Mutant enzyme combination, gene, recombinant plasmid, spermidine directed synthesis bacteria and application

Abstract

The application discloses a mutant enzyme combination, genes, a recombinant plasmid, spermidine directed synthesis bacteria and application, and relates to the technical field of biological engineering. A carboxyspermidine dehydrogenase mutant of the mutant enzyme combination is subjected to amino acid mutation on the basis of an amino acid sequence shown in SEQ ID NO:1, the amino acid mutation is single-point mutation or combined mutation of F158A and D262E, and a carboxyspermidine decarboxylase mutant is subjected to amino acid mutation on the basis of an amino acid sequence shown in SEQ ID NO:2, the amino acid mutation is single-point mutation of L270A. The mutant enzyme combination is formed by the carboxyspermidine dehydrogenase mutant and the carboxyspermidine decarboxylase mutant through specific site mutation, the catalytic efficiency and reaction matching of continuous conversion of butanediamine and aspartic acid semialdehyde to spermidine are improved, thereby butanediamine accumulation is reduced, and the yield and conversion efficiency of spermidine are improved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to mutant enzyme combinations, genes, recombinant plasmids, spermidine-directed synthesis bacteria, and their applications. Background Technology

[0002] Spermine is an important class of polyamine compounds with wide applications in biomedicine, agriculture, and fine chemicals. Compared with traditional chemical synthesis methods, biological synthesis of spermine has advantages such as mild reaction conditions, environmental friendliness, and suitability for integration with microbial metabolic engineering. Therefore, the synthesis of spermine using microbial cell factories or enzyme catalytic systems has become an important research direction in this field.

[0003] Currently, the biological synthesis of spermidine mainly includes three metabolic pathways: the first is the SAM (S-adenosylmethionine)-butanediamine pathway, which is widely found in eukaryotes and most bacteria. This pathway relies on the decarboxylation of S-adenosylmethionine to generate decarboxylated S-adenosylmethionine as an aminopropyl donor, which then reacts with butanediamine under the catalysis of spermidine synthase to generate spermidine. The second is the aspartic semialdehyde-butanediamine pathway, which is mainly found in bacteria. This pathway involves the condensation of butanediamine and aspartic semialdehyde under the action of carboxysemidine dehydrogenase to generate carboxysemidine, which is further catalyzed by carboxysemidine decarboxylase to generate spermidine. The third is the aspartic semialdehyde-guanidinediamine pathway, which exists in some cyanobacteria. This pathway is mediated by carboxypropylguanidinediamine dehydrogenase and carboxypropylguanidinediamine decarboxylase.

[0004] Of the pathways described above, the aspartate semialdehyde-butanediamine pathway does not rely on SAM as an aminopropyl donor, thus avoiding to some extent the problem of SAM being competitively consumed by multiple metabolic pathways as the main intracellular methyl donor, and therefore has good potential for engineering applications. In this pathway, carboxysemine dehydrogenase and carboxysemine decarboxylase catalyze the continuous conversion of butanediamine to carboxysemine and carboxysemine to spermine, respectively, and are the key enzymes determining the butanediamine consumption rate and spermine formation efficiency.

[0005] However, current technologies typically employ naturally derived carboxysemine dehydrogenases and carboxysemine decarboxylases directly, or only modify the metabolic pathways of microbial chassis strains, without paying sufficient attention to the catalytic performance of these two key enzymes and the matching relationship between the two consecutive reactions. Due to the limited substrate affinity, catalytic efficiency, or reaction adaptability of natural enzymes, butanediamine and aspartic semialdehyde cannot be efficiently and continuously converted into spermine, easily leading to the accumulation of butanediamine and increased precursor consumption. Simultaneously, butanediamine, as a key intermediate, exhibits certain cytotoxicity; its accumulation inhibits bacterial growth and metabolic activity, further limiting spermine yield and conversion efficiency.

[0006] Therefore, how to synergistically optimize the carboxyl spermidine dehydrogenase and carboxyl spermidine decarboxylase in the aspartic semialdehyde-butanediamine pathway to improve the continuous conversion efficiency of the two key catalytic reactions, reduce the accumulation of butanediamine, and promote the efficient synthesis of spermidine has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] One objective of the first aspect of this invention is to provide a mutant enzyme combination for the synthesis of spermidine, which solves the technical problem in the prior art that the catalytic efficiency and continuous reaction matching of carboxysperidine dehydrogenase and carboxysperidine decarboxylase in the spermidine biosynthesis system are insufficient, resulting in the accumulation of butanediamine, inhibition of cell activity, and limitation of spermidine yield and conversion efficiency.

[0008] Another objective of the first aspect of this invention is to achieve efficient and targeted conversion of ornithine to spermidine, thereby avoiding the accumulation of butanediamine in the system, effectively reducing its toxic and inhibitory effects on cells, and promoting a dynamic balance between precursor supply and product synthesis, thereby reducing the metabolic load of individual strains and thus increasing the yield and conversion efficiency of spermidine.

[0009] The second aspect of the present invention aims to provide a mutant enzyme combination encoding gene for the synthesis of spermidine as described above.

[0010] The third aspect of this invention aims to provide a recombinant plasmid containing the gene encoding the mutant enzyme combination for the synthesis of spermidine described above.

[0011] The fourth aspect of this invention aims to provide a spermidine-directed synthesis bacterium.

[0012] The fifth aspect of this invention aims to provide a method for synthesizing spermidine.

[0013] According to a first aspect of the present invention, the present invention provides a combination of mutant enzymes for the synthesis of spermidine, the combination of mutant enzymes comprising a carboxysemidine dehydrogenase mutant and a carboxysemidine decarboxylase mutant; The carboxysemine dehydrogenase mutant was obtained by mutating the carboxysemine dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of the carboxysemine dehydrogenase mutant were F158A and / or D262E. The carboxysemine decarboxylase mutant was obtained by mutating the carboxysemine decarboxylase with the amino acid sequence shown in SEQ ID NO: 2, and the mutation site of the carboxysemine decarboxylase mutant is L270A.

[0014] According to a second aspect of the present invention, the present invention also provides a mutant enzyme combination encoding gene for the synthesis of spermidine as described above.

