of items 1 to 19, wherein the dicarboxylate is octadecanedicarboxylate and / or the dicarboxylic acid is octadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine octadecanedicarboxylate.

[0082] 21. Cadaverine dicarboxylate, produced by the method according to any one of items 13 to 20.

[0083] 22. Use of cadaverine dicarboxylate, produced by the method according to any one of items 13 to 20, for the production of nylon.

[0084] 23. The use according to item 22, wherein the cadaverine dicarboxylate is cadaverine adipate and the nylon is nylon 5,6.

[0085] 24. A fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism has been engineered to produce lysine from a carbon source, and the medium has been modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, such that the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth employing inorganic anions instead of dicarboxylate anions in the buffer system.

[0086] 25. The fermentation broth according to item 24, wherein the fermentation broth is supplemented with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production, preferably to maintain the total ammonium concentration at 0.05% w / v to 0.5% w / v.

[0087] 26. The fermentation broth according to item 24 or 25, having a dicarboxylate salt ratio (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0088] 27. The fermentation broth according to item 24 or 26, wherein the microorganism engineered to produce lysine is as defined in item 11 or 12.

[0089] 28. A lysine dicarboxylate stream obtained from a lysine fermentation, comprising lysine cations, dicarboxylate anions, wherein the medium has been modified to include an ammonium dicarboxylate buffer system and preferably lacks non-essential inorganic ions, and wherein the stream has a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0090] 29. The fermentation broth according to any one of items 24 to 27, or the lysine dicarboxylate stream according to item 28, wherein the inorganic ions are or comprise phosphate anions, sulfate anions, and / or chloride anions.

[0091] 30. The fermentation broth according to any one of items 24 to 27 or 29, or the lysine dicarboxylate stream according to item 28 or 29, which does not contain a carbonate buffer system (e.g., ammonium carbonate and / or ammonium bicarbonate) and / or does not contain carbonate or carbonate anions as lysine counter anions. [Example]

[0092] Example 1 General Materials and Methods The following reference materials are used in the examples. Recombinant DNA manipulations generally follow the methods described in Sambrook et al. (2001). Restriction enzymes, T4 DNA ligase, Rapid DNA Ligation Kit, SanPrep Column DNA Gel Extraction Kit, Plasmid Mini-Prep Kit, and agarose were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). TE buffer contains 10 mM Tris-HCl (pH 8.0) and 1 mM Na2EDTA (pH 8.0). TAE buffer contains 40 mM Tris-acetate (pH 8.0) and 2 mM Na2EDTA.

[0093] In Example 2, restriction enzyme digestion was performed in buffers provided by Sangon Biotech.

[0094] A typical restriction enzyme digestion contains 0.8 μg of DNA, 2 μL of restriction enzyme buffer (10x concentration), 1 μL of bovine serum albumin (0.1 mg / mL), 1 μL of restriction enzyme, and 8 μL of TE. The reaction is incubated at 37°C for 1 hour and analyzed by agarose gel electrophoresis. DNA used in cloning experiments was digested and terminated by heating at 70°C for 15 minutes, after which DNA was extracted using a SanPrep Column DNA Gel Extraction Kit. The DNA concentration in the sample was determined as follows: An aliquot of DNA (10 μL) was diluted to 1 mL with TE, and the absorbance at 260 nm was measured relative to the absorbance of TE. The DNA concentration was calculated based on the fact that the absorbance at 260 nm of 50 μg / mL double-stranded DNA is 1.0.

[0095] Agarose gels typically contain 0.7% agarose (mass / volume) in TAE buffer. Ethidium bromide (0.5 μg / ml) was added to the agarose to allow visualization of DNA fragments under a UV lamp. Agarose gels were run in TAE buffer. DNA fragment sizes were determined using two sets of 1 kb Plus DNA Ladder (obtained from Sangon Biotech).

[0096] Example 2 Cloning, expression and activity testing of lysine decarboxylase expressed in Escherichia coli. The E. coli lysine decarboxylase kdc (2-keto acid decarboxylase) gene was synthesized and cloned into pET21a (Millipore Sigma, formerly Novagen). The wild-type kdc nucleic acid sequence from E. coli strain BW25113 (EC 4.1.1.18) is represented by SEQ ID NO: 1, and the amino acid sequence is represented by SEQ ID NO: 2, both of which are annotated as lysine decarboxylase.

[0097] A plasmid containing the kdc gene (SEQ ID NO: 1) was transformed into BL21(DE3) E. coli cells. The empty plasmid pET21a was also transformed as a negative control. For enzyme expression and characterization experiments, flasks containing 40 mL of TB were inoculated with 5% of the overnight culture and shaken. The flasks were incubated at 30°C with shaking at 250 rpm for 2 hours, after which protein production was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 30°C with shaking at 250 rpm for 4 hours, after which the incubation was terminated. Cells were harvested by centrifugation, and the collected wet cells were stored at -80°C.

[0098] KDC enzyme activity was assessed using a pH-based in vitro assay. Enzyme activity was tested using commercially available lysine-HCl salt. Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Cells were first lysed using a benchtop sonicator. The cell lysate was partially clarified by centrifugation (14,000 g for 5 minutes). The protein concentration of the resulting clarified lysate was measured using a Bradford Protein Assay Kit (Sangon Biotech). The lysate was normalized by dilution with 10 mM Tris buffer. The normalized lysate was then diluted 1:5 with 10 mM Tris buffer. For multiwell plate assays, 20 μL of lysate was added to each well. Each condition was performed in triplicate.

[0099] The reaction mixtures contained 15% lysine-HCl and 0.04% pyridoxal-5'-phosphate (PLP). The pH of each reaction mixture was adjusted to approximately 6.5 by adding 1 M H2SO4 and 1 N NaOH. The lysate was then added to the reaction mixture, constantly maintaining the pH at 6.5 by adding 1 M H2SO4. The amount of H2SO4 used was recorded and used to calculate the activity of the enzyme. The assay reaction was complete when no more H2SO4 was required to maintain the pH at 6.5.

[0100] Example 3 Fermentation of transformed E. coli overexpressing KDC For this example, growth media were prepared as follows: All solutions were prepared with distilled, deionized water. LB medium (1 L) contained Bacto™ tryptone (i.e., an enzymatic digest of casein) (10 g), Bacto™ yeast extract (i.e., the water-soluble portion of autolyzed yeast cells) (5 g), and NaCl (10 g). LB-glucose medium contained glucose (10 g), MgSO (0.12 g), and thiamine hydrochloride (0.001 g) in 1 L of LB medium. LB freezing buffer contained K2HPO4 (6.3 g), KH2PO4 (1.8 g), MgSO4 (1.0 g), (NH4)2SO4 (0.9 g), sodium citrate dihydrate (0.5 g), and glycerol (44 mL) in 1 L of LB medium. M9 salts (1 L) contained NaHPO (6 g), KHPO (3 g), NHCl (1 g), and NaCl (0.5 g). M9 minimal medium contained 1 L of M9 salts plus D-glucose (10 g), MgSO (0.12 g), and thiamine hydrochloride (0.001 g). Antibiotics were added as appropriate to final concentrations of the following: ampicillin (Ap), 50 μg / mL; chloramphenicol (Cm), 20 μg / mL; kanamycin (Kan), 50 μg / mL; and tetracycline (Tc), 12.5 μg / mL. Antibiotic stock solutions were prepared in water, except for chloramphenicol, which was prepared in 95% ethanol, and tetracycline, which was prepared in 50% aqueous ethanol. Aqueous stock solutions of IPTG were prepared at various concentrations.

[0101] Standard fermentation medium (1 L) contained KHPO (7.5 g), ammonium iron(III) citrate (0.3 g), citric acid monohydrate (2.1 g), and concentrated HSO (1.2 mL). Prior to autoclaving, the fermentation medium was adjusted to pH 7.0 by adding concentrated NHOH. Just before the start of fermentation, D-glucose, MgSO (0.24 g), potassium, and (NH)(MoO) were added. 24 A trace mineral supplement containing ZnSO₄·4H₂O (0.0037 g), ZnSO₄·7H₂O (0.0029 g), H₃BO₃ (0.0247 g), CuSO₄·5H₂O (0.0025 g), and MnCl₂·4H₂O (0.0158 g) was added. IPTG stock solution was added to the specified final concentration as needed (e.g., when the optical density at 600 nm was between 15 and 20). The glucose feed solution and MgSO₄ (1 M) solution were autoclaved separately. A glucose feed solution (650 g / L) was prepared by combining 300 g of glucose and 280 mL of H₂O. The trace mineral and IPTG solutions were sterilized through a 0.22 μm membrane. Antifoam (Sigma 204) was added to the fermentation broth as needed. Typical wet E. coli cell densities reached 120 g / L.

