Method for producing proheparin, heparan sulfate and heparin in yeast

Abstract

The present invention relates to a recombinant yeast cell that produces UDP-D-glucuronic acid and / or UDP-N-acetyl-D-glucosamine compared to a wild type cell of the recombinant yeast cell; and optionally, the yield of 3 '-phosphate-adenylate sulfuric acid is increased. The invention also relates to a method for producing heparan sulfate, which comprises a step of culturing the recombinant yeast cell, further relates to an application of the non-invasive recombinant yeast cell for producing heparan sulfate, and finally relates to heparan sulfate obtained by applying the method.

Description

Technical Field

[0001] The present invention relates to a recombinant yeast cell, which produces an increased production of UDP-D-glucuronic acid and / or UDP-N-acetyl-D-glucosamine, and optionally 3'-phospho-adenylic acid sulfate, compared to its wild-type cell. The present invention also relates to a method for producing heparan sulfate, the method comprising the step of culturing the recombinant yeast cell of the present invention, the present invention further relates to the use of the non-invasive recombinant yeast cell for producing heparan sulfate, and finally the present invention relates to the heparan sulfate obtained by applying the method of the present invention. Background Art

[0002] Heparin is a natural glycosaminoglycan stored in the secretory granules of mast cells. Although the intrinsic biological function of heparin has not been fully elucidated, it was discovered as early as the 1910s that heparin is an effective anticoagulant. Its anticoagulant properties are attributed to the high degree of sulfation of heparin and the resulting negative charge, which mediates the binding of factor Xa and thrombin to antithrombin. Therefore, heparin has been an effective and widely used thrombotic treatment for more than half a century.

[0003] In recent years, the global annual market size of heparin has been estimated at approximately $7 billion and is likely to increase substantially as new therapeutic applications for heparin and less sulfated heparan sulfate analogs are established. The precise sulfation pattern of glycosaminoglycans modulates their interactions with growth factors, enzymes, inhibitors, etc., making these well-defined heparan sulfates new anticancer, antibacterial, antidiabetic, anti-neurodegenerative and even better anticoagulant drugs.

[0004] Despite the obvious demand, the main source of heparin and its analogs is still animal tissue, especially pig intestine, which may lead to drug contamination and shortages in the event of animal disease outbreaks. The instability of traditional heparin sources, the difficulty of synthetic heparin production, and the huge market capacity of heparin and the potential applications of new heparan sulfates have prompted efforts to develop microbial cell factories for recombinant biosynthesis of heparin and its analogs.

[0005] Yeast is an efficient cell factory candidate for the production of heparin, heparan sulfate, and proheparin (the precursor of heparin and heparan sulfate), although this has not been demonstrated before. Although bacterial enzymes can be used to synthesize proheparin, all enzymes required for the modification of proheparin, i.e., the biosynthesis of heparan sulfate and heparin, come from vertebrates. Since enzymes derived from eukaryotic cells are generally more stable and more functional when expressed in eukaryotic hosts, yeasts such as S. cerevisiae are more suitable for the biosynthesis of heparin than bacterial systems.

[0006] Despite the clear advantages of yeast as cell factories for proheparin, heparan sulfate, and heparin, three distinct but closely related engineering challenges must be overcome: First, for yeast cell factories to become viable alternatives to traditional heparin sources, they must prove to be cost-effective and produce high titers of product. Therefore, yeast chassis with efficient substrate utilization and high precursor yields must be designed (Unit 1). Furthermore, these new efficient yeast strains must be engineered to enable proheparin production (Unit 2). Finally, proheparin-producing strains must be modified to biosynthesize heparin and other heparan sulfates with specific sulfation patterns (Unit 3).

[0007] The solutions to these obstacles presented in this article not only provide an excellent production platform for heparin, but also enable industrial-scale production of novel heparan sulfates for widespread medical applications. Summary of the invention

[0008] (1) Optimize chassis engineering

[0009] To establish yeast cell factories as an economically viable platform for the production of proheparin and heparan sulfate, it is critical to design a recombinant yeast chassis that can effectively utilize inexpensive substrates (e.g., D-glucose, (NH4)SO4) to provide high cytoplasmic precursor concentrations for proheparin synthesis (UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine) and modification (3'-phospho-adenylic acid sulfate). Figure 1a As shown, the biosynthetic pathways for UDP-sugar and 3'-phospho-adenylic acid sulfate (PAPS) are unconnected and can therefore be optimized separately via a modular pathway engineering approach.

