Expression cassettes, expression vectors, genetically engineered strains and their applications in the preparation of malonic acid

Abstract

This invention provides an expression cassette, an expression vector, a genetically engineered strain, and their application in the preparation of malonic acid. The expression cassette includes expression cassette 1, expression cassette 2, and expression cassette 3. Expression cassette 1 includes the gene for the mitochondrial 37S ribosomal protein mS47; expression cassette 2 includes the ACC1 gene, which incorporates a mitochondrial localization signal encoding gene at its 5' end; and expression cassette 3 includes the upstream homologous arm of an endogenous ACC1 gene, which incorporates a weak promoter at its 5' end. This invention, through the expression cassette, can promote an increase in the levels of mS47 and ACC1 in mitochondria, thereby promoting the synthesis pathway of acetyl-CoA-malonyl-CoA-malonic acid (ACEA), while simultaneously reducing the level of endogenous ACC1 in the cytoplasm, avoiding competitive effects on the mitochondrial ACEA synthesis pathway, and ultimately effectively increasing malonic acid production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more particularly to expression cassettes, expression vectors, genetically engineered strains, and their application in the preparation of malonic acid. Background Technology

[0002] Malonic acid is an organic acid with the chemical formula HOOCCH2COOH. It has a wide range of applications, including as a raw material for organic synthesis, in the pharmaceutical industry for the production of barbiturates and other drugs, in the manufacture of fragrances, adhesives, and bactericides, as well as as a surface treatment agent for leather and aluminum products, an electroplating polishing agent, a foaming agent for foamed plastics, a cleaning agent for nuclear reactors, a flux for hot welding, and an explosive controller.

[0003] Currently, malonic acid is mainly obtained through chemical synthesis, such as by hydrolyzing diethyl malonate, or by hydrolyzing calcium fluoroacetate in an aqueous solution of calcium hydroxide to obtain calcium malonate, followed by acidic hydrolysis to obtain malonic acid. These chemical synthesis methods typically suffer from complex reaction processes and high production costs. Therefore, microbial synthesis of malonic acid has emerged. Within microorganisms, malonic acid can be obtained via a series of enzymatic catalytic actions along the phosphoenolpyruvate / pyruvate-oxaloacetate-aspartic acid-β-alanine-malonic acid hemialdehyde-malonic acid pathway. Alternatively, malonic acid can be synthesized using malonyl-CoA as a precursor with fewer catalytic enzymes. This microbial method of malonic acid synthesis offers advantages of being green and sustainable, and represents a potential future approach for malonic acid synthesis.

[0004] However, there is currently limited research on microbial synthesis of malonic acid, resulting in lower yields compared to traditional chemical synthesis methods. Therefore, finding a microbial synthesis method that can increase malonic acid production is an urgent issue. Summary of the Invention

[0005] This invention provides an expression cassette that can promote the increase of mS47 and ACC1 content in mitochondria, thereby promoting the synthesis pathway of acetyl-CoA-malonyl-CoA-malonic acid, while reducing the content of endogenous ACC1 in the cytoplasm, avoiding competitive effects on the synthesis pathway of acetyl-CoA-malonyl-CoA-malonic acid in mitochondria, and ultimately effectively increasing malonic acid production.

[0006] The present invention provides an expression vector, including an expression cassette and other acceptable gene elements, which can also increase malonic acid production.

[0007] This invention provides a genetically engineered strain, including a recombinant Yersinia lipophila with an expression cassette or expression vector, which can also increase malonic acid production.

[0008] The present invention provides the application of the above-described expression cassette, expression vector, or genetically engineered strain in the preparation of malonic acid.

[0009] The present invention also provides a method for preparing malonic acid, which is simple and easy to implement, and the whole process is easy to control precisely by human intervention, and can stably produce a large amount of malonic acid.

[0010] The present invention provides an expression box, which includes expression box 1, expression box 2 and expression box 3;

[0011] Expression cassette 1 includes the gene encoding the mitochondrial 37S ribosomal protein mS47;

[0012] Expression cassette 2 includes the ACC1 gene, which incorporates a mitochondrial localization signal encoding gene at its 5' end;

[0013] Expression frame 3 includes the upstream homologous arm of the endogenous ACC1 gene, which is integrated into the weak promoter at the 5' end.

[0014] The expression cassette described above, wherein the gene encoding the mitochondrial 37S ribosomal protein mS47 has:

[0015] (a) A nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or

[0016] (b) A nucleotide sequence obtained by deleting, substituting, or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as (a); or

[0017] (c) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in (a) or (b) and has the same or similar function as (a).

[0018] The expression frame described above contains the nucleotide sequence of the gene encoding the mitochondrial localization signal, as shown in SEQ ID NO:3.

[0019] The expression box described above, wherein the weak promoter is FBA, and the nucleotide sequence of the weak promoter is shown in SEQ ID NO:4.

[0020] The expression box described above, wherein expression box 1 includes hp4d-mS47-ICL; and / or,

[0021] Expression box 2 includes TEF-MLS-ACC1-XPR2; and / or,

[0022] Expression cassette 3 includes the upstream homologous arm of the FBA-ACC1 gene.

[0023] The present invention provides an expression vector comprising the above-described expression cassette and other acceptable gene elements.

[0024] The present invention provides a genetically engineered strain, wherein the genetically engineered strain is a recombinant Yersinia lipolyticis, comprising the above-described expression cassette or the above-described expression vector.

[0025] The present invention provides the application of the above-described expression cassette, expression vector, or genetically engineered strain in the preparation of malonic acid.

[0026] The present invention provides a method for preparing malonic acid, which includes inoculating the above-mentioned genetically engineered strain into a culture medium for fermentation culture to obtain malonic acid.