[0102] Example 4A Crystallization of lysine sulfate from lysine fermentation supernatant with excess sulfuric acid (1.2 equivalents) Lysine was obtained by fermentation of a commercially available strain of Corynebacterium glutamicum engineered to produce lysine from carbohydrates. C. glutamicum was fermented while maintaining strict control of temperature (generally between 25°C and 37°C, with an optimum of approximately 30°C) and pH (between 7.0 and 7.4). Furthermore, because C. glutamicum is highly oxygen-demanding, the fermentation vessel was aerated during the exponential growth phase.

[0103] C. glutamicum was initially cultured in a seed medium containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride and transferred to three 1 L Erlenmeyer flasks in 400 mL volumes. The flasks were first sterilized by heating at 120°C for 20 minutes. After sterilization, the flasks were cooled to 30°C, and the medium was inoculated with a C. glutamicum colony grown on an LB plate. The resulting culture was incubated at 30°C and pH 7.0 for 9 hours with sufficient aeration and agitation.

[0104] After seed culture, C. glutamicum was then fermented at pH 7 in a medium containing 25 g / L glucose, 15 g / L ammonium sulfate, 1.5 g / L potassium dihydrogen phosphate, 5 g / L sodium glutamate, 1 g / L sodium chloride, and 0.5 g / L yeast extract. The medium was then introduced into a 15 L stainless steel fermenter in an 8 L volume (this volume increased over the course of the fermentation). The vessel was first sterilized by heating at 120 °C for 20 min. After sterilization, the vessel was cooled to 30 °C, and then 1 L of the seed culture was inoculated into the medium. The medium was incubated at 30 °C with aeration at 1 vvm and agitation at 800 rpm. pH was controlled by adding 25% ammonium hydroxide solution. When the glucose concentration in the medium dropped to 0.5-1.0%, a feed solution was continuously added to the medium to maintain the sugar level between 0.5 and 0.8%. The feed solution contained 110 g / L glucose, 8.9 g / L magnesium sulfate, 5.3 g / L potassium chloride, 4 g / L phosphoric acid, and 1.5 g / L yeast extract at pH 7.0. Ammonium sulfate solution (450 g / L) was replenished when the total ammonium level dropped to 0.15% to maintain the total ammonium level at 0.1-0.2%.

[0105] After fermentation was completed, glucose, ammonium hydroxide, and ammonium sulfate were added to increase the total volume of the fermentation broth to 10 L, and the cells were then separated from the spent medium by centrifugation. The cell-free supernatant was then clarified by microfiltration and ultrafiltration. The process stream was then passed through an ion exchange column for desalting and concentrated to obtain a solution containing 146 g / L of lysine. The product, lysine sulfate, was then crystallized by adding 1200 g (1.2 equivalents, 1200 g / L) of 98% sulfuric acid. The product was filtered and dried. The purity of the lysine sulfate (i.e., dilysine monosulfate) was then determined by HPLC analysis. The production of lysine by fermentation over time is shown in Figure 1. The lysine sulfate content was found to be 98.3%, with a yield of 82.6%.

[0106] Example 4B Crystallization of lysine hydrochloride from lysine fermentation supernatant with excess hydrochloric acid (1.2 equivalents) Lysine was obtained by fermentation of a commercially available C. glutamicum strain engineered to produce lysine from carbohydrates. C. glutamicum was fermented while maintaining strict control of temperature (generally between 25°C and 37°C, with an optimum temperature of approximately 15-30°C) and pH (between 7.0 and 7.4). Furthermore, because C. glutamicum is highly oxygen-requiring, the fermentation vessel was aerated during the exponential growth phase. C. glutamicum was initially cultured in a seed medium containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and introduced into three 1 L Erlenmeyer flasks in 400 mL volumes. The flasks were first sterilized by heating at 120°C for 20 minutes. After sterilization, the flasks were cooled to 30°C, and the medium was inoculated with a C. glutamicum colony grown on an LB plate. The resulting culture was incubated at 30°C and pH 7.0 for 9 hours with sufficient aeration and agitation.

[0107] After seed culture, C. glutamicum was then fermented at pH 7 in a medium containing 25 g / L glucose, 15 g / L ammonium chloride, 1.5 g / L potassium dihydrogen phosphate, 5 g / L sodium glutamate, 1 g / L sodium chloride, and 0.5 g / L yeast extract. The medium was then introduced into a 15 L stainless steel fermenter in an 8 L volume (this volume increased over the course of the fermentation). The vessel was first sterilized by heating at 120 °C for 20 min. After sterilization, the vessel was cooled to 30 °C, and then 1 L of the seed culture was inoculated into the medium. The medium was incubated at 30 °C with aeration at 1 vvm and agitation at 800 rpm. pH was controlled by adding 25% ammonium hydroxide solution. When the glucose concentration in the culture medium decreased to 0.5-1.0%, a feed solution was continuously added to the medium to maintain the sugar level between 0.5 and 0.8%. The feed solution contained 110 g / L glucose, 8.9 g / L magnesium sulfate, 5.3 g / L potassium chloride, 4 g / L phosphoric acid, and 1.5 g / L yeast extract at pH 7.0. Ammonium chloride solution (450 g / L) was replenished when the total ammonium level dropped to 0.15% to maintain the total ammonium level at 0.1-0.2%.

[0108] After fermentation was completed, glucose, ammonium hydroxide, and ammonium chloride were added to increase the total volume of the fermentation broth to 10 L, and the cells were then separated from the spent medium by centrifugation. The cell-free supernatant was then clarified by microfiltration and ultrafiltration. The process stream was then desalted through an ion exchange column and concentrated to obtain a solution containing 146 g / L of lysine. The product, lysine sulfate, was then crystallized by adding 1251 g (125.1 g / L; 1.2 equivalents) of 35% hydrochloric acid. The product was filtered and dried. The purity of the lysine hydrochloride was then determined by HPLC analysis. The lysine hydrochloride content was found to be 99.1%, with a yield of 87.4%.

[0109] Example 5A Crystallization of lysine adipate from lysine fermentation supernatant with excess adipic acid (1.2 equivalents) Although C. glutamicum can utilize a variety of carbon and nitrogen sources for production, employing a minimal medium with an ammonium adipate buffer system (instead of ammonium sulfate or ammonium chloride) streamlines downstream processing by reducing the inorganic anion content throughout the process and in downstream products.

[0110] After seed culture as described in Example 4A, C. glutamicum was then fermented at pH 7.0 in a medium containing 25 g / L glucose, 10 g / L ammonium adipate, 0.5 g / L potassium dihydrogen phosphate, 5 g / L sodium glutamate, 0.1 g / L sodium chloride, and 0.5 g / L yeast extract, and introduced into a 15 L stainless steel fermenter in an amount of 8 L (the volume increased during the fermentation process). The vessel was first sterilized by heating at 120°C for 20 minutes. Ammonium adipate was prepared by adding 3.2 kg of adipic acid to 7 L of a 25% aqueous solution of ammonium hydroxide (3 kg). The resulting solution was stirred at 50°C for 4 hours. After sterilization of the fermentation medium, the vessel was cooled to 30°C. 1 L of the seed culture was then inoculated into the medium and cultivated at 30°C with aeration at 1 vvm and stirring at 800 rpm. The pH was controlled by adding 25% ammonium hydroxide solution. When the glucose concentration in the culture medium dropped to 0.5-1.0%, a glucose source was continuously added to the medium to maintain the sugar level between 0.5 and 0.8%. When the total ammonium level dropped to 0.15%, ammonium adipate solution (39.7%) was supplemented to maintain the total ammonium level at 0.1-0.2%.