[0010] The first module of this modular pathway engineering approach aimed to achieve high cytoplasmic concentrations of UDP-D-glucuronic acid (UDP-GlcA) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) in a recombinant S. cerevisiae chassis. Unlike UDP-GlcNAc, S. cerevisiae does not produce UDP-GlcA endogenously. However, heterologous synthesis of UDP-GlcA has been achieved in yeast by integrating UDP-glucose dehydrogenase (AtUGD1) from Arabidopsis thaliana [1]. Therefore, the heterologous AtUGD1 was integrated into the genome of the S. cerevisiae S288C parent strain, resulting in a recombinant strain capable of producing UDP-GlcNAc and UDP-GlcA. Although the cytoplasmic pool of UDP-glucose, the direct precursor of UDP-GlcA, is already large in S. cerevisiae, in order to increase the concentration of cytoplasmic UDP-GlcA, the endogenous phosphoglucomutase PGM1 / 2 and UTP-glucose-1-phosphate uridyl transferase (UTP-glucose pyrophosphorylase) UGP1 were overexpressed in subsequent strains, which can directly convert D-glucose-6-P into UDP-glucose.

[0011] As the main precursor of chitin, UDP-GlcNAc is synthesized from fructose-6-P in S.cerevisiae in four steps: fructose-6-P is first induced to transaminize (transaminase encoded by GFA1) to glucosamine-6-P, using L-glutamine as a nitrogen donor. Subsequently, glucosamine-6-P is acetylated (acetylase encoded by GNA1) to N-acetylglucosamine-6-P, which is then converted to N-acetylglucosamine-1-P by P-acetylglucosamine mutase encoded by PCM1. In the last synthesis step, N-acetyl-glucosamine-1-P is converted to UDP-GlcNAc by UDP-GlcNAc pyrophosphorylase (encoded by QRI1). Studies have shown that S.cerevisiae cultures treated with mating pheromones induce increased transcription of GFA1 and PCM1. This dynamic change suggests that GFA1 and PCM1 are bottlenecks in the UDP-GlcNAc pathway. Therefore, the endogenous aminotransferase GFA1 and P-acetyl-glucosamine mutase PCM1 were overexpressed in subsequent S. cerevisiae strains to increase the cytoplasmic concentration of UDP-GlcNAc. To prevent the depletion of UDP-GlcNAc in the cytoplasm, the transcription of CHS3, encoding chitin synthase III, was also downregulated, as chitin synthase III synthesizes approximately 90% of chitin in S. cerevisiae. Downregulation was achieved by replacing the native CHS3 promoter with the weaker KEX2 promoter, which reduces CHS3 transcription. The resulting yeast strains (composed of the respective yeast cells) have increased cytoplasmic concentrations of UDP-GlcA and UDP-GlcNAc and can serve as a chassis for increased production of glycosaminoglycans such as heparinogen ( Figure 1b ).

[0012] The second module of this modular pathway engineering approach was designed to increase the cytoplasmic 3′-phospho-adenylic acid sulfate (PAPS) concentration of recombinant S. cerevisiae strains, in addition to the previously elevated UDP-sugar concentration. To this end, we engineered recombinant S. cerevisiae strains to overexpress the sulfate adenylate transferase (ATP-sulfatase) MET3, the sulfate adenylate kinase MET14, and the adenosine 3',5'-diphospho-nucleotidase MET22. Similar to the approach used in Escherichia coli (E. coli) [2], transcription of the endogenous 3'-phospho-adenylic acid sulfate reductase MET16 was downregulated to limit the consumption of PAPS.

[0013] Overexpression and downregulation of a gene can be achieved by any appropriate method known to those skilled in the art. In the present invention, in the two modules of the modular pathway engineering method, i.e., the UDP sugar module and the PAPS module, overexpression is achieved by integrating an extra gene copy with a strong promoter (pTDH3, pTEF1 or pPGK1) into the chromosome. Downregulation is achieved by exchanging the native promoter of the corresponding gene with the weak promoter KEX2 (pKEX2).