[0015] According to a third aspect of the present invention, the present invention also provides a recombinant plasmid containing the gene encoding the mutant enzyme combination for synthesizing spermidine as described above.

[0016] According to a fourth aspect of the present invention, the present invention also provides a spermidine-directed synthesizing bacterium, wherein the spermidine-directed synthesizing bacterium is a genetically engineered bacterium with Escherichia coli as the chassis strain, the spermidine-directed synthesizing bacterium contains the above-described mutant enzyme combination encoding gene for spermidine synthesis, the mutant enzyme combination encoding gene is integrated into the genome of the spermidine-directed synthesizing bacterium or located in the above-described recombinant plasmid, and the spermidine-directed synthesizing bacterium is capable of expressing the above-described mutant enzyme combination for spermidine synthesis.

[0017] According to a fifth aspect of the present invention, the present invention also provides a method for synthesizing spermidine, comprising the following steps: A first strain was obtained, which was used to synthesize and secrete ornithine; The spermidine-directed synthesizing bacteria described above were obtained as a second strain. The second strain synthesized aspartic semialdehyde using xylose as a carbon source and converted the ornithine into butanediamine using ornithine decarboxylase. The first strain and the second strain are co-cultured and fermented so that the ornithine synthesized and secreted by the first strain is converted into butanediamine by the second strain, and the butanediamine and the aspartic semialdehyde are synthesized into spermidine under the action of the mutant enzyme combination described above for the synthesis of spermidine.

[0018] Optionally, the first strain is an ornithine-producing bacterium, which synthesizes ornithine using glucose as a carbon source and secretes the ornithine into the extracellular space.

[0019] Optionally, the inoculation ratio of the first strain to the second strain is any value in (1-2):(2-4).

[0020] Optionally, the second strain is inoculated after the first strain has been cultured for a preset time, wherein the preset time is any value between 4h and 12h.

[0021] Optionally, the fermentation medium used in the co-culture fermentation includes 100g / L-130g / L of glucose and 50g / L-70g / L of xylose, and the initial pH of the fermentation medium is any value between 6.5 and 7.5.

[0022] Optionally, the spermidine-directed synthesizing bacteria are obtained by gene editing of Escherichia coli W3110, wherein the gene editing includes overexpression of the aspC gene, the lysC mutant gene, the ppc gene and the pyc gene, and knockout of the manZ gene and the ptsG gene.

[0023] This invention creates a mutant enzyme combination for spermidine synthesis by mutating a carboxysemine dehydrogenase from *Vibrio alginolyticus* to F158A and / or D262E, and a carboxysemine decarboxylase from *Herminium arsenogenum* to L270A. This mutant enzyme combination improves the substrate binding ability and catalytic efficiency of both carboxysemine dehydrogenase and carboxysemine decarboxylase, increasing the reaction efficiency of the continuous conversion of butanediamine and aspartic semialdehyde to spermidine via the carboxysemine intermediate. This allows butanediamine to be consumed more rapidly and converted to spermidine after formation, thereby reducing butanediamine accumulation and increasing the spermidine formation rate and synthesis yield.

[0024] Furthermore, this invention constructs a co-culture system of ornithine-producing bacteria and spermidine-synthesizing bacteria, and introduces carboxys-speridine dehydrogenase and carboxys-speridine decarboxylase optimized by site-directed mutagenesis into the spermidine-synthesizing bacteria. This allows butanediamine to rapidly enter subsequent reaction steps after synthesis, achieving efficient and directional conversion of ornithine to spermidine. This avoids the accumulation of butanediamine in the system, effectively weakens its toxic inhibitory effect on cells, and promotes a dynamic balance between precursor supply and product synthesis, thereby reducing the metabolic load of individual strains and thus improving the yield and conversion efficiency of spermidine.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0026] The following sections will describe some specific embodiments of the invention in a detailed manner by way of example and not limitation, with reference to the accompanying drawings. The same reference numerals in the drawings denote the same or similar parts or portions. Those skilled in the art should understand that these drawings are not necessarily drawn to scale. In the drawings: Figure 1 This is a schematic flowchart of a method for synthesizing spermidine according to an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the principle of a method for synthesizing spermidine according to an embodiment of the present invention. Detailed Implementation

[0027] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0028] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of this application. Furthermore, it should be noted that, for ease of description, only the parts relevant to this application are shown in the accompanying drawings, not the entire structure. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application.

[0029] The terms “comprising” and “having”, and any variations thereof, used in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] This invention provides a mutant enzyme combination for synthesizing spermidine. The mutant enzyme combination includes a carboxysemidine dehydrogenase mutant and a carboxysemidine decarboxylase mutant. The carboxysemidine dehydrogenase mutant is obtained by mutating the carboxysemidine dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1, and the mutation site of the carboxysemidine dehydrogenase mutant is F158A and / or D262E. The carboxysemidine decarboxylase mutant is obtained by mutating the carboxysemidine decarboxylase with the amino acid sequence shown in SEQ ID NO: 2, and the mutation site of the carboxysemidine decarboxylase mutant is L270A. Here, the amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of carboxysemine dehydrogenase derived from Vibrio alginolyticus; the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of carboxysemine decarboxylase derived from Herminiimonas arsenicoxydans.

[0032] In this embodiment, the F158A, D262E, and L270A mutations are all site-directed mutagenesis techniques used to substitute bases in the genes encoding carboxysemine dehydrogenase and carboxysemine decarboxylase, thereby achieving amino acid substitution at specific sites in the protein. These mutations improve the enzyme's affinity for substrates and catalytic efficiency by altering the spatial structure, charge distribution, and substrate binding ability of the enzyme's active site, thus promoting the efficient conversion of butanediamine to spermine. Specifically, the F158A mutant mutates phenylalanine at position 158 of the amino acid sequence of the carboxysemine dehydrogenase shown in SEQ ID NO:1 to alanine; the D262E mutant mutates aspartic acid at position 262 of the amino acid sequence of the carboxysemine dehydrogenase shown in SEQ ID NO:1 to glutamic acid; and the L270A mutant mutates leucine at position 270 of the amino acid sequence of the carboxysemine decarboxylase shown in SEQ ID NO:2 to alanine. Here, the amino acid sequence of the carboxysemine dehydrogenase mutant with both F158A and D262E double-site mutations is shown in SEQ ID NO:3, and the amino acid sequence of the carboxysemine decarboxylase mutant with the single-site mutation L270A is shown in SEQ ID NO:4.