[0111] After fermentation was completed, glucose, ammonium hydroxide, and ammonium adipate were added to increase the total volume of the fermentation broth to 10 L, and the cells were then separated from the spent medium by centrifugation. The cell-free supernatant was then clarified by microfiltration and ultrafiltration. At this point, the lysine concentration was 146 g / L, and 1752 g (1.2 equivalents, 175.2 g / L) of adipic acid was added to crystallize the product, lysine adipate. The product was filtered and dried. The purity of the lysine adipate was then determined by HPLC analysis. The lysine adipate content was found to be 99.3%, with a yield of 93.4%.

[0112] In this example, the adipate fraction was greater than 80%, and was calculated as follows: adipate fraction = [(molarity of adipate ions) × 2] / [molarity of monocationic lysine ions (Lys+)] × 100%. Advantageously, the lysine-adipate stream (separated from cells and cell debris) can then be employed directly as the starting material for the bioconversion of lysine adipate to PDA adipate shown in Examples 13-20, thereby avoiding the need for lysine purification by ion exchange prior to the bioconversion step.

[0113] Example 5B Crystallization of lysine adipate from lysine fermentation supernatant with 0.6 equivalents of adipic acid. Although C. glutamicum can utilize a variety of carbon and nitrogen sources for production, employing a minimal medium with an ammonium adipate buffer system (instead of ammonium sulfate or ammonium chloride) streamlines downstream processing by reducing the inorganic anion content throughout the process and in downstream products.

[0114] After seed culture as described in Example 4, C. glutamicum was then fermented at pH 7.0 in a medium containing 25 g / L glucose, 10 g / L ammonium adipate, 0.5 g / L potassium dihydrogen phosphate, 5 g / L sodium glutamate, 0.1 g / L sodium chloride, and 0.5 g / L yeast extract, and introduced into a 15 L stainless steel fermenter in an amount of 8 L (the volume increased during the fermentation process). The vessel was first sterilized by heating at 120°C for 20 minutes. Ammonium adipate was prepared by adding 3.2 kg of adipic acid to 7 L of a 25% aqueous solution of ammonium hydroxide (3 kg). The resulting solution was stirred at 50°C for 4 hours. After sterilization of the fermentation medium, the vessel was cooled to 30°C. 1 L of the seed culture was then inoculated into the medium and cultivated at 30°C with aeration at 1 vvm and stirring at 800 rpm. The pH was controlled by adding 25% ammonium hydroxide solution. When the glucose concentration in the culture medium dropped to 0.5-1.0%, a glucose source was continuously added to the medium to maintain the sugar level between 0.5 and 0.8%. When the total ammonium level dropped to 0.15%, ammonium adipate solution (39.7%) was supplemented to maintain the total ammonium level at 0.1-0.2%.

[0115] After fermentation was completed, glucose, ammonium hydroxide, and ammonium adipate were added to increase the total volume of the fermentation broth to 10 L, and the cells were then separated from the spent medium by centrifugation. The cell-free supernatant was then clarified by microfiltration and ultrafiltration. At this point, the lysine concentration was 146 g / L. 876 g (0.6 equivalents, 87.6 g / L) of adipic acid was added to crystallize the product, lysine adipate. The product was filtered and dried. The purity of the lysine adipate was then determined by HPLC analysis, and the lysine adipate content was found to be 99.4%, with a yield of 88.5%.

[0116] Example 5C Lysine fermentation with ammonium adipate without crystallization of lysine adipate. Although C. glutamicum can utilize a variety of carbon and nitrogen sources for production, employing a minimal medium with an ammonium adipate buffer system (instead of ammonium sulfate or ammonium chloride) streamlines downstream processing by reducing the inorganic anion content throughout the process and in downstream products.

[0117] After seed culture as described in Example 4, C. glutamicum was then fermented at pH 7.0 in a medium containing 25 g / L glucose, 10 g / L ammonium adipate, 0.5 g / L potassium dihydrogen phosphate, 5 g / L sodium glutamate, 0.1 g / L sodium chloride, and 0.5 g / L yeast extract, and introduced into a 15 L stainless steel fermenter in an 8 L volume. The vessel was first sterilized by heating at 120°C for 20 minutes. Ammonium adipate was prepared by adding 3.2 kg of adipic acid to 7 L of a 25% aqueous solution of ammonium hydroxide (3 kg). The resulting solution was stirred at 50°C for 4 hours. After sterilization of the fermentation medium, the vessel was cooled to 30°C, and then 1 L of the above seed culture was inoculated into the medium. The medium was cultivated at 30°C with aeration at 1 vvm and stirring at 800 rpm. The pH was controlled by adding 25% ammonium hydroxide solution. When the glucose concentration in the culture medium dropped to 0.5–1.0%, a glucose source was continuously added to the medium to maintain the sugar level between 0.5 and 0.8%. When the total ammonium level dropped to 0.15%, ammonium adipate solution (39.7%) was supplemented to maintain the total ammonium level at 0.1–0.2%.

[0118] After fermentation was completed, glucose, ammonium hydroxide, and ammonium adipate were added to increase the total volume of the fermentation broth to 10 L, and the cells were then separated from the spent medium by centrifugation. The cell-free supernatant was then clarified by microfiltration and ultrafiltration. The lysine concentration in the supernatant was 146 g / L, and the supernatant was used directly in the next reaction without crystallization purification.

[0119] Example 6 Conversion of lysine hydrochloride to PDA hydrochloride using whole cells expressing lysine decarboxylase For the production of pentamethylenediamine (PDA)-HCl, 2 g of engineered wet E. coli containing lysine decarboxylase (1 g wet cells corresponds to 0.1-0.15 g cells on a dry basis) was added to 1 L of a 200 g / L lysine hydrochloride solution containing 0.1 g / L PLP. The pH was maintained at 6.5 using HCl. The temperature of the solution was brought to 37°C. The reaction was then initiated and continued for 10 hours while maintaining the pH at 6.5. The lysine content was determined by high-performance liquid chromatography (HPLC) at the end of the reaction (<0.0% w / v).

[0120] The reaction mixture was passed through a 0.2 micron microfiltration membrane (to remove large particles, such as cells, bacterial fragments, and aggregates) and a 10 kDa ultrafiltration membrane (to remove proteins and other soluble macromolecules in the medium). The filtrate was concentrated under reduced pressure to ¼ of its original volume. Two volumes of methanol were added to the mixture, which was then crystallized at 15°C. The solid was then collected and dried. The mass of this white solid product was 174.67 g, and its PDA-HCl content was analyzed. The PDA-HCl content was found to be 99.3%, with a yield of 91.1%.

[0121] Example 7 Conversion of lysine hydrochloride to PDA hydrochloride using lysine decarboxylase from cell lysates The same method as in Example 6 was used to produce PDA-HCl, except that 2 g of lysate from engineered E. coli cells containing lysine decarboxylase was added instead of whole cells. To obtain a soluble cell extract, 2 g of engineered E. coli bacterial cells were added to 10 mL of phosphate buffer (pH 7.0) and stirred well. The cells were then disrupted by high-pressure homogenization and then centrifuged to obtain the soluble cell extract. The mass of the white solid remaining in the reaction was 172.9 g. The content of PDA-HCl was found to be 99.5%, and the yield was 90.2%.

[0122] Example 8 Crystallization of PDA hydrochloride from ethanol The same method as in Example 6 was used to prepare PDA-HCl, except that ethanol was added instead of methanol for crystallization at 15° C. The mass of the white solid remaining in the reaction was 177.35 g. The content of PDA-HCl was found to be 99.2%, with a yield of 92.5%.

[0123] Example 9 Crystallization of PDA hydrochloride from isopropanol The same method as in Example 6 was used to prepare PDA-HCl, except that three volumes of isopropyl alcohol were added instead of methanol for crystallization at 15°C. The mass of the white solid remaining in the reaction was 171.2 g. The content of PDA-HCl was found to be 99.4%, with a yield of 89.3%.

[0124] Example 10 Increased lysine hydrochloride concentration and adjustment to pH 7 The same method as in Example 6 was used to produce PDA-HCl, except that the concentration of lysine hydrochloride was 300 g / L instead of 200 g / L at pH 6.5, the pH was maintained at 7, 4 g of engineered wet E. coli was added instead of 2 g, PLP was 0.15 g / L instead of 0.1 g / L, and the reaction time was 13 hours instead of 10 hours. The mass of the white solid remaining in the reaction was 223.4 g. The content of PDA-HCl was found to be 99.3%, with a yield of 91.5%.