[0027] In the preparation method described above, the inoculum size of the genetically engineered strain is 4% (v / v) to 6% (v / v) of the culture medium volume; and / or,

[0028] Fermentation conditions include: temperature 25℃-35℃, aeration rate ≥1.5 vvm, and stirring speed 200-700 rpm; and / or,

[0029] When the glucose content in the culture medium is ≤10g / L, glucose should be added at a flow rate of 2-10g / L / h to maintain the glucose content in the culture medium ≥10g / L.

[0030] This invention provides an expression cassette, comprising expression cassette 1, expression cassette 2, and expression cassette 3. Expression cassette 1 includes the gene for the mitochondrial 37S ribosomal protein mS47. Expression cassette 2 includes the ACC1 gene, which incorporates a mitochondrial localization signal encoding gene at its 5' end. Expression cassette 3 includes the upstream homologous arm of the endogenous ACC1 gene, which incorporates a weak promoter at its 5' end. This invention promotes an increase in the content of mS47 in mitochondria through expression cassette 1, thereby promoting the catalysis of malonyl-CoA to malonic acid by mS47 in mitochondria. Expression cassette 2 promotes an increase in the content of ACC1 in mitochondria, thereby promoting the catalysis of acetyl-CoA to malonyl-CoA by ACC1 in mitochondria, supplementing the precursor of malonyl-CoA. Finally, expression cassette 3 can reduce the expression level of the endogenous ACC1 gene, reducing the content of endogenous ACC1 in the cytoplasm, thus avoiding competition between endogenous ACC1 in the cytoplasm and ACC1 in the mitochondria for acetyl-CoA precursor. The expression cassette of this invention can effectively increase malonic acid production through multiple mechanisms and pathways. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below. The specific embodiments listed below are merely descriptions of the principles and features of the present invention, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Malonic acid, with the chemical formula HOOCCH2COOH, is an organic acid with a wide range of applications. Currently, malonic acid can be obtained through chemical synthesis or microbial synthesis. For example, it can be synthesized by hydrolyzing diethyl malonate, or through microbial synthesis via a series of enzymatic catalysis along the pathway of phosphoenolpyruvate / pyruvate-oxaloacetic acid-aspartic acid-β-alanine-malonic acid semialdehyde-malonic acid. Chemical synthesis often suffers from complex reaction processes and high production costs, while microbial synthesis offers advantages in terms of green sustainability. Therefore, microbial synthesis of malonic acid may be a potential future method for its production. However, limited existing research has resulted in lower yields compared to traditional chemical synthesis methods. Therefore, finding a microbial synthesis method that can increase malonic acid production is a pressing issue.

[0033] To address the aforementioned problems, the present invention provides an expression cassette, comprising expression cassette 1, expression cassette 2, and expression cassette 3. Expression cassette 1 includes the gene encoding mitochondrial 37S ribosomal protein mS47, expression cassette 2 includes the ACC1 gene with a mitochondrial localization signal encoding gene integrated at its 5' end, and expression cassette 3 includes the upstream homologous arm of an endogenous ACC1 gene with a weak promoter integrated at its 5' end.

[0034] The gene encoding mitochondrial 37S ribosomal protein mS47 (mS47) encodes mitochondrial 37S ribosomal protein mS47, an endogenous enzyme in *Yarrowia lipolytica*, located in mitochondria, and possessing the potential to catalyze the synthesis of malonic acid from malonyl-CoA. Therefore, when expression frame 1 includes the gene encoding mitochondrial 37S ribosomal protein mS47, overexpression of this gene can increase the content of mS47 in mitochondria, thereby promoting the pathway of malonyl-CoA to malonic acid synthesis and ultimately increasing malonic acid production.

[0035] Within microorganisms, promoting only the malonyl-CoA pathway to malonic acid without correspondingly increasing malonyl-CoA synthesis or supplementing its source may lead to malonyl-CoA deficiency. Furthermore, as an intermediate metabolite, malonyl-CoA is used not only for malonic acid synthesis within cells but also participates in other metabolic pathways such as fatty acid biosynthesis. Therefore, malonyl-CoA deficiency can also affect other malonyl-CoA-dependent metabolic processes.

[0036] To avoid the above situation, this invention introduces expression frame 2, which includes the ACC1 gene, with a mitochondrial localization signal encoding gene integrated at its 5' end. The ACC1 gene encodes acetyl-CoA carboxylase 1 (ACC1), which catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA. Since acetyl-CoA is a key intermediate in cellular metabolism, participating in multiple important biochemical pathways and occupying a central position in the metabolic network, acetyl-CoA is usually relatively abundant in microorganisms. Therefore, using acetyl-CoA as a precursor to synthesize malonyl-CoA has minimal impact on other metabolic processes. Furthermore, ACC1 is generally located in the cytoplasm, and the malonyl-CoA it catalyzes is also located in the cytoplasm. However, mS47 is generally located in the mitochondria, and its catalytic synthesis of malonyl-CoA into malonic acid occurs in the mitochondria. Therefore, the malonyl-CoA synthesized by ACC1 needs to be transported from the cytoplasm to the mitochondria to serve as a catalytic raw material for mS47. This transport inevitably involves energy consumption and a decrease in catalytic efficiency. To address the issue of reduced catalytic efficiency caused by the different intracellular localization of ACC1 and mS47, this invention integrates a gene encoding a mitochondrial targeting signal into the 5' end of the ACC1 gene. The mitochondrial targeting signal (MTS) is a specific amino acid sequence that guides the transport of newly synthesized proteins from the cytoplasm to the mitochondria. Therefore, the ACC1 gene with the MTS-encoding gene integrated at the 5' end is actually a fusion gene with the MTS-encoding gene at the 5' end and the ACC1 gene at the 3' end. Transcription and translation of this fusion gene yield a fusion protein with the MTS-encoding gene at the N-terminus and ACC1 at the C-terminus. The MTS then guides the transport of ACC1 to the mitochondria. Therefore, when expression box 2 includes the ACC1 gene with the MTS-encoding gene integrated at the 5' end, overexpression of this gene increases the ACC1 content in the mitochondria, thereby promoting the pathway of acetyl-CoA to malonyl-CoA. Ultimately, it can synergistically work with expression box 1 to increase malonic acid production.