[0033] In this embodiment, a mutant enzyme combination for spermidine synthesis was formed by mutating the carboxysemine dehydrogenase from *Vibrio alginolyticus* to F158A and / or D262E, and the carboxysemine decarboxylase from *Herminium arsenogenidum* to L270A. This mutant enzyme combination improves the substrate binding ability and catalytic efficiency of both carboxysemine dehydrogenase and carboxysemine decarboxylase, increasing the reaction efficiency of the continuous conversion of butanediamine and aspartic semialdehyde to spermidine via the carboxysemine intermediate. This allows butanediamine to be consumed and converted to spermidine more rapidly after its formation, thereby reducing butanediamine accumulation and increasing the spermidine formation rate and synthesis yield.

[0034] In a preferred embodiment, the mutation sites of the carboxysemine dehydrogenase mutant are a double-site mutation at F158A and D262E, and its amino acid sequence is shown in SEQ ID NO:3. The mutation site of the carboxysemine decarboxylase mutant is L270A, and its amino acid sequence is shown in SEQ ID NO:4. By performing a double-site mutation at F158A and D262E on the carboxysemine dehydrogenase and a single-site mutation at L270A on the carboxysemine decarboxylase, the enzyme activities of the two key catalytic reactions are synergistically enhanced, thereby significantly improving the conversion efficiency of butanediamine to spermine. Because butanediamine can be rapidly converted into downstream products after its formation, its accumulation in the intracellular and extracellular spaces is significantly reduced, even approaching the minimum level, while the spermine formation rate is significantly increased, ultimately resulting in the highest spermine yield.

[0035] The present invention also provides a mutant enzyme combination encoding gene for synthesizing spermidine as described above, the mutant enzyme combination encoding gene including the gene sequence shown in SEQ ID NO: 5 corresponding to the amino acid sequence shown in SEQ ID NO: 1 and the gene sequence shown in SEQ ID NO: 6 corresponding to the amino acid sequence shown in SEQ ID NO: 2.

[0036] In this embodiment, the amino acid sequence shown in SEQ ID NO: 3 corresponds to the gene sequence shown in SEQ ID NO: 7, and the amino acid sequence shown in SEQ ID NO: 4 corresponds to the gene sequence shown in SEQ ID NO: 8.

[0037] This invention also provides a recombinant plasmid containing the aforementioned gene encoding a mutant enzyme combination for spermidine synthesis. This recombinant plasmid can serve as an expression vector for the mutant enzyme combination gene, enabling the introduction, replication, and expression of the gene encoding both the carboxysemidine dehydrogenase mutant and the carboxysemidine decarboxylase mutant in host cells, providing a genetic basis for the co-expression of the two mutant enzymes in the same host system. Through this recombinant plasmid, the two key mutant enzymes can be introduced as complementary enzyme catalytic modules into the spermidine synthesis system, thereby providing conditions for improving the continuous conversion efficiency of butanediamine and aspartic semialdehyde to spermidine.

[0038] This invention also provides a spermidine-directed synthesizing bacterium, which is a genetically engineered bacterium using *Escherichia coli* as a substrate strain. This spermidine-directed synthesizing bacterium contains the gene encoding the aforementioned mutant enzyme combination for spermidine synthesis. The gene encoding the mutant enzyme combination is integrated into the genome of the spermidine-directed synthesizing bacterium or located in the aforementioned recombinant plasmid. The spermidine-directed synthesizing bacterium can express the aforementioned mutant enzyme combination for spermidine synthesis, enabling the bacterium to express carboxysemidine dehydrogenase mutants and carboxysemidine decarboxylase mutants by integrating or carrying the aforementioned mutant enzyme combination encoding gene in the *E. coli* substrate strain. This allows the bacterium to form a key catalytic module in the aspartic semialdehyde-butanediamine pathway within the cell. This spermidine-synthesizing bacterium can further continuously convert butanediamine, formed from ornithine, into spermidine with aspartic semialdehyde, ensuring that butanediamine is consumed promptly after generation, reducing the inhibitory effect of butanediamine accumulation on bacterial activity, and improving the efficiency and yield of spermidine-directed synthesis.

[0039] like Figure 1 As shown, the present invention also provides a method for synthesizing spermidine, comprising the following steps: Step S100: Obtain the first strain, which is used to synthesize and secrete ornithine; Step S200: Obtain the above-mentioned spermidine-directed synthesis bacteria as the second strain. The second strain uses xylose as a carbon source to synthesize aspartic acid semialdehyde and uses ornithine decarboxylase to convert ornithine into butanediamine. Step S300: The first strain and the second strain are co-cultured and fermented so that the ornithine synthesized and secreted by the first strain is converted into butanediamine by the second strain, and the butanediamine and aspartic semialdehyde are combined with the mutant enzymes used to synthesize spermidine to synthesize spermidine.

[0040] In this embodiment, the spermidine synthesis method based on a dual-bacterial co-culture system achieves efficient spermidine synthesis by constructing two engineered bacteria with clearly defined functional divisions. First, an ornithine-producing bacterium is constructed as the first strain to efficiently synthesize and secrete ornithine using glucose as a carbon source. Second, a spermidine-directed synthesis bacterium based on *E. coli* is constructed as the second strain. This strain enhances the aspartic acid metabolic pathway to provide aspartic acid semialdehyde and utilizes ornithine to synthesize butanediamine. Carboxysemine dehydrogenase and carboxysemine decarboxylase are expressed in this strain. The carboxysemine dehydrogenase mutant is a single-point mutation or combination mutation of F158A and / or D262E on the amino acid sequence shown in SEQ ID NO:1. The carboxysemine decarboxylase mutant is... A single-point mutation L270A was performed on the amino acid sequence shown in NO:2. Finally, the first and second strains were co-cultured and fermented, allowing the ornithine produced by the first strain to be taken up by the second strain and converted into butanediamine. Under the synergistic action of aspartic semialdehyde and the aforementioned mutant enzyme, ornithine was further converted into spermidine, thus achieving a highly efficient and directional conversion of ornithine to spermidine. Here, the first strain is an ornithine-producing bacterium that synthesizes ornithine using glucose as a carbon source and secretes it extracellularly.