[0125] Example 11 Conversion of lysine sulfate to PDA sulfate using whole cells expressing lysine decarboxylase The same method as in Example 6 was used to produce PDA-sulfate, except that lysine hydrochloride was replaced with 200 g / L lysine sulfate, the reaction time was 12 hours instead of 10 hours, and crystallization was carried out in two volumes of ethanol at 15°C. The pH was maintained at 6.5 using sulfate. The mass of the white solid remaining in the reaction was 201.88 g. The content of PDA-sulfate was found to be 99.1%, with a yield of 92.1%.

[0126] Example 12 Conversion of lysine acetate to PDA acetate using whole cells expressing lysine decarboxylase The same method as in Example 6 was used to produce PDA-acetate, except that lysine hydrochloride was replaced with 200 g / L lysine acetate, the reaction time was 12 hours instead of 10 hours, and crystallization was carried out in two volumes of isopropyl alcohol at 15°C. The pH was maintained at 6.5 using acetate. The mass of the white solid remaining in the reaction was 259.4 g. The content of PDA-acetate was found to be 99.2%, with a yield of 85.3%.

[0127] Example 13 Conversion of lysine adipate to PDA adipate using whole cells expressing lysine decarboxylase The same method as in Example 6 was used to produce PDA-adipate, except that lysine hydrochloride was replaced with a 200 g / L solution of lysine adipate crystals obtained in Example 5A, the reaction time was 12 hours instead of 10 hours, and crystallization was carried out at 15°C. The pH was maintained at 6.5 using adipate. The mass of the white solid remaining in the reaction was 306.8 g. The PDA-adipate content was found to be 99.2%, with a yield of 90.3%.

[0128] Example 14A Conversion of lysine adipate to PDA adipate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to produce PDA-adipate, except that 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed thoroughly and then added dropwise from a height of 10 cm using a peristaltic pump to a cross-linker containing 2% calcium chloride and 3% boric acid. This was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using adipate. The mass of the white solid remaining in the reaction was 313.56 g. The PDA-adipate content was found to be 99.2%, and the yield was 92.3%.

[0129] Example 14B Conversion of lysine adipate to PDA adipate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to produce PDA-adipate, except that lysine hydrochloride was replaced with a 200 g / L solution of lysine adipate crystals obtained in Example 5B, and 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed thoroughly and then added dropwise from a height of 10 cm using a peristaltic pump to a crosslinker containing 25.2% calcium chloride and 3% boric acid. This was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using adipate. The mass of the white solid remaining in the reaction was 309.82 g. The PDA-adipate content was found to be 99.0%, and the yield was 91.3%.

[0130] Example 14C Conversion of lysine adipate to PDA adipate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to prepare the PDA-adipate fermentation reaction solution, except that lysine hydrochloride was replaced with the supernatant of the lysine-adipate fermentation broth obtained in Example 5C, the lysine-adipate concentration was adjusted to 200 g / L, and 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed well and then added dropwise from a height of 10 cm using a peristaltic pump to a cross-linking agent containing 25.2% calcium chloride and 3% boric acid. The solution was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using adipate. The mass of the white solid remaining in the reaction was 299.97 g. The content of PDA-adipate was found to be 98.6% and the yield was 88.3%.

[0131] Example 15 Conversion of lysine adipate to PDA adipate using lysine decarboxylase from cell lysates The same method as in Example 13 was used to produce PDA-adipate, except that a cell lysate from lysine decarboxylase-containing E. coli cells was used instead of whole cells. After the reaction, the mass of the white solid remaining in the reaction was 308.8 g. The PDA-adipate content was found to be 99.1%, and the yield was 90.9%.

[0132] Example 16 Crystallization of PDA-adipate using ethanol instead of methanol The same method as in Example 13 was used to prepare PDA-adipate, except that ethanol was added instead of methanol for crystallization at 15°C. The mass of the white solid remaining in the reaction was 311 g. The PDA-adipate content was found to be 99.5%, with a yield of 91.5%.

[0133] Example 17 Increased pH to 7.5 instead of 6.5 The same method as in Example 16 was used to produce PDA-adipate, except that the pH was controlled at 7.5 with adipic acid instead of 6.5, and the reaction was carried out for 16 hours instead of 12 hours. The mass of the white solid remaining in the reaction was 309.2 g. The content of PDA-adipate was found to be 99.1%, and the yield was 91.0%.

[0134] Example 18 Lysine-adipate concentration increased from 200g / L to 400g / L The same method as in Example 16 was used to produce PDA-adipate, except that the starting concentration of lysine adipate was 400 g / L instead of 200 g / L, and the reaction was carried out for 18 hours instead of 12 hours. The mass of the white solid remaining in the reaction was 615 g. The content of PDA-adipate was found to be 99.2%, with a yield of 90.5%.

[0135] Example 19A Crystallization of PDA-adipate using isopropanol instead of methanol The same method as in Example 13 was used to prepare PDA-adipate, except that three times the volume of isopropanol was added instead of methanol for crystallization at 15° C. The mass of the white solid remaining in the reaction was 312.7 g. The content of PDA-adipate was found to be 99.1%, with a yield of 92.0%.

[0136] Example 19B Crystallization of PDA-adipate using n-propanol instead of methanol The same method as in Example 13 was used to prepare PDA-adipate, except that three times the volume of n-propanol was added instead of methanol for crystallization at 15°C. The mass of the white solid remaining in the reaction was 274.0 g. The content of PDA-adipate was found to be 99.3%, with a yield of 80.6%.

[0137] Example 20 Crystallization of PDA-adipate by direct crystallization in water The same method as in Example 13 was used to produce PDA-adipate, but the PDA-adipate was crystallized directly in water (without adding organic solvents such as methanol, ethanol, or isopropanol). The reaction solution was slowly cooled to 4°C, and 1 g of PDA-adipate seed crystals was added. The crystals were stirred at a low speed. The solution was then dried by suction filtration for 10 hours. The mass of the white solid remaining in the reaction was 153.9 g. The PDA-adipate content was found to be 99.1%, with a yield of only 45.3%.

[0138] Example 21A Lysine fermentation with ammonium suberate combined with crystallization with excess suberic acid The same method as in Example 5A was used, except that an ammonium suberate buffer system (sebacic acid / ammonium sebacate) was used instead of an ammonium adipate buffer system. After fermentation was completed, separation, and filtration, an excess amount of suberic acid was added to crystallize the product, lysine suberate. The product was filtered and dried. The purity of the lysine suberate was then determined by HPLC analysis. The lysine suberate content was found to be 99.3%, and the yield was 92.5%.

[0139] Example 21B Lysine fermentation with ammonium azelaate combined with crystallization with excess azelaic acid The same method as in Example 5A was used, except that an ammonium azelaate buffer system (azelaic acid / ammonium azelaate) was used instead of an ammonium adipate buffer system. After fermentation was completed, separation, and filtration, an excess amount of azelaic acid was added to crystallize the product lysine azelaate. The product was filtered and dried. The purity of the lysine azelaate was then determined by HPLC analysis. The content of lysine azelaate was found to be 99.0%, and the yield was 91.2%.

[0140] Example 21C Lysine fermentation with ammonium dodecanedicarboxylate combined with crystallization with excess dodecanedicarboxylic acid The same method as in Example 5A was used, except that an ammonium dodecanedicarboxylate buffer system (dodecanedicarboxylic acid / ammonium dodecanedicarboxylate) was used instead of an ammonium adipate buffer system. After fermentation was completed, separation, and filtration, an excess of dodecanedicarboxylic acid was added to crystallize the product lysine dodecanedicarboxylate. The product was filtered and dried. The purity of the lysine dodecanedicarboxylate was then determined by HPLC analysis. The content of lysine dodecanedicarboxylate was found to be 98.6%, and the yield was 88.7%.

[0141] Example 22A Conversion of lysine suberate to PDA suberate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to produce PDA-suberate, except that lysine hydrochloride was replaced with a 200 g / L solution of lysine suberate crystals obtained in Example 21A, and 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed well and then added dropwise from a height of 10 cm using a peristaltic pump to a cross-linking agent containing 2% calcium chloride and 3% boric acid. This was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using suberic acid. The mass of the white solid remaining in the reaction was 340.6 g. The PDA suberate content was found to be 99.6%, and the yield was 90.3%.