[0037] During the experiment, overexpression of the gene encoding the mitochondrial localization signal integrated at the 5' end in expression frame 2 did indeed increase the content of ACC1 in mitochondria. However, the inventors subsequently discovered that endogenous ACC1 in the cytoplasm competes with mitochondrial ACC1 for acetyl-CoA precursors, resulting in a decrease in the efficiency of mitochondrial ACC1 in catalyzing the conversion of acetyl-CoA to malonyl-CoA. Therefore, this invention also introduces expression frame 3 to solve the above problems. Expression frame 3 includes an upstream homologous arm of the endogenous ACC1 gene with a weak promoter integrated at the 5' end. The endogenous ACC1 gene encodes endogenous ACC1 located in the cytoplasm. To reduce the expression level of the endogenous ACC1 gene, a DNA fragment of approximately 150-500 bp can be selected upstream of the endogenous ACC1 gene as an upstream homologous arm, and a weak promoter can be integrated at the 5' end of this upstream homologous arm to obtain an upstream homologous arm of the endogenous ACC1 gene with a weak promoter integrated at the 5' end. Therefore, the upstream homologous arm of the endogenous ACC1 gene with a weak promoter integrated at the 5' end is actually a fusion gene with a weak promoter at the 5' end and an upstream homologous arm of the endogenous ACC1 gene at the 3' end. In the microorganism, this fusion gene locates the endogenous ACC1 gene through its upstream homologous arm, undergoes homologous recombination with the endogenous ACC1 gene, and introduces the weak promoter into the 5' end of the endogenous ACC1 gene, thereby reducing the expression level of the endogenous ACC1 gene and thus reducing the amount of endogenous ACC1 in the cytoplasm. It is understandable that although the ACC1 gene in expression frame 2 has the same sequence as the endogenous ACC1 gene, because the ACC1 gene in expression frame 2 lacks an upstream homologous arm of the endogenous ACC1 gene, expression frame 3 will only reduce the expression level of the endogenous ACC1 gene and will not affect the overexpression of the ACC1 gene in expression frame 2 with the mitochondrial localization signal encoding gene integrated at the 5' end.

[0038] In summary, the expression cassettes provided by this invention can promote an increase in the content of mitochondrial mS47 by overexpressing the gene encoding the mitochondrial 37S ribosomal protein mS47 through expression cassette 1, thereby promoting the catalysis of malonyl-CoA to malonic acid by mS47. Simultaneously, by overexpressing the ACC1 gene, which integrates into the mitochondrial localization signal gene at the 5' end, through expression cassette 2, the content of ACC1 in the mitochondria is increased, thereby promoting the catalysis of acetyl-CoA to malonic acid by ACC1 in the mitochondria to supplement malonyl-CoA. Finally, by utilizing the upstream homologous arm of the endogenous ACC1 gene, which integrates a weak promoter at the 5' end, in expression cassette 3, the expression level of the endogenous ACC1 gene is reduced, resulting in a decrease in the content of endogenous ACC1 in the cytoplasm, thus avoiding competition between endogenous ACC1 in the cytoplasm and ACC1 in the mitochondria for acetyl-CoA precursors. The expression cassettes of this invention can effectively increase malonic acid production through multiple mechanisms and pathways.

[0039] Furthermore, the gene encoding the mitochondrial 37S ribosomal protein mS47 has the following characteristics:

[0040] (a) A nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or

[0041] (b) A nucleotide sequence obtained by deleting, substituting, or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as (a); or

[0042] (c) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in (a) or (b) and has the same or similar function as (a).

[0043] This invention, through sequence analysis of the *Yarrowia lipolytica* genome, determined that the gene encoding the mitochondrial 37S ribosomal protein mS47 encodes mS47, which catalyzes the synthesis of malonic acid from malonyl-CoA. Specifically, the gene encoding mitochondrial 37S ribosomal protein mS47 can be the wild-type mitochondrial 37S ribosomal protein mS47, and this gene is YALI1_D07981g, with the nucleotide sequence shown in SEQ ID NO:1. The gene encoding the mitochondrial 37S ribosomal protein mS47 can also be a modified sequence as shown in SEQ ID NO:1, specifically as shown in SEQ ID NO:2. This modified sequence replaces the three bases TTC at positions 346-348 with ATC, and the three bases GAA at positions 355-357 with AGC. This results in a mutation at position 116 from phenylalanine (Phe, F) to isoleucine (Ile, I), and at position 119 from glutamic acid (Glu, E) to serine (Ser, S). The mitochondrial 37S ribosomal protein mS47 encoded by the nucleotide sequence shown in SEQ ID NO:2 exhibits a stronger ability to catalyze the synthesis of malonic acid than the wild-type mS47.

[0044] In the technical solution of this invention, to promote a good match between the mitochondrial localization signal encoding gene and the ACC1 gene, and to increase the content of ACC1 in mitochondria, this invention can also regulate the mitochondrial localization signal. Specifically, when the nucleotide sequence of the mitochondrial localization signal encoding gene is as shown in SEQ ID NO:3, the production of malonic acid can be increased.