[0041] In this embodiment, a co-culture system of ornithine-producing bacteria and spermidine-synthesizing bacteria was constructed. Carboxy-speridine dehydrogenase and carboxy-speridine decarboxylase optimized through site-directed mutagenesis were introduced into the spermidine-synthesizing bacteria. This allowed butanediamine to rapidly enter subsequent reaction steps after synthesis, achieving efficient and directional conversion of ornithine to spermidine. This avoided the accumulation of butanediamine in the system, effectively reducing its toxic inhibitory effect on cells, and simultaneously promoting a dynamic balance between precursor supply and product synthesis. This reduced the metabolic load on individual strains, thereby increasing spermidine yield and conversion efficiency.

[0042] In step S100, W3110 *E. coli* is subjected to targeted metabolic engineering to enhance the glucose-to-ornithine synthesis pathway and block the conversion of ornithine to downstream butanediamine and spermidine, thereby constructing a donor strain with only ornithine synthesis and secretion functions. By increasing the flux of the glutamate-to-ornithine pathway and weakening competing branches, carbon flux is highly concentrated in ornithine production, and extracellular secretion of ornithine is achieved through transport or membrane permeability regulation.

[0043] like Figure 2 As shown, the reaction mechanism of step S100 is as follows: glucose undergoes glycolysis and the tricarboxylic acid cycle to generate α-ketoglutarate, which is further converted into glutamate. Subsequently, ornithine is generated through the ornithine biosynthesis pathway, namely, the catalytic conversion by enzyme genes such as ArgB, ArgC, ArgD, ArgE, and ArgF. Because the downstream decarboxylation pathway is blocked, ornithine accumulates intracellularly and is transported extracellularly, thus forming a stable precursor supply pool. This achieves high-throughput output of the precursor in a single direction, avoiding the diversion of intermediate metabolites and the generation of toxic substances.

[0044] In step S200, a spermidine-synthesizing bacterium based on *E. coli* W3110 was constructed. This was achieved by enhancing the aspartate metabolic pathway to increase the endogenous supply of aspartate semialdehyde. Simultaneously, ornithine decarboxylase (SpeC) was introduced to convert ornithine to butanediamine. Furthermore, site-directed mutagenesis-optimized carboxysemidine dehydrogenase (VaCASDH) and carboxysemidine decarboxylase (HaCASDC) were introduced to construct a complete spermidine synthesis module. In addition, by weakening glucose utilization, the strain preferentially utilizes xylose, thereby achieving carbon source specialization with the donor bacteria.

[0045] The reaction mechanism in step S200 involves three consecutive reaction processes: First, exogenous or extracellular ornithine undergoes decarboxylation under the action of SpeC to generate butanediamine; second, butanediamine and aspartic semialdehyde, generated from the metabolism of aspartic acid, undergo condensation and reduction under the catalysis of VaCASDH to generate carboxysemine; finally, carboxysemine undergoes decarboxylation under the action of HaCASDC to generate spermidine. By performing site-directed mutagenesis on VaCASDH and HaCASDC, the substrate affinity and catalytic efficiency of the enzyme are improved, enabling the rapid conversion of butanediamine, thereby achieving low accumulation or even instantaneous consumption of the intermediate.

[0046] In step S300, a synergistic metabolic network for precursor supply and product conversion is established by co-culturing the first and second strains in the same system. By setting different carbon sources, namely glucose for the first strain and xylose for the second strain, the first strain continuously and stably releases ornithine into the system, while the second strain simultaneously and efficiently converts ornithine into spermidine, thereby achieving dynamic matching of metabolic flux.

[0047] In a further embodiment, the inoculation ratio of the first strain to the second strain is any value in (1-2):(2-4), that is, the inoculation ratio of the first strain and the second strain can be 1:2, 1:3, 1:4, 2:2, or 2:3, or any other value in (1-2):(2-4), achieving an effective match between precursor supply and product conversion. The inoculation ratio within the above range keeps the ornithine production rate of the first strain and the consumption rate of the second strain in a dynamic balance, avoiding both the waste of resources caused by excessive accumulation of the precursor ornithine and the limitation of product synthesis by insufficient precursor supply, thereby increasing spermidine yield and reducing the accumulation of intermediate products.

[0048] In a further embodiment, the second strain is inoculated after the first strain has been cultured for a preset time, which is any value between 4h and 12h. That is, the second strain is inoculated after the first strain has been cultured for 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, or 12h. In this embodiment, by limiting the inoculation time of the second strain to within the 4h-12h time window of the first strain's culture, the ornithine-producing bacteria can fully establish an ornithine accumulation base in the early stage. Subsequently, the spermidine-synthesizing bacteria participate in the transformation process under conditions of sufficient precursor supply, thereby achieving temporal synergistic control of the metabolic process. This temporal regulation avoids the limited transformation efficiency caused by insufficient precursor supply and prevents metabolic competition and substrate deficiency caused by premature introduction of spermidine-synthesizing bacteria, significantly improving the metabolic matching degree of the dual-strain system and the overall spermidine yield.

[0049] In a further embodiment, the fermentation medium used in the co-culture fermentation includes 100 g / L-130 g / L of glucose and 50 g / L-70 g / L of xylose. The initial pH of the fermentation medium is any value between 6.5 and 7.5. That is, the concentration of glucose in the fermentation medium can be 100 g / L, 110 g / L, 120 g / L, 125 g / L, or 130 g / L, or any other value between 100 g / L and 130 g / L. The concentration of xylose can be 50 g / L, 55 g / L, 60 g / L, 65 g / L, or 70 g / L, or any other value between 50 g / L and 70 g / L. The initial pH is 6.5, 7.0, or 7.5, or any other value between 6.5 and 7.5. This allows the first strain and the second strain to preferentially utilize different carbon sources, thereby achieving division of labor and synergy in carbon source utilization between the two strains. The above culture conditions not only ensured that the first strain had sufficient carbon source to continuously synthesize and secrete ornithine, but also provided a suitable metabolic environment for the second strain to efficiently convert ornithine into spermidine, while avoiding carbon source competition and metabolic inhibition. Thus, in synergy with the inoculation ratio and inoculation sequence, the yield of spermidine was further increased and the accumulation of intermediate products was reduced.