[0142] Example 22B Conversion of lysine azelaate to PDA azelaate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to produce PDA-azelate, except that lysine hydrochloride was replaced with a 200 g / L solution of lysine azelaate crystals obtained in Example 21B, and 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed thoroughly and then added dropwise from a height of 10 cm using a peristaltic pump to a crosslinker containing 2% calcium chloride and 3% boric acid. The solution was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using azelaic acid. The mass of the white solid remaining in the reaction was 366.05 g. The PDA azelaate content was found to be 99.1%, and the yield was 91.9%.

[0143] Example 22C Conversion of lysine dodecanedicarboxylate to PDA dodecanedicarboxylate using immobilized whole cells expressing lysine decarboxylase. The same method as in Example 13 was used to produce PDA-dodecanedicarboxylate, except that lysine hydrochloride was replaced with a 200 g / L solution of lysine dodecanedicarboxylate crystals obtained in Example 21C, and 2 g of immobilized lysine decarboxylase-containing E. coli cells were used instead of whole cells. To immobilize the cells, 20 mL of a mixed solution of 8% polyvinyl alcohol and 2.5% sodium alginate was added to 2.5 g of E. coli cells. The solution was mixed thoroughly and then added dropwise from a height of 10 cm using a peristaltic pump to a crosslinker containing 2% calcium chloride and 3% boric acid. This was then allowed to solidify for 8 hours, filtered, and repeatedly rinsed with distilled water (3-4 times). The immobilized cells were ready for use and could be reused. The pH of the reaction was maintained at 6.5 using dodecanedicarboxylic acid. The mass of the white solid remaining in the reaction was 399.46 g. The content of PDA dodecanedicarboxylate was found to be 98.1% and the yield was 87.2%.

[0144] Summary and Conclusion Crystallization of lysine dicarboxylate from spent lysine fermentation supernatant. Examples 4A, 4B, 5A, 5B, 21A, 21B, and 21C compare various approaches for obtaining lysine salt crystals from the cell-free supernatant of spent lysine fermentation broth. In Examples 4A and 4B, lysine fermentation was carried out in the presence of an ammonium inorganic anion buffer system (containing ammonium sulfate or ammonium chloride), after which the resulting cell-free supernatant was first desalted through an ion exchange column before crystallization of the lysine inorganic salt (e.g., as lysine sulfate or lysine hydrochloride) was induced by the addition of excess sulfuric acid (Example 4A) or hydrochloric acid (Example 4B). In contrast, the lysine fermentations described in Examples 5A, 5B, 21A, 21B, and 21C were carried out in the presence of an ammonium organic anion buffer system (i.e., containing ammonium dicarboxylate), and then crystallization of the lysine organic salt was induced directly from the cell-free supernatant (i.e., without desalting via an ion exchange column) by addition of the corresponding dicarboxylic acid, i.e., adipic acid (Examples 5A and 5B), suberic acid (Example 21A), azelaic acid (Example 21B), or dodecanedicarboxylic acid (Example 21C). The yields and purities of the resulting lysine salt crystals are summarized in Table 1 below.

[0145] [Table 1]

[0146] As shown in Table 1, when an ammonium dicarboxylic acid buffer system was employed in lysine fermentation and when lysine salt crystallization was induced by the addition of the corresponding dicarboxylic acid (Examples 5A, 5B, 21A, 21B, and 21C), higher yields and / or purities of lysine salt crystals were generally obtained compared to when an ammonium inorganic anion buffer system was used (Examples 4A and 4B). Surprisingly, the yields / purities of lysine salt crystals obtained using dicarboxylic acids (Examples 5A, 5B, 21A, 21B, and 21C) were comparable to or higher than those obtained using inorganic anions / acids (Examples 4A and 4B), even though the latter method included an additional desalting step by passage through an ion exchange column. Furthermore, the results of Examples 5A and 5B suggest that using an equivalent or excess amount of dicarboxylic acid to induce lysine salt crystallization further promotes crystal formation and results in increased yields compared to when a limited amount (e.g., 0.6 equivalents) of organic dicarboxylic acid is used. Interestingly, among the various conditions shown in Table 1, crystallization of lysine dicarboxylate with excess adipic acid presented the highest combination of both yield and purity.

[0147] Bioconversion of lysine dicarboxylate to PDA dicarboxylate. In conventional industrial fermentation processes, streamlined, so-called "one-pot" procedures are highly favored, minimizing intermediate purification steps (e.g., intermediate purification steps) to improve overall efficiency and reduce operating costs; costly purification steps are typically deferred until the final product. The impact of introducing an additional lysine salt crystallization step on the yield and purity of the final product (PDA adipate) was evaluated and compared with a more conventional streamlined procedure lacking the additional lysine salt crystallization step. More specifically, the bioconversion of lysine adipate to PDA adipate in Examples 14A and 14B utilized the lysine adipate crystals of Examples 5A and 5B as the starting material, while the bioconversion in Example 14C utilized the lysine adipate fermentation supernatant of Example 5C as the starting material. A summary of the yield and purity of the PDA adipate produced is shown in Table 2 below.

[0148] [Table 2]

[0149] As shown in Table 2, the conventional streamlined production method, in which bioconversion was performed directly on lysine fermentation supernatant, resulted in a PDA-adipate yield of 88.3% and an overall purity of 98.6%. Among the impurities detected were significant amounts of organic impurities, such as acetic acid, protein, lactic acid, and pyruvate. Upon heating at 250 °C for 8 h, the adverse effects of these organic impurities became visible in the form of yellowing of the PDA-adipate product and were quantified as a decrease in light transmittance upon dissolution. In contrast, when bioconversion was performed on resolubilized lysine-adipate crystals isolated by adding adipic acid to lysine fermentation supernatant, the resulting PDA-adipate product was not only more pure (99.0–99.2%) but also in significantly higher yields (91.3–92.3%). The reduction in organic impurities was visible in the form of a whiter PDA-adipate product and was quantified as an increase in light transmittance upon dissolution. Interestingly, the use of lysine adipate crystals isolated by adding excess adipic acid to the lysine fermentation supernatant resulted in a higher yield and purity of PDA adipate compared to lysine adipate crystals isolated by adding 0.6 equivalents of adipic acid.

[0150] The applicability of this production method with dicarboxylic acids with longer carbon chain lengths than adipic acid (C6) was also explored in Example 22A (suberic acid, C8), Example 22B (azelaic acid, C9), and Example 22C (dodecanedicarboxylic acid, C12). A summary of the yields and purities of the PDA dicarboxylates produced is shown in Table 3 below.

[0151] [Table 3]

[0152] As can be seen from the results in Table 3, employing dicarboxylic acids with longer carbon chain lengths resulted in higher yields and purities of PDA dicarboxylates, but a decrease in yield and purity was observed when using dicarboxylic acids with 12 carbons.

[0153] Alcohol-induced crystallization of PDA dicarboxylates. The inventors further unexpectedly discovered that the yield of PDA dicarboxylate products can be greatly increased by adding alcohol to induce crystallization after bioconversion. While all alcohols tested increased yields, methanol, ethanol, and isopropanol proved particularly effective at inducing crystallization of various PDA dicarboxylates. Interestingly, the highest yield of PDA adipate was obtained with isopropanol (92% yield, Example 19A), while the poorest performing alcohol was n-propanol (80.6% yield, Example 19B). Furthermore, high yields of PDA dicarboxylate crystals were obtained by cooling to approximately 15°C instead of 4°C in the absence of alcohol (Example 20).