[0045] In the technical solution of this invention, to promote the reduction of endogenous ACC1 gene expression level, facilitate good matching between the upstream homologous arm of the endogenous ACC1 gene and the weak promoter, and further solve the problem of competition between endogenous ACC1 in the cytoplasm and ACC1 in the mitochondria for acetyl-CoA precursor, this invention can also regulate the weak promoter. Specifically, when the weak promoter is FBA and the nucleotide sequence of the weak promoter is as shown in SEQ ID NO:4, malonic acid production can be increased.

[0046] In one specific embodiment, expression frame 1 may include hp4d-mS47-ICL, wherein hp4d is a constitutive strong promoter formed by tandemly connecting four upstream activation sequences UAS1B of pXPR2 with the most concise LEU2 promoter, particularly suitable for *Yersinia lipolytica*; mS47 is the gene encoding the mitochondrial 37S ribosomal protein mS47, which encodes mS47 and catalyzes the production of malonic acid from malonyl-CoA; ICL refers to the terminator of the gene encoding the endogenous isocitrate lyase in *Yersinia lipolytica*. By regulating the gene elements of expression frame 1, introducing specific promoter elements at the 5' end and specific terminator elements at the 3' end of the mS47 encoding gene, good matching of gene elements in expression frame 1 can be promoted, thereby promoting the overexpression of mS47 in expression frame 1. In one embodiment of the present invention, the nucleotide sequence of hp4d-mS47-ICL is shown in SEQ ID NO:5 or SEQ ID NO:6.

[0047] Expression box 2 may include TEF-MLS-ACC1-XPR2, where TEF refers to the promoter sequence of a transcription elongation factor (TEF) gene derived from fungi, commonly used in gene expression systems of fungi such as yeast, and can drive high levels of gene expression; MLS is the gene encoding mitochondrial targeting signal (MTS), which, after being translated into an amino acid sequence, can guide the transport of newly synthesized proteins from the cytoplasm to the mitochondria. Specifically, the nucleotide sequence of MLS may be as shown in SEQ ID NO:3; ACC1 is the gene encoding acetyl-CoA carboxylase 1 (ACC1), which can catalyze the carboxylation of acetyl-CoA to produce malonyl-CoA; and XPR2 is a terminator sequence derived from yeast strains such as Pichia pastoris. By regulating the gene elements in expression cassette 2, the MLS gene element is fused to the 5' end of the ACC1 gene element, allowing the ACC1 protein encoded by the ACC1 gene element to be transported to the mitochondria. Simultaneously, introducing a specific promoter element at the 5' end and a specific terminator element at the 3' end of the fusion gene encoded by MLS-ACC1 promotes good matching of the gene elements in expression cassette 2, thereby promoting the overexpression of MLS-ACC1 in expression cassette 2. In one embodiment of the present invention, the nucleotide sequence of TEF-MLS-ACC1-XPR2 is shown in SEQ ID NO:7.

[0048] Expression cassette 3 may include the upstream homology arm of the FBA-ACC1 gene, where FBA refers to the promoter sequence derived from the gene encoding fructose-1,6-bisphosphate aldolase (FBA). Existing research indicates that FBA can function as a weak promoter. By regulating the gene elements in expression cassette 3 and fusing the FBA promoter element to the 5' end of the upstream homology arm element of the ACC1 gene, the expression level of endogenous ACC1 can be reduced, thereby decreasing the content of endogenous ACC1 in the cytoplasm and preventing endogenous ACC1 in the cytoplasm from competing with ACC1 in the mitochondria for acetyl-CoA precursors. In one embodiment of the present invention, the nucleotide sequence of the FBA-ACC1 Up homology arm is shown in SEQ ID NO:8.

[0049] It is understandable that if any one of expression frames 1, 2, or 3 meets the above conditions, malonic acid production can be increased. If any two or all of expression frames 1, 2, or 3 meet the above conditions, since the promoter and terminator sequences of each expression frame are different, there will be no crosstalk between gene expression frames 1, 2, and 3. This ensures independent regulation of each expression frame, reduces competition for transcription and translation resources, thereby improving gene expression efficiency, and also reduces the risk of recombination or mutation, thus improving the stability of the entire genetic construct.

[0050] In one feasible embodiment of the present invention, hp4d in hp4d-mS47-ICL in expression box 1 or TEF in TEF-MLS-ACC1-XPR2 in expression box 2 can be replaced with other strong promoters, such as ut8, GPD, etc.; ICL in hp4d-mS47-ICL in expression box 1 or XPR2 in TEF-MLS-ACC1-XPR2 in expression box 2 can be replaced with other terminators, such as CYC1, etc.

[0051] Furthermore, to achieve high-throughput screening of strains with high gene expression, different screening markers can be introduced into expression frames 1, 2, and 3 respectively. In one embodiment of the present invention, expression frame 1 can be fused with green fluorescent protein (GFP) to obtain the fused expression frame hp4d-mS47-ICL-FBA-GFP-CYC; expression frame 2 can be fused with red fluorescent protein (RFP) to obtain the fused expression frame TEF-MLS-ACC1-XPR2-FBA-RFP-CYC; and expression frame 3 can be fused with leucine (Leu, L) to obtain the fused expression frame TEF-Leu-CYC-FBA-ACC1 upstream homologous arm; wherein, CYC is a terminator element. Subsequently, flow cytometry is used to screen genetically engineered strains with high GFP fluorescence intensity and RFP fluorescence intensity to obtain genetically engineered strains that highly express expression frames 1 and 2. Then, by using leucine-deficient culture medium, genetically engineered strains that fail to express expression frame 3 can be screened out, and finally genetically engineered strains that highly express the expression frames provided in the first aspect of the present invention can be obtained.

[0052] Based on the above research, a second aspect of the present invention provides an expression vector, including the expression cassette provided in the first aspect of the present invention and other acceptable gene elements.