[0050] Furthermore, by controlling the initial pH of the fermentation medium within the range of 6.5-7.5, the culture environment is kept in a near-neutral suitable condition. This simultaneously satisfies the optimal operating environment for the growth of the first strain and the ornithine synthesis-related enzymes, as well as the key enzymes for spermidine synthesis in the second strain. This ensures a synergistic improvement in cell activity and enzyme catalytic efficiency, and effectively avoids the decrease in enzyme activity or changes in cell membrane permeability caused by pH deviation. This further improves precursor conversion efficiency, increases spermidine yield, and reduces the accumulation of intermediate products.

[0051] like Figure 2 As shown, in a further embodiment, the spermidine-directed synthesizing bacterium was obtained by gene editing of *E. coli* W3110. The gene editing included overexpression of the aspC gene, the lysC mutant gene, the ppc gene, and the pyc gene, as well as knockout of the manZ gene and the ptsG gene. Through this gene editing modification, the metabolic flux of the second strain was directionally enhanced towards aspartic acid and its derivatives, while simultaneously relieving feedback inhibition of the LysC enzyme and increasing the biosynthetic flux of aspartic acid semialdehyde. Furthermore, by overexpressing the ppc and pyc genes, the ability of carbon flux to be converted from central metabolism to precursor substances was enhanced, thereby providing a sufficient precursor supply for spermidine synthesis. Simultaneously, by knocking out the glucose catabolism pathway, i.e., knocking out the manZ and ptsG genes, the consumption of glucose by the second strain was reduced, decreasing carbon source competition with the first strain, allowing the two strains to efficiently utilize different carbon sources and achieve metabolic specialization in the co-culture system. Therefore, the second strain can significantly improve precursor generation efficiency and enhance the metabolic synergy of the two-strain system, thereby increasing the overall spermidine yield and reducing byproduct accumulation. Here, the single-point mutation position of the LysC mutant (from Corynebacterium glutamicum, whose gene sequence is shown in SEQ ID NO: 9) is C932T, which is a single-base substitution mutation in the 932nd base of the LysC gene, in which cytosine is mutated to thymine, in order to obtain an enzyme related to the synthesis of aspartic semialdehyde with feedback resistance.

[0052] like Figure 2 As shown, the gene editing further includes overexpression of the speC gene. Overexpression of speC enhances the expression level of ornithine decarboxylase in cells, thereby increasing the conversion efficiency of ornithine to butanediamine and further promoting the flux of subsequent spermidine synthesis-related metabolic pathways. This overexpression measure effectively strengthens the key rate-limiting steps of the polyamine synthesis pathway, improves the conversion efficiency of precursor substances, reduces ornithine accumulation in cells, and simultaneously increases the production rate and yield of the target product spermidine. Furthermore, in synergy with other metabolic engineering modifications, such as LysC mutation and carbon flux optimization, it further enhances the coordination and stability of overall metabolic flux.

[0053] like Figure 2 As shown, gene editing further includes weakening the thrA and dapA genes and replacing their start codons from ATG to GTG. By weakening the expression levels of the thrA and dapA genes and replacing their start codons from ATG to GTG, the metabolic flux of the intracellular homoserine and lysine synthesis pathways is reduced, thereby reducing the consumption of carbon flux in the branched metabolic pathways and allowing more carbon sources and metabolic intermediates to preferentially flow to the polyamine synthesis pathway.

[0054] Furthermore, the first strain was obtained through CRISPR / Cas9 gene editing of E. coli W3110, enabling efficient and precise site-specific editing of target genes, including gene knockout, insertion, or point mutation, thereby improving the controllability and accuracy of gene modification. This technology has the advantages of simple operation, high editing efficiency, and strong specificity, effectively avoiding the non-specific mutations and random integration problems existing in traditional gene editing methods, thus constructing an engineered strain with a clear genetic background and stable performance.

[0055] Furthermore, the first strain constructed based on CRISPR / Cas9 can more stably perform precursor generation or metabolic regulation functions, forming a highly efficient synergy with the second strain, thereby improving the overall product generation efficiency and stability of the fermentation system.

[0056] The technical solution of this application will be further described below with reference to specific embodiments.

[0057] Example 1 A spermidine-synthesizing bacterium, comprising a mutant enzyme combination for spermidine synthesis, was obtained. Specifically, a carboxysylspermine dehydrogenase mutant with a combined F158A and D262E mutation on the amino acid sequence shown in SEQ ID NO: 1, and a carboxysylspermine decarboxylase mutant with a single L270A mutation on the amino acid sequence shown in SEQ ID NO: 2. The above spermidine-synthesizing bacteria were activated by inoculating at 1% in LB tubes at 37°C and 220 rpm for 12 h. After 12 h, the bacteria were transferred to 100 mL LB shake flasks and incubated at 37°C and 220 rpm for another 8 h. Then, the culture was transferred to M9 basal medium (see CN120608108A). 60 g / L xylose and 50 g / L ornithine were added, and the culture was continued at 37°C and 220 rpm for 48 h to synthesize spermidine.

[0058] The synthesis method of spermidine-synthesizing bacteria is as follows: Sangon Biotech (Shanghai) Co., Ltd. was commissioned to design and synthesize corresponding site-directed mutagenesis primers. Using the gene sequences encoding wild-type VaCASDH and HaCASDC as templates, single or multiple mutation sites were introduced at the target sites using Gene Splicing by Overlap Extension PCR (SOE PCR). Specifically, the introduction of F158A and D262E mutations into the amino acid sequence shown in SEQ ID NO:1 yielded the amino acid sequence of the VaCASDH mutant shown in SEQ ID NO:3, thereby obtaining the gene sequence SEQ ID NO:7 encoding the amino acid sequence shown in SEQ ID NO:3. The introduction of L270A mutations into the amino acid sequence shown in SEQ ID NO:2 yielded the amino acid sequence of the HaCASDC mutant shown in SEQ ID NO:4, thereby obtaining the gene sequence SEQ ID NO:8 encoding the amino acid sequence shown in SEQ ID NO:4. The gene sequences SEQ ID NO:7 and SEQ ID NO:8 encoding VaCASDH mutant and HaCASDC mutant, respectively, were cloned into expression vectors to obtain the corresponding recombinant plasmids. The recombinant plasmids were transformed into W3110 Escherichia coli, and metabolic pathway engineering was performed on W3110 Escherichia coli, including overexpression of the aspC gene, the lysC mutant gene (C932T, lysC from Corynebacterium glutamicum), the ppc gene and the pyc gene, overexpression of the speC gene, weakening of the thrA gene and the dapA gene, and knockout of the key genes manZ and ptsG related to glucose uptake or metabolism, in order to enhance the supply of aspartic semialdehyde, increase the production of butanediamine and its conversion efficiency to spermidine, and reduce branching metabolic consumption, thereby obtaining spermidine-synthesizing bacteria.