[0154] [Table 4A]

[0155] [Table 4B]

[0156] In summary, by using a medium modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, the method described herein avoids the problem of generating large amounts of inorganic ions typically produced in conventional biological fermentation methods for preparing cadaverine. By reducing the amount of inorganic anions used in the medium, the subsequent purification steps required to remove the inorganic ions can be avoided, thereby significantly reducing production costs and the adverse environmental impacts associated with the disposal of excess inorganic ions. The inventors surprisingly discovered that adding a dicarboxylic acid to crystallize the intermediate lysine salt and then returning the lysine dicarboxylate salt crystals to the production process can significantly increase the yield and purity of the final cadaverine salt, as well as increase light transmittance and avoid product yellowing, compared to methods for producing cadaverine by one-pot fermentation. When an organic dicarboxylic acid is used for crystallization, the purity and yield of lysine salt crystals obtained using an excess amount of acid are higher than the corresponding results obtained using 0.6 equivalents of acid. Furthermore, the present inventors have surprisingly discovered that by using the highly active lysine decarboxylase described herein, not only can the amount of enzyme used be significantly reduced, thereby reducing costs, but also the yield and purity of the product cadaverine salt can be improved. Furthermore, the present inventors have surprisingly discovered that by crystallizing the final product cadaverine salt using a specific alcoholic solvent, the yield and / or purity of the product cadaverine salt can also be improved. Furthermore, the present invention also preferably uses a C6-C9 organic dicarboxylic acid to form the salt, and particularly preferably uses adipic acid to form the cadaverine salt.

[0157] Embodiment Embodiment 1. A method for producing cadaverine dicarboxylate having reduced inorganic ion content, comprising: (a) providing a fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism has been engineered to produce lysine from a carbon source, and the medium has been modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; (c) obtaining a lysine dicarboxylate stream from the fermentation broth, wherein the lysine dicarboxylate stream has a reduced inorganic ion content compared to a lysine inorganic salt stream obtained by a corresponding method that uses inorganic anions instead of dicarboxylate anions in the buffer system; (d) subjecting the lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the lysine dicarboxylate stream at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system; and adding a highly active lysine decarboxylase at a lysine decarboxylase addition ratio that is significantly reduced compared to the prior art, thereby producing a solution containing cadaverine dicarboxylate, wherein the lysine decarboxylase addition ratio is defined as the ratio of the added mass of lysine decarboxylase, calculated on a dry basis of lysine decarboxylase cells, to the mass of lysine in the lysine fermentation broth, based on the molecular weight of lysine dicarboxylate; (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution; A method comprising or consisting essentially of:

[0158] Embodiment 2. A method for producing cadaverine dicarboxylate having reduced inorganic ion content, comprising: (a) providing a fermentation broth comprising a microorganism immersed in a modified medium, the microorganism being engineered to produce lysine from a carbon source, the medium being modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; (c) adding an equal equivalent or excess of dicarboxylic acid to obtain lysine dicarboxylate crystals and dissolving the crystals in an aqueous solution to obtain a lysine dicarboxylate stream from the fermentation broth, the lysine dicarboxylate stream having a reduced inorganic ion content compared to a lysine inorganic salt stream obtained by a corresponding method using inorganic anions instead of dicarboxylate anions in the buffer system; (d) subjecting the lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system to said solution, thereby producing a solution comprising cadaverine dicarboxylate; (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution; A method comprising or consisting essentially of:

[0159] Embodiment 3. A highly active lysine decarboxylase is prepared by the steps of: (1) A plasmid containing the kdc gene of lysine decarboxylase represented by SEQ ID NO: 1 is transformed into Escherichia coli cells; (2) Selecting a positive single colony of the transformed strain and inoculating it into LB test tube medium, inoculating the medium overnight at 30°C and 180 RPM, the LB test tube medium containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride; (3) A flask containing TB medium was inoculated with a 5% inoculum dose from an overnight culture, the TB medium comprising 12 g / L peptone, 24 g / L yeast extract, and 4 g / L glycerol; (4) Place the flask in a shaker and incubate at 30°C and 250 RPM for approximately 2 hours. (5) Protein expression was induced with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) under induction conditions of 30°C and 250 RPM for 4 hours. (6) Centrifuge the fermentation broth to harvest the cells, and store the wet cells at -80°C. 2. The method of embodiment 1, wherein the lysine decarboxylase is treated with

[0160] Embodiment 4. In step (i), the E. coli is BL21(DE3) E. coli, or In step (ii), the incubation conditions are incubation at 30°C, 180 RPM, and shaking for 16 hours; or In step (iii), a flask containing TB medium is inoculated with a 5% inoculum dose from 40 mL of overnight cultured LB medium fermentation broth, or In step (iv), the incubation time is 2 hours, or In step (v), the concentration of IPTG is 0.2 mM, or In step (v), incubation is carried out for a further 4 hours at 30°C with shaking. 4. The method of embodiment 3.

[0161] Embodiment 5. The method of any one of embodiments 1, 3, or 4, wherein the lysine decarboxylase addition ratio of highly active lysine decarboxylase is (1:600) to (1:1000).

[0162] Embodiment 6 The method of embodiment 2, wherein in step (c), an excess of dicarboxylic acid is added.

[0163] Embodiment 7. The method of any one of embodiments 1 to 6, wherein step (b) further comprises supplementing the fermentation broth with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production, preferably to maintain the total ammonium concentration at 0.05% w / v to 0.5% w / v.

[0164] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the lysine dicarboxylate stream has a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molarity of dicarboxylate ions) x 2] / [molarity of monocationic lysine (Lys+) ions] x 100%.

[0165] Embodiment 9. (i) does not include the addition of any further source of non-essential inorganic anions, thereby minimizing the amount of inorganic anions present in the lysine dicarboxylate stream; and / or (ii) does not include a purification step to remove inorganic ions from the fermentation broth and / or lysine dicarboxylate stream; 9. The method of any one of embodiments 1 to 8.

[0166] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the inorganic ions are or include phosphate, sulfate, and / or chloride anions.

[0167] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the method does not include the use of a carbonate buffer system and / or does not include carbonate or carbonate anion as the lysine counter anion, wherein the carbonate buffer system includes ammonium carbonate and / or ammonium bicarbonate.

[0168] Embodiment 12 The method of any one of embodiments 1 to 11, wherein the method does not include a distillation step to purify the lysine from the lysine dicarboxylate stream.

[0169] Embodiment 13. Before step (a), (i) a growth medium formulated to contain inorganic salts, or (ii) a growth medium formulated to lack non-essential inorganic salts; 13. The method of any one of embodiments 1 to 12, wherein the microorganism is cultured until a desired cell mass is reached, and then the growth medium is replaced with the modified medium comprising an ammonium dicarboxylate buffer system and lacking non-essential inorganic ions.

[0170] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the microorganism engineered to produce lysine is immobilized to facilitate medium exchange and / or lysine dicarboxylate stream processing.

[0171] Embodiment 15 The method of any one of embodiments 1 to 14, wherein the microorganism engineered to produce lysine is a bacterium.

[0172] Embodiment 16. The method of any one of embodiments 1 to 15, wherein step (e) of crystallizing cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution comprises adding a sufficient amount of alcohol solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals by at least 20% compared to a corresponding crystallization method without the addition of an organic solvent.

[0173] Embodiment 17 The method of embodiment 16, wherein the increase in yield is at least 25%, or at least 30%, or at least 35%, or at least 40%.

[0174] Embodiment 18 The method of embodiment 16 or 17, wherein the alcohol solvent is methanol, ethanol, or isopropanol.

[0175] Embodiment 19 The method of embodiment 18, wherein the alcohol solvent is isopropanol.

[0176] Embodiment 20 The method of any one of embodiments 1 to 19, wherein the enzymatic decarboxylation is carried out by subjecting the lysine in the lysine dicarboxylate stream to viable or intact cells of a microorganism expressing a lysine decarboxylase.

[0177] Embodiment 21 The method of embodiment 20, wherein viable or intact cells of the microorganism expressing lysine decarboxylase are immobilized.

[0178] Embodiment 22 The method of any one of embodiments 1 to 21, wherein the enzymatic decarboxylation is carried out by subjecting the lysine in the lysine dicarboxylate stream to a cell lysate of a microorganism expressing a lysine decarboxylase.

[0179] Embodiment 23 The method of any one of embodiments 1 to 22, wherein the dicarboxylate and / or dicarboxylic acid contains 4 to 18 carbons.

[0180] Embodiment 24 The method of any one of embodiments 1 to 22, wherein the dicarboxylate and / or dicarboxylic acid contains 6 to 9 carbons.