[0014] (2) Engineering of producing heparinogenic yeast strains

[0015] Heparinogen is a glycosaminoglycan composed of repeating disaccharide units (-GlcA-β1,4-GlcNAc-ɑ1,4-) and is the precursor of unmodified GAGs of heparin and other heparan sulfates. In mast cells, the biosynthesis of heparinogen is initiated by the formation of a tetrasaccharide linker (-GlcA-β1,3-Gal-β1,3-Gal-β1,4-Xyl-O-Ser) on specific Ser residues of the sericin core protein. The formation of this linker is catalyzed by four enzymes, each of which elongates the linker by one residue from its respective UDP-sugar. The tetrasaccharide linker serves as a primer for the subsequent synthesis of heparinogen ([-GlcA-β1,4-GlcNAc-ɑ1,4-]n) by alternating addition of glucuronic acid residues and N-acetylglucosamine residues from their UDP-sugars. This polymerization is performed by isomeric complexes of the exonuclease family of GlcA / GlcNAc transferase II EXT1 and EXT2. This complex eukaryotic heparan synthesis pathway, which requires multiple different enzymes and UDP-sugars to synthesize heparanogen, can be replaced by a bacterial heparanogen synthetase that requires only UDP-GlcA and UDP-GlcNAcd. Escherichia coli K5 expresses two genes, KfiC and KfiA, encoding UDP-GlcA glucosyltransferase and UDP-GlcNAc glucosyltransferase, respectively, while Pasteurella multocida (P. multocida) expresses two highly homologous dual-acting heparanogen synthases, PmHS1 and PmHS2. Although both enzymes of P. multocida have glucuronidation and hexasaccharide transfer activities (dual action), their different catalytic preferences suggest that they play different roles in heparanogen synthesis: PmHS2 is less dependent on polysaccharide primers, while PmHS1 elongates heparanogen sugar chains three times faster than PmHS2, thereby producing polymers with larger molecular weights. When PmHS2 was heterologously expressed in Bacillus megaterium, the heparinogen titer in shake flasks was approximately 250 mg / L and the heparinogen titer in bioreactors was 2.74 g / L [3].

[0016] In this work, genes encoding PmHS1 and PmHS2 were integrated into the genome of an engineered S.cerevisiae strain capable of UDP-GlcA and UDP-GlcNAc biosynthesis, resulting in a recombinant yeast strain capable of producing proheparin. However, it was observed that the bacterial enzymes (PmHS1 and PmHS2) tend to localize in the yeast nucleus, hampering the efficient biosynthesis of proheparin. To counteract nuclear localization, the nuclear export sequence (NES) of the Hsp70 protein of S.cerevisiae was fused to the C-terminus of the proheparin synthase. When PmHS1_NES and PmHS2_NES were expressed in S.cerevisiae, an increase in the production of proheparin was observed, especially in the strain expressing PmHS1_NES. Co-expression of PmHS1_NES and PmHS2_NES further increased the titer of proheparin, which may be due to the different properties of the two isoforms: PmHS2 is better at synthesizing proheparin primers, while PmHS1 is a super proheparin synthetase that excels at elongating proheparin sugar chains. S.cerevisiae (see unit (1) "Optimizing chassis engineering") To PmHS1_NES and PmHS2_NES, The strain with the best heparinogen production can be obtained.