[0059] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the carboxysemine dehydrogenase mutant in the mutant enzyme combination introduces a combination mutation of F158A and D262E on the amino acid sequence shown in SEQ ID NO: 1, while the carboxysemine decarboxylase mutant is a single point mutation that introduces F39A on the amino acid sequence shown in SEQ ID NO: 2.

[0060] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that the carboxysemine dehydrogenase mutant in the mutant enzyme combination is a combination mutation of F158A and D262E introduced into the amino acid sequence shown in SEQ ID NO: 1, and the carboxysemine decarboxylase mutant is a single point mutation of L156A introduced into the amino acid sequence shown in SEQ ID NO: 2.

[0061] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that the mutant of the carboxysemine dehydrogenase in the mutant enzyme combination is a combination mutation of F158A and D262E introduced into the amino acid sequence shown in SEQ ID NO: 1, and no site mutation was introduced into the carboxysemine decarboxylase.

[0062] Comparative Example 4 The only difference between Comparative Example 4 and Example 1 is that the mutant carboxysemine dehydrogenase mutant in the mutant enzyme combination is a single-point mutation that introduces F158A into the amino acid sequence shown in SEQ ID NO: 1, and no site mutation is introduced into the carboxysemine decarboxylase.

[0063] Comparative Example 5 The only difference between Comparative Example 5 and Example 1 is that the mutant carboxysemine dehydrogenase mutant in the mutant enzyme combination is a single-point mutation that introduces D262E into the amino acid sequence shown in SEQ ID NO: 1, and no site mutation is introduced into the carboxysemine decarboxylase.

[0064] Comparative Example 6 The only difference between Comparative Example 6 and Example 1 is that the carboxysemine dehydrogenase mutant in the mutant enzyme combination is a combination mutation that introduces L299A into the amino acid sequence shown in SEQ ID NO: 1, and no site mutation is introduced into the carboxysemine decarboxylase.

[0065] Comparative Example 7 The only difference between Comparative Example 7 and Example 1 is that no site mutations were introduced in either carboxysemine dehydrogenase or carboxysemine decarboxylase.

[0066] The spermidine production and intracellular and extracellular butanediamine accumulation in Example 1 and Comparative Examples 1-7 were tested respectively, and the test results are shown in Table 1.

[0067] Table 1. Results of spermidine yield tests in Example 1 and Comparative Examples 1-7

[0068] As shown in Table 1, Example 1 used a carboxysylspermine dehydrogenase mutant with a combined F158A and D262E mutation on the amino acid sequence shown in SEQ ID NO: 1, and combined it with a carboxysylspermine decarboxylase mutant with a single L270A mutation on the amino acid sequence shown in SEQ ID NO: 2. When used in combination, the spermidine yield reached 28.6 g / L, and the butanediamine accumulation was only 0.18 g / L. This indicates that the mutant enzyme combination can effectively promote the conversion of butanediamine to spermidine and significantly reduce the accumulation of butanediamine intermediates.

[0069] Compared with Comparative Example 7, where no site mutations were introduced in either the carboxysemine dehydrogenase or the carboxysemine decarboxylase, the spermidine yield in Example 1 increased from 12.5 g / L to 28.6 g / L, while the butanediamine accumulation decreased from 15.1 g / L to 0.18 g / L. This indicates that the catalytic efficiency of the natural carboxysemine dehydrogenase and the natural carboxysemine decarboxylase is limited, making it difficult to achieve efficient and continuous conversion of butanediamine. In contrast, the specific site-mutated enzyme combination of this application can significantly improve the spermidine synthesis efficiency.

[0070] Comparative Examples 4 and 5, which introduced the F158A or D262E single-point mutation into carboxysemine dehydrogenase, respectively, yielded spermine yields of 19.2 g / L and 18.7 g / L, and butanediamine accumulations of 6.9 g / L and 7.2 g / L, respectively. These results indicate that single-point mutations of F158A or D262E can improve the catalytic performance of carboxysemine dehydrogenase to some extent, but the effect of individual mutations on increasing butanediamine consumption and spermine production remains limited. Comparative Example 3 introduced a combined mutation of F158A and D262E into the carboxysemine dehydrogenase, but did not introduce a site mutation into the carboxysemine decarboxylase. Its spermine yield increased to 24.5 g / L, and the accumulation of butanediamine decreased to 4.1 g / L. This indicates that the combined mutation of F158A and D262E can further improve the ability of carboxysemine dehydrogenase to catalyze the reaction of butanediamine with aspartic semialdehyde to produce carboxysemine. However, if the carboxysemine decarboxylase is not optimized simultaneously, the subsequent conversion of carboxysemine to spermine will still be limited.

[0071] Comparative Examples 1 and 2 introduced the F39A or L156A mutation into the carboxysemine decarboxylase, respectively. Their spermidine yields were 24.3 g / L and 24.8 g / L, and their butanediamine accumulations were 4.3 g / L and 3.9 g / L, respectively, showing no significant improvement compared to Comparative Example 3. However, in Example 1, after introducing the L270A mutation into the carboxysemine decarboxylase, the spermidine yield was further increased to 28.6 g / L, and the butanediamine accumulation was further reduced to 0.18 g / L. These results indicate that not all mutation sites in the carboxysemine decarboxylase can improve spermidine synthesis. The L270A mutation can form a better catalytic match with the F158A and D262E combined mutations of the carboxysemine dehydrogenase, thereby promoting the synergistic progression of the two consecutive reactions. In Comparative Example 6, after introducing non-target mutations, spermidine production was only 13.1 g / L, while butanediamine accumulation was as high as 14.9 g / L, further demonstrating that the F158A, D262E, and L270A mutation sites selected in this application are specific and irreplaceable.