[0181] Embodiment 25. The method of any one of embodiments 1 to 22, wherein the dicarboxylate is adipate and / or the dicarboxylic acid is adipic acid, thereby producing a lysine dicarboxylate stream that is a lysine adipate stream.

[0182] Embodiment 26 The method of embodiment 9, wherein the purification step is desalting and / or ion exchange.

[0183] Embodiment 27 The method of embodiment 15, wherein the bacterium belongs to the genus Corynebacterium or Brevibacterium.

[0184] Embodiment 28 The method of embodiment 27, wherein the bacterium belongs to the genus Corynebacterium and is Corynebacterium glutamicum.

[0185] Embodiment 29 The method of embodiment 27, wherein the bacterium belongs to the genus Brevibacterium and is Brevibacterium flavum or Brevibacterium lactofermentum.

[0186] Embodiment 30. Cadaverine dicarboxylate prepared by the method of any one of embodiments 1 to 29.

[0187] Embodiment 31 Use of cadaverine dicarboxylate, produced by the method of any one of embodiments 1 to 29, for the production of nylon.

[0188] Embodiment 32 The use of embodiment 31, wherein the cadaverine dicarboxylate is cadaverine adipate and the nylon is nylon 5,6.

[0189] Embodiment 33. A method for producing lysine dicarboxylate crystals having reduced inorganic impurities, comprising: (a) providing a fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism has been engineered to produce lysine from a carbon source, and the medium has been modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; (c) adding a sufficient amount of dicarboxylic acid to the spent fermentation broth to induce the formation of lysine dicarboxylate crystals, wherein the inorganic ion content of the lysine dicarboxylate crystals is reduced compared to the lysine inorganic salt obtained by a corresponding method that substitutes inorganic anions for dicarboxylic acid anions in the buffer system. 10. A method comprising or consisting essentially of:

[0190] Embodiment 34. The method of embodiment 33, wherein the sufficient amount of dicarboxylic acid added in step (c) corresponds to at least 1 equivalent per mole of lysine in the spent fermentation broth.

[0191] Embodiment 35. The method of embodiment 33, wherein the sufficient amount of dicarboxylic acid added in step (c) corresponds to an excess equivalent relative to the moles of lysine in the spent fermentation broth.

[0192] Embodiment 36. The method of any one of embodiments 33 to 35, wherein step (b) further comprises supplementing the fermentation broth with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production, preferably to maintain the total ammonium concentration at 0.05% w / v to 0.5% w / v.

[0193] Embodiment 37. The method of any one of embodiments 33 to 36, wherein prior to crystallization, the spent fermentation broth has a dicarboxylate ratio (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the formula: DSR = [(molarity of dicarboxylate ions) × 2] / [molarity of monocationic lysine (Lys+) ions] × 100%.

[0194] Embodiment 38. (i) does not include the addition of any further source of non-essential inorganic anions, thereby minimizing the amount of inorganic anions present in the lysine dicarboxylate stream; and / or (ii) does not include a purification step (e.g., desalting and / or ion exchange) to remove inorganic ions from the fermentation broth and / or lysine dicarboxylate stream; 38. The method of any one of embodiments 33 to 37.

[0195] Embodiment 39. The method of any one of embodiments 33 to 38, wherein the inorganic ions are or include phosphate, sulfate, and / or chloride anions.

[0196] Embodiment 40 The method of any one of embodiments 33 to 39, which does not include the use of a carbonate buffer system (e.g., ammonium carbonate and / or ammonium bicarbonate) and / or does not include carbonate or carbonate anions as lysine counter anions.

[0197] Embodiment 41 The method of any one of embodiments 33 to 40, which does not include a distillation step to purify the lysine.

[0198] Embodiment 42. Before step (a), (i) a growth medium formulated to contain inorganic salts, or (ii) a growth medium formulated to lack non-essential inorganic salts; 42. The method of any one of embodiments 33 to 41, wherein the microorganism is cultured until a desired cell mass is reached, and then the growth medium is replaced with the modified medium comprising an ammonium dicarboxylate buffer system and lacking non-essential inorganic ions.

[0199] Embodiment 43 The method of any one of embodiments 33 to 42, wherein the microorganism engineered to produce lysine is immobilized to facilitate medium exchange and / or lysine dicarboxylate stream processing.

[0200] Embodiment 44. The method of any one of embodiments 33 to 43, wherein the microorganism engineered to produce lysine is a bacterium, preferably belonging to the genus Corynebacterium (e.g., Corynebacterium glutamicum) or Brevibacterium (e.g., Brevibacterium flavum or Brevibacterium lactofermentum).

[0201] Embodiment 45. A method for producing cadaverine dicarboxylate having reduced organic and inorganic impurities, comprising or consisting essentially of steps (a)-(c) as defined in any one of embodiments 33-44; (d) dissolving the lysine dicarboxylate crystals isolated from step (c) in an aqueous solution and subjecting the lysine to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for the reaction to occur by adding a dicarboxylic acid to the solution, thereby producing a solution containing cadaverine dicarboxylate; (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, the organic solvent preferably being an alcohol, such as methanol, ethanol, or isopropanol; The method further comprises:

[0202] Embodiment 46. A method for producing cadaverine dicarboxylate having reduced organic and inorganic impurities, comprising: (i) providing an aqueous solution containing lysine dicarboxylate; (ii) subjecting lysine dicarboxylate to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for said reaction to occur by adding a dicarboxylic acid to said solution, thereby producing a solution comprising cadaverine dicarboxylate; (iii) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals (e.g., by at least 20%, 25%, 30%, 35%, or 40%) compared to a corresponding crystallization method without the addition of an organic solvent, wherein the organic solvent is preferably an alcohol, such as methanol, ethanol, or isopropanol; 10. A method comprising or consisting essentially of:

[0203] Embodiment 47 The method of embodiment 45 or 46, wherein the enzymatic decarboxylation is carried out by providing lysine to viable or intact cells of a microorganism that expresses lysine decarboxylase.

[0204] Embodiment 48 The method of embodiment 47, wherein viable or intact cells of the microorganism expressing lysine decarboxylase are immobilized.

[0205] Embodiment 49 The method of embodiment 47 or 48, wherein the enzymatic decarboxylation is carried out by providing lysine to a cell lysate of a microorganism expressing lysine decarboxylase.

[0206] Embodiment 50. The method of any one of embodiments 47 to 49, wherein the lysine decarboxylase is the lysine decarboxylase of SEQ ID NO: 2 or a variant thereof having lysine decarboxylase activity comprising an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2.

[0207] Embodiment 51. The method of any one of embodiments 33 to 50, wherein the dicarboxylate and / or dicarboxylic acid contains 4 to 18 carbons, 4 to 16 carbons, 4 to 14 carbons, 4 to 12 carbons, 4 to 10 carbons, 4 to 9 carbons, 6 to 10 carbons, or 6 to 9 carbons.

[0208] Embodiment 52. - the dicarboxylate is succinate and / or the dicarboxylic acid is succinic acid, thereby producing a lysine dicarboxylate that is lysine succinate; - the dicarboxylate is glutarate and / or the dicarboxylic acid is glutaric acid, thereby producing a lysine dicarboxylate that is lysine glutarate; - the dicarboxylate is adipate and / or the dicarboxylic acid is adipic acid, thereby producing a lysine dicarboxylate that is lysine adipate; - the dicarboxylate is pimelate and / or the dicarboxylic acid is pimelic acid, thereby producing a lysine dicarboxylate that is lysine pimelate; - the dicarboxylic acid salt is suberate and / or the dicarboxylic acid is suberic acid, thereby producing a lysine dicarboxylate which is lysine suberate; - the dicarboxylic acid salt is azelaic acid salt and / or the dicarboxylic acid salt is azelaic acid, thereby producing a lysine dicarboxylate salt that is lysine azelaate; - the dicarboxylate is sebacate and / or the dicarboxylic acid is sebacic acid, thereby producing a lysine dicarboxylate that is lysine sebacate; - the dicarboxylate is undecanedicarboxylate and / or the dicarboxylic acid is undecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine undecanedicarboxylate; - the dicarboxylate is dodecanedicarboxylate and / or the dicarboxylic acid is dodecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine dodecanedicarboxylate; - the dicarboxylate is tridecanedicarboxylate and / or the dicarboxylic acid is tridecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine tridecanedicarboxylate; - the dicarboxylate is tetradecanedicarboxylate and / or the dicarboxylic acid is tetradecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine tetradecanedicarboxylate; - the dicarboxylate is pentadecanedicarboxylate and / or the dicarboxylic acid is pentadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine pentadecanedicarboxylate; - the dicarboxylate is hexadecanedicarboxylate and / or the dicarboxylic acid is hexadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine hexadecanedicarboxylate; - the dicarboxylate is heptadecanedicarboxylate and / or the dicarboxylic acid is heptadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine heptadecanedicarboxylate; - the dicarboxylate is octadecanedicarboxylate and / or the dicarboxylic acid is octadecanedicarboxylic acid, thereby producing a lysine dicarboxylate that is lysine octadecanedicarboxylate; 52. The method of any one of embodiments 33 to 51.