[0017] (3) Engineered yeast strains producing heparan sulfate and heparin

[0018] In the biosynthesis of heparinogen, polysaccharides are modified by various enzymes to produce various heparan sulfates with different biological functions ( Figure 2a). The first two heparinogen modification steps, N-deacetylation and PAPS-dependent N-sulfation of N-acetylglucosamine, are carried out by dual-acting enzymes called N-deacetylase-N-sulfotransferases (NDSTs). Although four highly homologous vertebrate NDST isoforms are known, only NDST1 and NDST2 have high N-deacetylation and N-sulfation activities. In contrast, the NDST3 isoform has high N-deacetylation activity but low N-sulfation activity. N-sulfated glucosamine (GlcNS) is required for the subsequent C5-epimerization of the adjacent D-glucuronic acid (GlcA) residue to L-iduronic acid (IdoA) by C5-isomerase (C5-epi). The epimerized L-iduronic acid increases the affinity of the residue for heparan sulfate 2-O-sulfotransferase (HS2ST) by 5-fold, facilitating the transfer of sulfate from PAPS to C2 of the L-iduronic residue. In addition to the modification of proheparin by N-deacetylation, N-sulfation, C5-epimeterization, and 2-O-sulfation, three isoforms of heparan sulfate 6-O-sulfotransferase (HS6ST) have been reported to catalyze the transfer of sulfate from PAPS to the glucosamine residue C6 of proheparin. All three isoforms have a clear preference for GlcNS / GlcNAc residues immediately downstream of the IdoA residue. The final modification of heparan sulfate, the transfer of sulfate to the 3-OH of the glucosamine unit, is rare in nature but is critical to many of the biological functions of the resulting GAGs. The seven known heparan sulfate 3-O-sulfotransferase (HS3ST) isoforms can be classified by their ability to transfer a sulfate group to the non-reducing end of a glucosamine bond attached to GlcA, IdoA, IdoA2S, or a combination thereof ( Figure 2b ). For example, HS3ST isoform 1 (HS3ST1) only catalyzes sulfate transfer to the 3-OH of glucosamine linked to GlcA or IdoA. Unlike HS3ST1, HS3ST3 requires an adjacent 2-O-thio IdoA (IdoA2S) residue for sulfate transfer. The activity of HS3ST5 is independent of the attached uric acid residue [4].

[0019] The large number of possible modifications of proheparin, namely N-deacetylation, N-sulfation, C5-epimerization, 2-O-sulfation, 6-O-sulfation, 3-O-sulfation and combinations of modifications, has led to a large number of different proheparin derivatives. The possible modifications of proheparin under enzyme catalysis and the resulting modifications produced by their combination are further complicated by the different substrate preferences of the enzyme subtypes. This is particularly true for the subtypes of HS3ST, whose different substrate preferences mediate a variety of different sulfation patterns (SP). This wealth of structural information translates into a large number of biological functions that are yet to be clarified. Since heparin is used as an anticoagulant, the sulfation pattern (SP) that leads to the binding of antithrombin to heparan sulfate (GlcNS6S-GlcA-GlcnS3S6s-IdoA2S-GlcNS6S) has been well characterized. However, the biological functions of heparan sulfates with different sulfation patterns and their mechanisms of action have only recently begun to be studied. Initial studies on the topic have shown that heparan sulfates with specific modification and sulfation patterns can mediate a variety of biological functions, enabling their therapeutic use as anticancer, anti-inflammatory, antimicrobial, antidiabetic or anti-neurodegenerative agents ( Figure 3 ). Research into the biological functions and therapeutic applications of well-defined heparan sulfates has been hampered by a lack of production capacity. To date, most heparan sulfates have been derived from animal tissues, which do not allow for control over the type and extent of enzymatic modifications that form the heterogeneity of heparan sulfates. Cell factories, particularly yeast cell factories, offer a solution for industrial production of homogeneous, well-defined heparan sulfates. This is because enzyme expression can be tightly controlled, allowing yeast cell factories to precisely control the type and extent of enzymatic modifications of the proheparin.

[0020] In order to obtain a yeast cell factory capable of producing heparan sulfate with different modifications and sulfation patterns, the heterologous genes required for modifying heparinogen need to be stably integrated into the yeast genome. Since many of the required enzymes are natively localized in the endoplasmic reticulum and Golgi apparatus, the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragments of the genes encoding the corresponding enzymes are truncated to achieve cytoplasmic localization. In order to design a yeast strain (or yeast cell) capable of industrially producing heparan sulfate (i.e., heparin) with a sulfation pattern that binds to antithrombin, NDST1-ΔMD, C5-epimerase-ΔMD, HS2ST1-ΔMD, HS6ST1-ΔMD, and HS3ST1-ΔSD were integrated into the genome of the previously designed ST_GNS-016. To achieve the correct sulfation pattern, especially 3-O-sulfation, it is essential to integrate HS3ST1, HS3ST3, HS3ST5, or HS3ST1-similar, HS3ST-similar, HS3ST5-similar sulfotransferases. Heparan sulfates characterized by N-, 3-, and 6-O-sulfation have been shown to bind well to Tau, a protein implicated in the development of Alzheimer's disease. Therefore, N-, 3-, and 6-O-sulfated heparan sulfates may be a viable therapeutic approach to combat the disease [5]. To engineer a yeast strain that can industrially produce N-, 3-, and 6-O-sulfated heparan sulfates, NDST1-ΔMD, HS6ST1-ΔMD, and HS3ST5-ΔMD were stably integrated into the previously engineered strain ST_GNS-016. Similar to the well-defined heparan sulfates that mediate binding to antithrombin or to Tau, many heparan sulfates with other modifications and sulfation patterns can also mediate specific binding to cytokines, growth factors, and other proteins. Yeast strains were engineered to produce heparan sulfates with these specific modifications and sulfation patterns ( Figure 3 ).