[0072] Example 2 The method for spermidine synthesis using spermidine-directed synthetic bacteria includes the following steps: Step S100: Obtain the first strain, which is used to synthesize and secrete ornithine; Step S200: Prepare a second strain using the mutant enzyme combination from Example 1; Step S300: After culturing the first strain for 6 hours, the second strain is inoculated into the culture system containing the first strain at a ratio of 1:3. The first and second strains are then co-cultured and fermented to convert ornithine synthesized and secreted by the first strain into butanediamine via the second strain. The butanediamine then reacts with aspartic semialdehyde under the action of the aforementioned mutant enzyme combination used for spermidine synthesis to synthesize spermidine. The culture medium used for co-culture fermentation is M9 medium (see CN120608108A), with an initial pH of 7.0, a basic carbon source of 120 g / L glucose and 60 g / L xylose, a fixed total inoculation amount of the first and second strains at an OD600 of 0.2, a fermentation temperature of 37℃, a shaking speed of 220 rpm, and a fermentation period of 48 hours, resulting in the synthesis of spermidine.

[0073] The synthesis method of the second strain is the same as that of the spermidine-synthesizing bacteria in Example 1.

[0074] Example 3 The only difference between Example 3 and Example 2 is that the inoculation ratio of the first strain and the second strain is 1:2, and the second strain and the first strain are inoculated at the same time.

[0075] Example 4 The only difference between Example 4 and Example 2 is that the inoculation ratio of the first strain and the second strain is 1:4, and the second strain and the first strain are inoculated at the same time.

[0076] Example 5 The only difference between Example 5 and Example 2 is that the second strain was inoculated 4 hours after the first strain.

[0077] Example 6 The only difference between Example 6 and Example 2 is that the second strain was inoculated 12 hours after the first strain.

[0078] Example 7 The only difference between Example 7 and Example 2 is that the initial pH of the fermentation medium is 6.5.

[0079] Example 8 The only difference between Example 8 and Example 2 is that the initial pH of the fermentation medium is 7.5.

[0080] Comparative Example 8 The only difference between Comparative Example 8 and Example 2 is that the inoculation ratio of the first strain to the second strain is 1:1.

[0081] Comparative Example 9 The only difference between Comparative Example 9 and Example 2 is that the inoculation ratio of the first strain to the second strain is 2:1.

[0082] Comparative Example 10 The only difference between Comparative Example 10 and Example 2 is that the second strain was inoculated at the same time as the first strain.

[0083] Comparative Example 11 The only difference between Comparative Example 11 and Example 2 is that the second strain was inoculated 18 hours after the first strain.

[0084] Comparative Example 12 The only difference between Comparative Example 12 and Example 2 is that the second strain was inoculated 24 hours after the first strain.

[0085] Comparative Example 13 The only difference between Comparative Example 13 and Example 2 is that the initial pH of the fermentation medium is 6.0.

[0086] Comparative Example 14 The only difference between Comparative Example 14 and Example 2 is that the initial pH of the fermentation medium is 8.0.

[0087] The spermidine production and intracellular and extracellular butanediamine accumulation in Examples 2-8 and Comparative Examples 8-14 were tested, and the results are shown in Table 3. The testing methods for spermidine production and intracellular and extracellular butanediamine accumulation are as follows.

[0088] Determination of spermidine / butanediamine content: 200 µL of a reaction solution containing spermidine and butanediamine was added, followed by 40 µL of 2M NaOH solution to make the reaction solution alkaline. Then, 60 µL of saturated NaHCO3 solution was added for buffering. Finally, 400 µL of 10 mg / mL dansyl chloride solution was added, with acetone as the solvent. The reaction was carried out at 40 °C in the dark for 45 min. After the reaction was completed, 20 µL of 25% ammonia water was added to stop the reaction. After standing for 30 min, the volume was adjusted to 1 mL with acetonitrile. Finally, the solution was centrifuged at 4000 rpm for 5 min at 4 °C and filtered through a 0.22 µm organic filter membrane for high-performance liquid chromatography (HPLC) analysis.

[0089] The chromatograph was a Waters 2689 system, the column was a Waters Sunfire C18 (4.6×250mm, 5mm), the flow rate was 1mL / min, the UV detection wavelength was 254nm, the injection volume was 10µL, the column temperature was 35℃, the mobile phase A was water and B was acetonitrile, gradient elution was used, and the elution program is shown in Table 2.

[0090]

[0091] Table 3. Results of spermidine yield tests in Examples 2-8 and Comparative Examples 8-14

[0092] As shown in Table 3, in Example 2, a second strain was constructed using the mutant enzyme combination from Example 1. The first and second strains were co-cultured at an inoculation ratio of 1:3. When the second strain was inoculated 6 hours after the first strain was cultured, the spermidine yield reached 36.5 g / L, and the butanediamine accumulation decreased to 0.03 g / L. Compared with the system in Example 1 where ornithine was added exogenously for fermentation, the spermidine yield in Example 2 was further increased, and the butanediamine accumulation was further reduced. This indicates that, based on the improved key catalytic efficiency of the mutant enzyme combination, the co-culture division of labor between the first and second strains can further achieve dynamic matching between precursor supply and product conversion, thereby improving the overall spermidine synthesis efficiency.

[0093] Regarding the inoculation ratio, in Examples 3 and 4, when the inoculation ratio of the first strain to the second strain was 1:2 and 1:4, respectively, the spermidine yield was 30.5 g / L and 29.7 g / L, and the butanediamine accumulation was 0.15 g / L and 0.21 g / L, respectively, both maintaining good spermidine synthesis effects. However, in Comparative Examples 8 and 9, when the inoculation ratio was 1:1 and 2:1, respectively, the spermidine yield decreased to 26.8 g / L and 24.4 g / L, respectively, and the butanediamine accumulation increased to 0.18 g / L and 0.48 g / L, respectively. These results indicate that the inoculation ratio of the first strain to the second strain affects the balance between the ornithine supply rate and the subsequent butanediamine conversion rate. When the proportion of the second strain is insufficient, the conversion ability of ornithine to butanediamine and spermidine is limited, easily leading to intermediate accumulation. When the ratio of the two strains is within the range of (1-2):(2-4), it is more conducive to maintaining the coordinated relationship between precursor supply and product conversion. In Example 2, the 1:3 inoculation ratio showed the best performance, indicating that this ratio can more fully utilize the second strain's ability to continuously convert ornithine and butanediamine.