[0053] This invention does not limit the specific type of expression vector; it can be a plasmid. Plasmids have the advantages of stability, reliability, and ease of operation. They can self-replicate and maintain a stable copy number in daughter cells. Therefore, plasmids can be constructed by recombining the above expression cassette into a plasmid vector.

[0054] This invention does not limit the method of recombining the expression cassette into the plasmid; those skilled in the art can choose the method as needed. Optionally, the expression cassette and plasmid vector can be digested with restriction endonucleases to obtain an expression cassette and linear plasmid vector with corresponding sticky ends. Then, DNA ligase can be used to ligate the expression cassette and linear plasmid vector with corresponding sticky ends to obtain the plasmid. The digestion can be single or double digestion, i.e., one or two restriction endonucleases can be used. Optionally, Gibson assembly technology can also be used to introduce terminal homologous sequences into the expression cassette and plasmid vector, and then the expression cassette and plasmid vector can be ligated to obtain the plasmid.

[0055] It is understood that the expression frames of the present invention include expression frame 1, expression frame 2, and expression frame 3. These three expression frames can be constructed on three expression carriers respectively; they can also be constructed on two expression carriers, for example, expression frame 1 and expression frame 2 are constructed on one expression carrier, and expression frame 3 is constructed on another expression carrier; or they can be constructed on one expression carrier, for example, expression frame 1, expression frame 2, and expression frame 3 are all constructed on one expression carrier. In one embodiment of the present invention, expression frame 1, expression frame 2, and expression frame 3 are constructed on three expression carriers respectively for use.

[0056] Furthermore, a third aspect of the present invention provides a genetically engineered strain, which is a recombinant Yersinia lipolyticis, comprising the expression frame provided in the first aspect of the present invention or the expression vector provided in the second aspect of the present invention.

[0057] Yarrowia lipolytica is an important industrial microbial strain, widely recognized as a food-grade safe microorganism. In recent years, with the rapid development of synthetic biology and gene editing technology, scientists have successfully constructed Yarrowia lipolytica cell factories capable of producing a variety of high-value-added industrial products, including biochemicals, biofuels, fragrances, pharmaceuticals, industrial enzymes, and pharmaceutical proteins. This gives the yeast enormous application potential in the food, pharmaceutical, and bioenergy sectors. Yarrowia lipolytica possesses a high acetyl-CoA metabolic flux and excellent fatty acid synthesis capabilities, making it an ideal microbial host for malonic acid synthesis.

[0058] In this invention, Yersinia lipophila PO1f can be used as a chassis strain, and an expression vector carrying the expression cassette provided in the first aspect of this invention can be transformed into it to induce overexpression of the target gene in the expression cassette in PO1f, thereby obtaining the genetically engineered strain provided by this invention.

[0059] This invention does not specify the method of transforming the expression vector into the chassis strain. Those skilled in the art can perform the transformation according to conventional transformation methods, such as the lithium acetate-polyethylene glycol method or the electroconversion method.

[0060] Furthermore, to obtain genetically engineered strains with high gene expression, this invention allows for repeated transformations, with screening performed after each transformation. Through multiple rounds of integration, genetically engineered strains producing high levels of malonic acid can ultimately be screened.

[0061] The fourth aspect of the present invention provides the application of the above-described expression cassette, expression vector, or genetically engineered strain in the preparation of malonic acid.

[0062] This invention promotes the increase of mS47 and ACC1 levels in mitochondria by regulating the expression cassette, thereby promoting the synthesis pathway of acetyl-CoA-malonyl-CoA-malonic acid (ACE). Simultaneously, it reduces the content of endogenous ACC1 in the cytoplasm, avoiding competitive effects on the mitochondrial ACE synthesis pathway, ultimately effectively increasing malonic acid production. It is understood that the aforementioned expression vector or genetically engineered strain, because it includes the expression cassette of this invention, can also increase malonic acid production and can therefore be used in malonic acid preparation.

[0063] The fifth aspect of this invention provides a method for preparing malonic acid, comprising inoculating the aforementioned genetically engineered strain into a culture medium for fermentation to obtain malonic acid. This method is simple and easy to implement, the entire process is easily and precisely controlled, and it can stably produce large quantities of malonic acid.

[0064] In one embodiment of the present invention, the genetically engineered strain of the present invention can be inoculated into a yeast extract peptone glucose culture medium at a certain inoculum amount for fermentation culture, and cultured for 80-100 hours under certain fermentation temperature, fermenter aeration rate, and stirring speed, during which the glucose content in the fermentation broth is controlled to be ≥10 g / L. After the fermentation culture is completed, malonic acid can be separated and purified to obtain malic acid.

[0065] Specifically, the inoculum size of the genetically engineered strain can be 4% (v / v) to 6% (v / v) of the culture medium volume. Inoculum size refers to the percentage of the volume of the seed culture transferred in to the volume of the culture medium after inoculation. That is, an inoculum size of 4% (v / v) to 6% (v / v) means that the volume of the genetically engineered strain seed culture transferred in is 4% to 6% of the total volume of the seed culture and the yeast extract peptone glucose culture medium after inoculation. It can be understood that the inoculum size can be selected from any value within the range of 4% to 6%, for example, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, or any value within a range thereof. The method for preparing the seed culture of the genetically engineered strain can be as follows: the genetically engineered strain stored at -80℃ is streaked onto yeast extract peptone glucose solid medium, cultured at 30℃ for 24h, a single colony is picked and inoculated into 5ml of liquid yeast extract peptone glucose medium, cultured at 30℃ for 24h, and the culture is inoculated into 50ml of liquid yeast extract peptone glucose medium, cultured at 30℃ for 24h to obtain the seed culture.

[0066] In addition, fermentation conditions may include: a temperature of 25℃-35℃, an aeration rate of ≥1.5vvm in the fermenter, and a stirring speed of 200-700rpm.