[0021] The present invention provides the following aspects, themes and preferred embodiments, which, individually or in combination, contribute to solving the objectives of the present invention:

[0022] (1) A recombinant yeast cell, characterized in that, compared with its wild-type cell, the recombinant yeast cell exhibits, preferably, increased production of UDP-GlcA (UDP-D-glucuronic acid) and / or UDP-GlcNAc (UDP-N-acetyl-D-glucosamine), and optionally PAPS (3′-phospho-adenosine sulfate).

[0023] Yeast cell can be any type of yeast cell. In a preferred embodiment, the genus of yeast cell is selected from the group consisting of following yeast genus: Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporium, Cryptococcus, Spore yeast and liposome yeast, such as Saccharomyces cerevisiae, Saccharomyces boulardi, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica. In a more preferred embodiment, the yeast cell is selected from the genus Saccharomyces. In a particularly preferred embodiment, the yeast cell is S. cerevisiae.

[0024] In a preferred embodiment, the recombinant yeast cell exhibits increased production of UDP-GlcA (UDP-D-glucuronic acid) and UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and optionally PAPS (3'-phospho-adenosine sulfate) compared to its wild-type cell (or its wild-type strain).

[0025] In another preferred embodiment, the recombinant yeast cell exhibits increased production of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3'-phospho-adenosine sulfate) compared to its wild-type strain.

[0026] Whether the recombinant yeast cells express or produce more of the corresponding substance (such as a precursor) can be determined by methods such as HPLC analysis. When determining the amount of a substance expressed / produced by two cell lines, both cell lines are subjected to the same procedure to ensure comparability of the results obtained.

[0027] In a preferred embodiment, the increase in the production of UDP-GlcA (UDP-D-glucuronic acid) and / or UDP-GlcNAc (UDP-N-acetyl-D-glucosamine); and / or PAPS (3'-phospho-adenosine sulfate) is achieved by regulating the genes involved in the biosynthetic pathway of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and / or PAPS (3'-phospho-adenosine sulfate), depending on the genes that need to be regulated. Figure 1a This process is described in . Regulation of a gene can result in overexpression or downregulation of the gene. Generally speaking, overexpression results in an increase in the gene product, while downregulation results in a decrease in the gene product. Downregulation and overexpression of a gene can be achieved by any suitable method known to those skilled in the art.

[0028] (2) The recombinant yeast cell according to item (1), characterized in that, compared with its wild-type cell, the recombinant yeast cell exhibits, preferably, an increased production of one or more enzymes selected from the following group: UTP-glucose-1-phosphate-uridylyl transferase (UDP-glucose pyrophosphorylase), phosphoglucomutase, glutamine-fructose-6-phosphate transaminase, N-acetyl-glucosamine-phosphomutase, and optionally ATP-sulfurylase, adenylate sulfate kinase and bisphospho-3'-nucleotidase.

[0029] In a preferred embodiment, the UDP-glucose pyrophosphorylase is encoded by a nucleotide (nt) sequence, which includes, preferably consists of, the nucleotide (nt) sequence SEQ ID NO: 30. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 29.

[0030] In a preferred embodiment, the phosphoglucomutase is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 26 or SEQ ID NO: 28, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 26 or SEQ ID NO: 28, respectively. The corresponding amino acid sequences (protein sequences) are SEQ ID NO: 25 and SEQ ID NO: 27, respectively.

[0031] In a preferred embodiment, the glutamine-fructose-6-phosphate aminotransferase is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 32, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 32. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 31.

[0032] In a preferred embodiment, the N-acetyl-glucosamine-phosphomutase is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 34. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 33.

[0033] In a preferred embodiment, the ATP-sulfurylase is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 36. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 35.