[0094] Regarding the inoculation sequence, in Examples 5 and 6, the second strain was inoculated 4 h and 12 h after the first strain, respectively. The spermidine yield was 35.8 g / L and 34.8 g / L, and the butanediamine accumulation was 0.04 g / L and 0.05 g / L, respectively, both close to the effect of Example 2. In contrast, in Comparative Example 10, when the first and second strains were inoculated simultaneously, the spermidine yield was 33.2 g / L and the butanediamine accumulation was 0.07 g / L. In Comparative Examples 11 and 12, when the second strain was inoculated 18 h and 24 h after the first strain, respectively, the spermidine yield decreased to 32.7 g / L and 29.9 g / L, respectively, and the butanediamine accumulation increased to 0.12 g / L and 0.25 g / L, respectively. These results indicate that inoculating the second strain 4-12 h after the first strain allows the first strain to establish a certain ornithine supply base before the second strain performs subsequent transformation, thereby avoiding the decrease in transformation efficiency caused by insufficient substrate in the early stage or asynchronous metabolism in the later stage. The second strain showed the best results when inoculated 6 hours after the first strain was cultured.

[0095] Regarding fermentation pH, in Examples 7 and 8, when the initial pH of the fermentation medium was 6.5 and 7.5, respectively, the spermidine yield was 32.5 g / L and 33.3 g / L, and the butanediamine accumulation was 0.11 g / L and 0.18 g / L, respectively. However, in Comparative Examples 13 and 14, when the initial pH was 6.0 and 8.0, respectively, the spermidine yield decreased to 26.7 g / L and 27.6 g / L, respectively, and the butanediamine accumulation increased to 0.35 g / L and 0.42 g / L, respectively. These results indicate that the initial pH of the fermentation system affects the cell growth state, precursor supply, and catalytic efficiency of the mutant enzyme combination. Maintaining the initial pH within the range of 6.5-7.5 is beneficial for maintaining the metabolic activity and enzyme catalytic efficiency of the dual-cell system, with pH 7.0 showing the best overall effect.

[0096] In summary, the F158A, D262E, and L270A mutation sites selected in this application form an effective continuous catalytic match between carboxysemine dehydrogenase and carboxysemine decarboxylase, which can significantly improve the conversion efficiency of butanediamine to spermine and reduce butanediamine accumulation. Furthermore, based on the above mutant enzyme combination, by setting the inoculation ratio of the first and second strains, the inoculation sequence of the second strain, and the initial pH of the fermentation medium, a dynamic balance between ornithine supply, butanediamine production, and spermine synthesis can be further achieved, thereby obtaining higher spermine yield and lower butanediamine accumulation.

[0097] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A mutant enzyme combination for synthesizing spermidine, characterized in that, The mutant enzyme combination includes a carboxysemine dehydrogenase mutant and a carboxysemine decarboxylase mutant; The carboxysemine dehydrogenase mutant was obtained by mutating the carboxysemine dehydrogenase with the amino acid sequence shown in SEQ ID NO: 1, and the mutation sites of the carboxysemine dehydrogenase mutant are F158A and D262E. The carboxysemine decarboxylase mutant was obtained by mutating the carboxysemine decarboxylase with the amino acid sequence shown in SEQ ID NO: 2, and the mutation site of the carboxysemine decarboxylase mutant is L270A; wherein, The carboxysemine dehydrogenase is derived from Vibrio alginolyticus, and the carboxysemine decarboxylase is derived from Herminium arsenogenum.

2. The mutant enzyme combination encoding gene for synthesizing spermidine as described in claim 1.

3. A recombinant plasmid containing the gene encoding the mutant enzyme combination for synthesizing spermidine as described in claim 2.

4. A spermidine-directed synthesis bacterium, characterized in that, The spermidine-directed synthesizing bacterium is a genetically engineered bacterium with Escherichia coli as the chassis strain. The spermidine-directed synthesizing bacterium contains the mutant enzyme combination encoding gene for spermidine synthesis as described in claim 2. The mutant enzyme combination encoding gene is integrated into the genome of the spermidine-directed synthesizing bacterium or located in the recombinant plasmid as described in claim 3, and the spermidine-directed synthesizing bacterium can express the mutant enzyme combination for spermidine synthesis as described in claim 1.

5. A method for synthesizing spermidine, characterized in that, Includes the following steps: A first strain was obtained, which was used to synthesize and secrete ornithine; The spermidine-directed synthesis bacteria of claim 4 are obtained as a second strain. The second strain synthesizes aspartic semialdehyde using xylose as a carbon source and converts the ornithine into butanediamine using ornithine decarboxylase. The first strain and the second strain are co-cultured and fermented, so that the ornithine synthesized and secreted by the first strain is converted into butanediamine by the second strain, and the butanediamine and the aspartic semialdehyde are subjected to the action of the mutant enzyme combination for synthesizing spermidine as described in claim 1 to synthesize spermidine; wherein... The second strain is inoculated 4-12 hours after the first strain is cultured, and the inoculation ratio of the first strain to the second strain is any value in (1-2):(2-4), and the initial pH of the fermentation medium is any value in 6.5-7.

5.

6. The method for synthesizing spermidine according to claim 5, characterized in that, The first strain is an ornithine-producing bacterium. The first strain synthesizes ornithine using glucose as a carbon source and secretes the ornithine into the extracellular space.

7. The method for synthesizing spermidine according to claim 5, characterized in that, The fermentation medium used in the co-culture fermentation comprises 100 g / L-130 g / L of glucose and 50 g / L-70 g / L of xylose.

8. The method for synthesizing spermidine according to claim 5, characterized in that, The spermidine-directed synthesizing bacteria were obtained by gene editing of Escherichia coli W3110. The gene editing included overexpression of the aspC gene, the lysC mutant gene, the ppc gene, and the pyc gene, as well as knockout of the manZ gene and the ptsG gene.

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