[0067] When the glucose content in the culture medium is ≤10g / L, glucose should be added at a flow rate of 2-10g / L / h to maintain the glucose content in the culture medium ≥10g / L.

[0068] It is understood that in the preparation method of the present invention, the yield of malonic acid can be increased if any one of the following conditions is met: inoculum size, fermentation temperature, fermenter aeration rate, stirring speed, glucose supplementation method, and flow rate. The more conditions are met, the better the effect of increasing the yield of malonic acid. The effect of increasing the yield of malonic acid is optimal when all of the following conditions are met: inoculum size, fermentation temperature, fermenter aeration rate, stirring speed, glucose supplementation method, and flow rate.

[0069] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions or as recommended by the manufacturer. All reagents and vectors used, unless otherwise specified, are commercially available or publicly available. Primer sequences, fragment sequences, and fusion expression cassette sequences used in the following examples are shown in Table 1, Table 2, and Table 3, respectively.

[0070] Table 1

[0071]

[0072] Table 2

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Table 3

[0079]

[0080]

[0081]

[0082]

[0083] Example 1: Obtaining the target fragment

[0084] (1) Obtain the target fragments mS47 and mmS47

[0085] Using the W29 wild-type Yersinia lipolyticis genome as a DNA template, fragment mS47 was amplified by polymerase chain reaction (PCR) using primer pairs 999-F and 999-R. The nucleotide sequence of fragment mS47 is shown in SEQ ID NO:1.

[0086] Using the W29 wild-type *Yersinia lipolyticis* genome as a DNA template, the 5' end fragment 999-up of fragment mS47 was amplified by PCR using primer pairs 999-F and 999-up-R. Using the W29 wild-type *Yersinia lipolyticis* genome as a DNA template, the 3' end fragment 999-down of fragment mS47 was amplified by PCR using primer pairs 999-down-F and 999-R. Fragments 999-up and 999-down were then fused by PCR using primer pairs 999-F and 999-R to obtain fragment mmS47 with mutations at two amino acid sites, F116I and E119S. The nucleotide sequence of fragment mmS47 is shown in SEQ ID NO:2.

[0087] (2) Obtain the target fragment MLS-ACC1

[0088] Using the genome of wild-type Yersinia lipolytica W29 as a DNA template, the MLS sequence was introduced into the 5' end of fragment ACC1 using PCR technology with primer pairs ACC1-F and ACC1-R, thereby amplifying fragment MLS-ACC1. The nucleotide sequence of the MLS sequence is shown in SEQ ID NO:3, and the nucleotide sequence of fragment MLS-ACC1 is shown in SEQ ID NO:9.

[0089] (3) Obtain the target fragments GFP and RFP

[0090] The nucleotide sequence of green fluorescent protein (GFP) was optimized based on the codon preference of *Yarrowia lipolytica* to obtain GFP fragment, which was then synthesized by Genewiz Biotechnology Co., Ltd. The nucleotide sequence of red fluorescent protein (RFP) was also optimized based on the codon preference of *Yarrowia lipolytica* to obtain RFP fragment, which was then synthesized by Genewiz Biotechnology Co., Ltd. The nucleotide sequence of GFP fragment is shown in SEQ ID NO:10, and the nucleotide sequence of RFP fragment is shown in SEQ ID NO:11.

[0091] (4) Obtain the target promoter and terminator segments.

[0092] Using the genome of wild-type *Yarrowia lipolyticis* W29 as a DNA template, the promoter fragment TEF (nucleotide sequence shown in SEQ ID NO:12) was amplified by PCR using primer pairs TEF-F and TEF-R. The promoter fragment FBA was also amplified by PCR using primer pairs FBA-F and FBA-R, and its nucleotide sequence is shown in SEQ ID NO:4. The nucleotide sequence of the promoter fragment hp4d (SEQ ID NO:13) was obtained from publicly available literature and synthesized by Genewiz Biotechnology Co., Ltd.

[0093] Using the genome of wild-type *Yersinia lipolytica* W29 as a DNA template, the terminator fragment CYC (nucleotide sequence shown in SEQ ID NO:14) was amplified by PCR using primer pairs CYC-F and CYC-R. The terminator fragment XPR2 (nucleotide sequence shown in SEQ ID NO:15) was amplified by PCR using primer pairs XPR2-F and XPR2-R. The terminator fragment ICL (nucleotide sequence shown in SEQ ID NO:16) was amplified by PCR using primer pairs ICL-F and ICL-R.

[0094] (5) Obtain the target segment Leu

[0095] Using the genome of wild-type Yersinia lipolyticis W29 as a DNA template, the fragment Leu (nucleotide sequence shown in SEQ ID NO:17) was amplified by PCR using primer pairs Leu-F and Leu-R.

[0096] (6) Obtain the target segment ACC1A

[0097] Using the genome of wild-type Yersinia lipolytica W29 as a DNA template, the upstream homologous arm of the ACC1 gene (denoted as ACC1A) was amplified by PCR using primer pairs ACC1-AF and ACC1-AR (nucleotide sequence shown in SEQ ID NO:18).

[0098] Example 2: Obtaining the target fusion expression box

[0099] (1) Obtain the fusion expression frame hp4d-mS47-ICL or hp4d-mmS47-ICL

[0100] Using fusion PCR, the promoter fragment hp4d, fragment mS47 or mmS47, and terminator fragment ICL are fused through primer pair hp4d-F and ICL-R to obtain fusion expression cassette hp4d-mS47-ICL or fusion expression cassette hp4d-mmS47-ICL. The nucleotide sequence of fusion expression cassette hp4d-mS47-ICL is shown in SEQ ID NO:5, and the nucleotide sequence of fusion expression cassette hp4d-mmS47-ICL is shown in SEQ ID NO:6.