[0034] In a preferred embodiment, adenylate sulfate kinase is encoded by a nucleotide (nt) sequence, wherein the nucleotide (nt) The sequence comprises the nucleotide (nt) sequence SEQ ID NO: 38, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 38 The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 37.

[0035] In a preferred embodiment, the bisphospho-3'-nucleotidase is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 40. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 39.

[0036] In a preferred embodiment, the recombinant yeast cell expresses increased amounts of UTP-glucose-1-phosphate-uridylyl transferase (UTP-glucose pyrophosphorylase), phosphoglucomutase, glutamine-fructose-6-phosphate aminotransferase, N-acetyl-glucosamine-phosphomutase, ATP-sulfurylase, adenylate sulfate kinase, and bisphospho-3'-nucleotidase compared to its wild-type strain.

[0037] (3) The recombinant yeast cell according to item (1), characterized in that

[0038] Overexpression

[0039] (i) one or more genes involved in the synthesis of UDP-GlcA; and / or

[0040] (ii) one or more genes involved in the synthesis of UDP-GlcNAc; and optionally

[0041] (iii) one or more genes involved in PAPS synthesis; and

[0042] Expression of a gene encoding a heterologous UDP-glucose dehydrogenase;

[0043] This results in the recombinant yeast cell expressing increased production of UDP-GlcA, UDP-GlcNAc and optionally PAPS compared to the expression of the corresponding genes in its wild-type strain.

[0044] In order to determine whether a defined gene is overexpressed compared to the expression level of the gene in wild-type cells, any suitable method known to the skilled person can be used. For example, the mRNA level can be determined by methods such as transcriptional analysis disclosed in other parts of this article.

[0045] In a preferred embodiment,

[0046] Overexpression

[0047] (i) one or more genes involved in the synthesis of UDP-GlcA; and

[0048] (ii) one or more genes involved in the synthesis of UDP-GlcNAc; and optionally

[0049] (iii) one or more genes involved in PAPS synthesis; and

[0050] Expression of a gene encoding a heterologous UDP-glucose dehydrogenase;

[0051] This results in the recombinant yeast cell expressing increased production of UDP-GlcA, UDP-GlcNAc and optionally PAPS compared to its wild-type strain.

[0052] In another preferred embodiment,

[0053] Overexpression

[0054] (i) one or more genes involved in the synthesis of UDP-GlcA; and

[0055] (ii) one or more genes involved in the synthesis of UDP-GlcNAc; and

[0056] (iii) one or more genes involved in PAPS synthesis; and

[0057] Expression of a gene encoding a heterologous UDP-glucose dehydrogenase;

[0058] As a result, the recombinant yeast cell expresses increased production of UDP-GlcA, UDP-GlcNAc and PAPS compared to its wild-type strain.

[0059] (4) The recombinant yeast cell according to item (3), characterized in that

[0060] The genes involved in (i) UDP-GlcA synthesis are genes encoding phosphoglucomutase, preferably phosphoglucomutase 1 / 2 (PGM1 / 2); and genes encoding UTP-glucose-1-phosphate-uridylyl transferase (UTP-glucose pyrophosphorylase), preferably UDP-glucose pyrophosphorylase 1 (UGP1);

[0061] The gene involved in (ii) UDP-GlcNAc synthesis is a gene encoding glutamine-fructose-6-phosphate aminotransferase, preferably glutamine-fructose-6-phosphate aminotransferase 1 (GFA1); and a gene encoding N-acetyl-glucosamine-phosphomutase, preferably phosphoacetylglucosamine mutase 1 (PCM1); and / or

[0062] The genes involved in (iii) PAPS synthesis are genes encoding sulfate adenylyltransferase (ATP-sulfurylase), preferably methionine-requiring protein 3 (MET3); adenylate sulfate kinase, preferably methionine-requiring protein 14 (MET14); and bisphospho-3'-nucleotidase, preferably methionine-requiring protein 22 (MET22).

[0063] In a preferred embodiment, phosphoglucomutase 1 / 2 (PGM1 / 2) is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, nucleotide (nt) sequence SEQ ID NO: 26 or SEQ ID NO: 28. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 25 or SEQ ID NO: 27, respectively.

[0064] In a preferred embodiment, UDP-glucose pyrophosphorylase 1 (UGP1) is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 30, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 30. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 29.