[0209] Embodiment 53 Cadaverine dicarboxylate prepared by the method of any one of embodiments 45 to 52.

[0210] Embodiment 54 Use of cadaverine dicarboxylate, produced by the method of any one of embodiments 45 to 52, for the production of nylon.

[0211] Embodiment 55. The use of embodiment 54, wherein the cadaverine dicarboxylate is cadaverine adipate and the nylon is nylon 5,6.

[0212] Embodiment 56. A fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, and the medium is modified to include an ammonium dicarboxylate buffer system and preferably lacking non-essential inorganic ions, wherein the inorganic ion content of the fermentation broth is reduced compared to a corresponding fermentation broth employing inorganic anions instead of dicarboxylate anions in the buffer system.

[0213] Embodiment 57. The fermentation broth of embodiment 56, wherein the fermentation broth is supplemented with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production, preferably to maintain the total ammonium concentration at 0.05% w / v to 0.5% w / v.

[0214] Embodiment 58. The fermentation broth of embodiment 56 or 57, having a dicarboxylate ratio (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the formula: DSR = [(molar concentration of dicarboxylate ions) × 2] / [molar concentration of monocationic lysine (Lys+) ions] × 100%.

[0215] Embodiment 59. The fermentation broth of embodiment 56 or 58, wherein the microorganism engineered to produce lysine is as defined in embodiment 43 or 44.

[0216] Embodiment 60. A lysine dicarboxylate stream obtained from lysine fermentation, comprising lysine cations, dicarboxylate anions, wherein the medium has been modified to include an ammonium dicarboxylate buffer system and preferably lacks non-essential inorganic ions, and wherein the stream has a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the formula: DSR = [(molarity of dicarboxylate ions) x 2] / [molarity of monocationic lysine (Lys+) ions] x 100%.

[0217] Embodiment 61. The fermentation broth of any one of embodiments 56 to 59, or the lysine dicarboxylate stream of embodiment 26, wherein the inorganic ions are or comprise phosphate, sulfate, and / or chloride anions.

[0218] Embodiment 62. The fermentation broth of any one of embodiments 56 to 59 or 61, or the lysine dicarboxylate stream of embodiment 28 or 29, which does not contain a carbonate buffer system (e.g., ammonium carbonate and / or ammonium bicarbonate) and / or does not contain carbonate or carbonate anions as lysine counter anions.

[0219] This application contains a sequence listing in computer readable format approximately 9 kb in size, created on February 24, 2020. The computer readable format is incorporated herein by reference.

[0220] [Table 5]

[0221] (References) TIFF0007776434000007.tif106170

Claims

1. A method for producing cadaverine dicarboxylate having reduced organic and inorganic impurities, comprising: (a) providing a fermentation broth comprising a microorganism immersed in a modified medium, wherein the microorganism is engineered to produce lysine from a carbon source, and the medium is modified to include an ammonium dicarboxylate buffer system; (b) fermenting the microorganism in the presence of a carbon source under culture conditions that permit lysine production while controlling the pH of the fermentation broth by the addition of ammonium hydroxide to maintain the pH in a range that promotes lysine production; (c) adding an equal or excess equivalent amount of dicarboxylic acid to obtain lysine dicarboxylate crystals and dissolving the crystals in an aqueous solution to obtain a lysine dicarboxylate stream from the fermentation broth, wherein the lysine dicarboxylate stream has a reduced inorganic ion content compared to a lysine inorganic salt stream obtained by a corresponding method using inorganic anions instead of dicarboxylate anions in the buffer system; (d) subjecting the lysine dicarboxylate in the lysine dicarboxylate stream to an enzymatic decarboxylation reaction while maintaining the pH of the solution at a level sufficient for said reaction to occur by adding an ammonium dicarboxylate buffer system to said solution, thereby producing a solution comprising cadaverine dicarboxylate; (e) crystallizing the cadaverine dicarboxylate by adding a sufficient volume of organic solvent to the solution; A method comprising:

2. The modified medium of step (a) does not contain non-essential inorganic ions.

2. The method of claim 1, wherein the inorganic ions are or include phosphate anions, sulfate anions, chloride anions, or any combination thereof.

3. 3. The method of claim 1 or 2, wherein step (b) further comprises supplementing the fermentation broth with an ammonium dicarboxylate solution to maintain the total ammonium concentration at a level conducive to lysine production, the total ammonium concentration being between 0.05% w / v and 0.5% w / v.

4. 4. The method of any one of claims 1 to 3, wherein the lysine dicarboxylate stream has a dicarboxylate fraction (DSR) of at least 50%, 55%, 60%, 65%, 70%, 75%, or 80%, calculated using the following formula: DSR = [(molar concentration of dicarboxylate ions) x 2] / [molar concentration of monocationic lysine (Lys+) ions] x 100%.

5. (i) does not include the addition of any further source of non-essential inorganic anions, thereby minimizing the amount of inorganic anions present in the lysine dicarboxylate stream; (ii) does not include a purification step to remove inorganic ions from the fermentation broth and / or lysine dicarboxylate stream; (iii) does not include the use of a carbonate buffer system and / or does not include carbonate or carbonate anion as the lysine counter anion, and the carbonate buffer system includes ammonium carbonate and / or ammonium bicarbonate; (iv) does not include a distillation step to purify lysine from the lysine dicarboxylate stream; or (v) defined by any combination of (i) to (iv); 5. The method according to any one of claims 1 to 4.

6. Prior to step (a), a growth medium formulated to include inorganic salts, 6. The method of any one of claims 1 to 5, wherein the microorganism is cultured until a desired cell mass is reached, after which the growth medium is replaced with the modified medium comprising an ammonium dicarboxylate buffer system and lacking non-essential inorganic ions.

7. 7. The method of any one of claims 1 to 6, wherein the microorganism engineered to produce lysine is immobilized to facilitate medium exchange and / or lysine dicarboxylate stream processing.

8. 8. The method of any one of claims 1 to 7, wherein the microorganism engineered to produce lysine is a bacterium.

9. 9. The method of claim 1, wherein step (e) of crystallizing the cadaverine dicarboxylate by adding a sufficient amount of organic solvent to the solution comprises adding a sufficient amount of alcohol solvent to the solution to increase the yield of recovered cadaverine dicarboxylate crystals by at least 20%, at least 25%, or at least 30%, or at least 35%, or at least 40% compared to a corresponding crystallization method without the addition of an organic solvent.

10. 10. The method of claim 9, wherein the alcohol solvent is methanol, ethanol, or isopropanol.

11. 11. The method of any one of claims 1 to 10, wherein the enzymatic decarboxylation is carried out by subjecting the lysine in the lysine dicarboxylate stream to viable or intact cells of a microorganism expressing a lysine decarboxylase.

12. 12. The method of claim 11, wherein viable or intact cells of the microorganism expressing lysine decarboxylase are immobilized.

13. 11. The method according to any one of claims 1 to 10, wherein the enzymatic decarboxylation is carried out by subjecting the lysine in the lysine dicarboxylate stream to a cell lysate of a microorganism expressing a lysine decarboxylase.

14. 14. The method of any one of claims 1 to 13, wherein the dicarboxylate and / or dicarboxylic acid contains 4 to 18 or 6 to 9 carbons.

15. 15. The method of any one of claims 1 to 14, wherein the dicarboxylate is adipate and / or the dicarboxylic acid is adipic acid, thereby producing a lysine dicarboxylate stream that is a lysine adipate stream.

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