[0101] (2) Obtain the fused expression frame TEF-MLS-ACC1-XPR2

[0102] Using fusion PCR, the promoter fragment TEF, the fragment MLS-ACC1, and the terminator fragment XPR2 were fused using primer pair TEF-F and XPR2-R to obtain the fusion expression cassette TEF-MLS-ACC1-XPR2, the nucleotide sequence of which is shown in SEQ ID NO:7.

[0103] (3) Obtain the fusion expression cassettes FBA-GFP-CYC and FBA-RFP-CYC

[0104] Using fusion PCR, the promoter fragment FBA, the GFP fragment, and the terminator fragment CYC were fused using primer pair FBA-F and CYC-R to obtain the fusion expression cassette FBA-GFP-CYC (nucleotide sequence shown in SEQ ID NO:19).

[0105] Using fusion PCR, the promoter fragment FBA, the fragment RFP, and the terminator fragment CYC were fused using primer pairs FBA-F and CYC-R to obtain the fusion expression cassette FBA-RFP-CYC (nucleotide sequence shown in SEQ ID NO:20).

[0106] (4) Obtain the fusion expression cassette mS47-GFP or mmS47-GFP

[0107] Using fusion PCR, the fusion expression cassette hp4d-mS47-ICL or hp4d-mmS47-ICL was fused with the fusion expression cassette FBA-GFP-CYC via primer pair hp4d-F and CYC-R, resulting in either the fusion expression cassette hp4d-mS47-ICL-FBA-GFP-CYC (denoted as mS47-GFP) or the fusion expression cassette hp4d-mmS47-ICL-FBA-GFP-CYC (denoted as mmS47-GFP). The nucleotide sequence of mS47-GFP is composed of the nucleotide sequences of the fusion expression cassette hp4d-mS47-ICL and the fusion expression cassette FBA-GFP-CYC, sequentially from the 5' end to the 3' end. Specifically, it consists of the nucleotide sequences shown in SEQ ID NO:5 and SEQ ID NO:19, sequentially from the 5' end to the 3' end. The nucleotide sequence of mmS47-GFP consists of the nucleotide sequence of the fusion expression cassette hp4d-mmS47-ICL and the nucleotide sequence of the fusion expression cassette FBA-GFP-CYC from the 5' end to the 3' end, that is, it consists of the nucleotide sequence shown in SEQ ID NO:6 and the nucleotide sequence shown in SEQ ID NO:19 from the 5' end to the 3' end.

[0108] (5) Obtain the fused expression frame MLS-ACC1-RFP

[0109] Using fusion PCR, the fusion expression cassettes TEF-MLS-ACC1-XPR2 and FBA-RFP-CYC were fused via primer pair TEF-F and CYC-R to obtain the fusion expression cassette TEF-MLS-ACC1-XPR2-FBA-RFP-CYC (denoted as MLS-ACC1-RFP). The nucleotide sequence of MLS-ACC1-RFP consists of the nucleotide sequences of fusion expression cassettes TEF-MLS-ACC1-XPR2 and FBA-RFP-CYC from the 5' end to the 3' end, specifically the nucleotide sequences shown in SEQ ID NO:7 and SEQ ID NO:20 from the 5' end to the 3' end.

[0110] (6) Obtain the fusion expression box Leu-ACC1A

[0111] Using fusion PCR, the promoter fragment TEF, fragment Leu, terminator fragment CYC, promoter fragment FBA, and fragment ACC1A were fused using primer pair TEF-F and ACC1-AR to obtain the fusion expression cassette TEF-Leu-CYC-FBA-ACC1A (denoted as Leu-ACC1A). The nucleotide sequence of Leu-ACC1A is shown in SEQ ID NO:8.

[0112] Example 3: Obtaining high-expression strains

[0113] (1) The experiment was divided into two groups, named the mS47 group and the mmS47 group, respectively. The only difference between the mS47 group and the mmS47 group was that the mS47 group used the fusion expression cassette mS47-GFP, while the mmS47 group used the fusion expression cassette mmS47-GFP. 3 μg of either the mS47-GFP or mmS47-GFP fusion expression cassette mS47-GFP and 3 μg of the MLS-ACC1-RFP fusion expression cassette were simultaneously transformed into Yeast Extract Peptonedextrose Medium (YPD) strain. After transformation, the transformed strain was cultured in liquid Yeast Extract Peptonedextrose Medium (YPD) to obtain the transformed strain. The liquid YPD contained 20 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose.

[0114] (2) The transformed strains were sorted using a flow cytometer to obtain the bacterial groups with the strongest GFR and RFP fluorescence intensity, and single colonies were sorted into YPD solid medium. The YPD solid medium consisted of 2 g / L peptone, 10 g / L yeast extract, 20 g / L glucose and 20 g / L agar powder.

[0115] (3) Randomly select the above single colonies into a 300mL Erlenmeyer flask containing 50mL liquid YPD for shake-flask fermentation, and screen out the strain with the highest malonic acid production, which is recorded as F1.

[0116] (4) The fusion expression cassette Leu-ACC1A was transformed into the F1 strain. After transformation, the strain was cultured in leucine-deficient medium to obtain a secondary transformed strain. The secondary transformed strain was sequenced, and strains that successfully underwent homologous recombination were screened. This strain was designated as F2.