[0065] In a preferred embodiment, glutamine-fructose-6-phosphate aminotransferase 1 (GFA1) is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 32, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 32. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 31.

[0066] In a preferred embodiment, phosphoacetylglucosamine mutase 1 (PCM1) is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 34, preferably consists of the nucleotide (nt) sequence SEQ ID NO: 34. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 33.

[0067] In a preferred embodiment, the methionine-requiring protein 3 (MET3) is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 36. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 35.

[0068] In a preferred embodiment, the methionine-requiring protein 14 (MET14) is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 38. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 37.

[0069] In a preferred embodiment, the methionine-requiring protein 22 (MET22) is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, the nucleotide (nt) sequence SEQ ID NO: 40. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 39.

[0070] In a preferred embodiment, overexpression of phosphoglucomutase (PGM) can be achieved by integrating an additional gene copy encoding phosphoglucomutase (PGM) and a strong promoter pTEF1 upstream of the gene into the chromosome.

[0071] In a preferred embodiment, overexpression of UDP-glucose pyrophosphorylase (UGP) can be achieved by integrating an additional gene copy encoding UTP-glucose-1-phosphate-uridylyl transferase (UGP) and a strong promoter pTDH3 upstream of the gene into the chromosome.

[0072] In a preferred embodiment, overexpression of glutamine-fructose-6-phosphate aminotransferase (GFA) can be achieved by integrating an additional copy of the gene encoding UTP-glucose-1-phosphate uridylyltransferase (GFA) and a strong promoter pTEF1 upstream of the gene into the chromosome.

[0073] In a preferred embodiment, overexpression of phosphoglucosamine mutase (PCM) can be achieved by chromosomal integration of an additional gene copy encoding UTP-glucose-1-phosphate-uridylyl transferase (PCM) and a strong promoter pTDH3 upstream of the gene.

[0074] In a preferred embodiment, overexpression of methionine-requiring protein 3 (MET3) can be achieved by integrating an additional gene copy encoding MET3 and a strong promoter pTDH3 upstream of the gene into the chromosome.

[0075] In a preferred embodiment, overexpression of methionine-requiring protein 14 (MET14) can be achieved by integrating an additional copy of the MET14 gene and a strong promoter pTEF1 upstream of the gene into the chromosome.

[0076] In a preferred embodiment, overexpression of methionine-requiring protein 22 (MET22) can be achieved by integrating an additional gene copy of MET22 and a strong promoter pPGK1 upstream of the gene into the chromosome.

[0077] (5) The recombinant yeast cell according to any one of the preceding items, characterized in that

[0078] Down-regulating the gene encoding chitin synthase, preferably chitin synthase 2 / 3 (CHS2 / 3), so that the production of chitin synthase is reduced compared to the wild-type strain; and / or

[0079] Down-regulating a gene encoding a 3′-phospho-adenylate sulphate reductase, preferably MET16, such that the production of the corresponding enzyme encoded by the down-regulated gene is reduced compared to the wild-type strain.

[0080] In a preferred embodiment, chitin synthase 2 / 3 (CHS2 / 3) is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 42 or SEQ ID NO: 44, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 42 or SEQ ID NO: 44, respectively. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 41 or SEQ ID NO: 43, respectively.

[0081] In a preferred embodiment, 3'-phospho-adenylate sulfate reductase (MET16) is encoded by a nucleotide (nt) sequence comprising, preferably consisting of, SEQ ID NO: 46. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 45.

[0082] (6) The recombinant yeast cell according to item (5), wherein down-regulation of the corresponding gene is achieved by reducing transcription of the gene.

[0083] (7) The recombinant yeast cell according to item (6), characterized in that the reduction of transcription is achieved by replacing the native promoter of the gene to be downregulated with a relatively weaker promoter.

[0084] (8) The recombinant yeast cell of any one of item (6) or (7), characterized in that the reduction of transcription is achieved by replacing the native promoter of CHS2 / 3 with the promoter KEX2 (pKEX2) and / or replacing the native promoter of MET16 with the promoter KEX2 (pKEX2).

[0085] In a preferred embodiment, the native promoter of MET16 is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO:48, preferably consists of the nucleotide (nt) sequence SEQ ID NO:48.