[0117] (5) Take 3 μg of the fusion expression cassette mS47-GFP or mmS47-GFP and 3 μg of the fusion expression cassette MLS-ACC1-RFP and transform them into the F2 strain. Then repeat steps (1)-(3) to screen out the strain with the highest malonic acid production, which is recorded as F3. Repeat this operation 5 times to obtain strains F3, F4, F5, F6 and F7 respectively. The results showed that the shake-flask level malonic acid yield of F1 in the mS47 group was 0.1 g / L; the shake-flask level malonic acid yield of F1 in the mmS47 group reached 0.3 g / L, F2 reached 1.2 g / L, F5 reached 1.5 g / L, F6 reached 1.7 g / L, and F7 reached 1.8 g / L; the wild-type Yersinia lipolytica Po1f strain, serving as the blank control group, had a shake-flask level malonic acid yield of 0 g / L under the same shake-flask fermentation conditions.

[0118] Example 4: Detection of malonic acid yield by strain F7 in a fermenter

[0119] (1) The above-mentioned F7 strain, stored at -80℃, was streaked onto YPD solid medium and cultured at 30℃ for 24 h. A single colony was then picked and inoculated into 5 ml of liquid YPD and cultured at 30℃ for 24 h. The culture was then inoculated into 50 ml of liquid YPD and cultured at 30℃ for 24 h to obtain the seed culture. The seed culture was inoculated into a 5L scale fermenter containing 4L of liquid YPD at inoculation rates of 1%, 3%, 5%, and 7% (v / v), respectively, for fermentation culture. The initial glucose concentration of the liquid YPD was 100 g / L, and the other components remained unchanged. Fermentation culture was carried out at a fermentation temperature of 30℃, a fermenter aeration rate of 1.5 vvm, and a stirring speed of 500 rpm for 96 h. During the fermentation, when the glucose content in the fermentation broth was ≤10 g / L, the glucose concentration was supplemented to 100 g / L to control the glucose content in the fermentation broth to be ≥10 g / L. After fermentation, the yields of malonic acid in the fermenter were measured to be 11.9 g / L, 13.2 g / L, 16.6 g / L, and 15.9 g / L, respectively. These results indicate that the optimal inoculum size is 5% (v / v).

[0120] (2) Other fermentation conditions were the same as in step (1), except that the aeration rate of the fermenter was changed. The aeration rates were set to 0.5 vvm, 1.0 vvm, 1.5 vvm, and 2.0 vvm, respectively. After 96 hours of fermentation, the yields of malonic acid in the fermenter were measured to be 8.3 g / L, 11.2 g / L, 14.8 g / L, and 19.1 g / L, respectively. This result indicates that the optimal aeration rate of the fermenter is 2.0 vvm.

[0121] (3) Other fermentation conditions were the same as in step (1), except for the feeding method, which was changed to two feeding methods: batch feeding and fed-batch feeding. The batch feeding method involved adding glucose to a concentration of 100 g / L when the glucose content in the fermentation broth was ≤10 g / L. The fed-batch feeding method involved continuously adding glucose at a flow rate of 2-10 g / L / h when the glucose content in the fermentation broth was ≤10 g / L. After 96 h of fermentation, the malonic acid yields in the fermenter were measured to be 18.8 g / L and 21.3 g / L, respectively. This result indicates that the fed-batch feeding method is preferred, possibly because it maintains the carbon-to-nitrogen ratio in the fermentation broth at a level suitable for the F7 strain to ferment and decompose.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An expression box, characterized in that, Includes expression box 1, expression box 2, and expression box 3; The expression frame 1 includes the gene encoding the mitochondrial 37S ribosomal protein mS47; The expression frame 2 includes the ACC1 gene, which incorporates a mitochondrial localization signal coding gene at its 5' end; The expression frame 3 includes the upstream homologous arm of the endogenous ACC1 gene, into which a weak promoter is integrated at the 5' end.

2. The expression cassette of claim 1, wherein, The gene encoding the mitochondrial 37S ribosomal protein mS47 has the following characteristics: (a) A nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or (b) A nucleotide sequence obtained by deleting, substituting, or adding one or more bases to the nucleotide sequence shown in (a), and having the same or similar function as (a); or (c) A nucleotide sequence that has at least 80% homology with the nucleotide sequence shown in (a) or (b) and has the same or similar function as (a).

3. The expression cassette of claim 1 or 2, wherein, The nucleotide sequence of the gene encoding the mitochondrial localization signal is shown in SEQ ID NO:

3.

4. The expression cassette of any one of claims 1-3, wherein, The weak promoter is FBA, and the nucleotide sequence of the weak promoter is shown in SEQ ID NO:

4.

5. The expression cassette of any one of claims 1-4, wherein, The expression box 1 includes hp4d-mS47-ICL; and / or, The expression box 2 includes TEF-MLS-ACC1-XPR2; and / or, The expression frame 3 includes the upstream homologous arm of the FBA-ACC1 gene.

6. An expression vector, characterized by, It includes the expression cassette as described in any one of claims 1-5 and other acceptable gene elements.

7. A genetically engineered strain, characterized in that, The genetically engineered strain is a recombinant Yersinia lipophila, which includes the expression cassette as described in any one of claims 1-5 or the expression vector as described in claim 6.

8. The use of the expression cassette according to any one of claims 1-5, the expression vector according to claim 6, or the genetically engineered strain according to claim 7 in the preparation of malonic acid.

9. A method for preparing malonic acid, characterized in that, This includes inoculating the genetically engineered strain of claim 7 into a culture medium for fermentation culture to obtain the malonic acid.

10. The preparation method according to claim 9, characterized in that, The inoculation amount of the genetically engineered strain is 4% (v / v) to 6% (v / v) of the culture medium volume; and / or, The fermentation conditions include: a temperature of 25℃-35℃, an aeration rate ≥1.5 vvm, and a stirring speed of 200-700 rpm; and / or, When the glucose content in the culture medium is ≤10g / L, glucose is added at a flow rate of 2-10g / L / h to maintain the glucose content in the culture medium ≥10g / L.

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