[0086] In a preferred embodiment, the native promoter of CHS3 is encoded by a nucleotide (nt) sequence, which includes the nucleotide (nt) sequence SEQ ID NO:47, and preferably consists of the nucleotide (nt) sequence SEQ ID NO:47.

[0087] In a preferred embodiment, the KEX2 promoter is encoded by a nucleotide (nt) sequence comprising the nucleotide (nt) sequence SEQ ID NO:49, preferably consisting of the nucleotide (nt) sequence SEQ ID NO:49.

[0088] The present invention also relates to any of the above-mentioned recombinant yeast cells, characterized in that the recombinant yeast cell additionally expresses a gene encoding UDP-glucose dehydrogenase, thereby being able to produce UDP-GlcA (UDP-D-glucuronic acid).

[0089] In a preferred embodiment, the UDP-glucose dehydrogenase is encoded by a nucleotide (nt) sequence, which includes, preferably consists of, the nucleotide (nt) sequence SEQ ID NO: 1. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 2.

[0090] (9) The recombinant yeast cell according to any of the preceding items, characterized in that, compared with its wild-type strain, the recombinant yeast cell exhibits increased production of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3'-phospho-adenosine sulfate).

[0091] (10) The recombinant yeast cell according to any of the preceding items, characterized in that the recombinant yeast cell additionally comprises a gene encoding a glycosaminoglycan synthase, thereby being able to produce glycosaminoglycans, such as heparinogen, hyaluronic acid, dermatan, chondroitin or their sulfated analogs; preferably, the recombinant yeast cell additionally expresses a gene encoding a glycosaminoglycan synthase, thereby being able to produce glycosaminoglycans, such as heparinogen, hyaluronic acid, dermatan, chondroitin or their sulfated analogs.

[0092] (11) The recombinant yeast cell according to any of the preceding items, characterized in that the recombinant yeast cell additionally comprises a gene encoding proheparin synthetase, preferably proheparin synthetase 1 and / or proheparin synthetase 2, thereby being able to produce proheparin; preferably, the recombinant yeast cell additionally expresses a gene encoding proheparin synthetase, preferably proheparin synthetase 1 and / or proheparin synthetase 2, thereby producing proheparin.

[0093] The recombinant yeast cells described in items (10) and (11) express genes encoding glycosaminoglycan synthase and proheparin synthase, and preferably, express an increased amount of glycosaminoglycan synthase and proheparin synthase, compared to their wild-type strains.

[0094] The genes encoding glycosaminoglycan synthase and heparinogen synthase, respectively, are heterologously expressed in recombinant yeast cells capable of UDP-GlcA and UDP-GlcNAc biosynthesis. To this end, the corresponding genes are integrated into the genome of the recombinant yeast cells according to items (1) to (9) by applying appropriate techniques known to the skilled person, as described elsewhere herein.

[0095] (12) The recombinant yeast cell according to item (11), characterized in that the proheparin synthetase is proheparin synthetase 1 (PmHS1) or a similar form of PmHS1, and / or proheparin synthetase 2 (PmHS2) or a similar form of PmHS2, preferably PmHS1 and PmHS2 are expressed.

[0096] In a preferred embodiment, the proheparin synthase 1 (PmHS1) is encoded by a nucleotide (nt) sequence, which includes, preferably consists of, the nucleotide (nt) sequence SEQ ID NO: 4. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 3.

[0097] In a preferred embodiment, the proheparin synthase 2 (PmHS2) is encoded by a nucleotide (nt) sequence, which includes, preferably consists of, the nucleotide (nt) sequence SEQ ID NO: 6. The corresponding amino acid sequence (protein sequence) is SEQ ID NO: 5.

[0098] (13) A recombinant yeast cell strain according to item (12), characterized in that the heparinogen synthetase PmHS1 and / or PmHS2 is fused with the nuclear export sequence (NES) of the Hsp70-type chaperone SSB1 of S. cerevisiae yeast at the C-terminus of the heparinogen synthetase, preferably, the NES nuclear export sequence is encoded by a nucleotide (nt) sequence, the nucleotide (nt) sequence includes the sequence SEQ ID NO: 54, or preferably consists of the nucleotide (nt) sequence SEQ ID NO: 54, so that the heparinogen synthetase PmHS1 and / or PmHS2 is specifically expressed in the cytoplasm instead of being localized in the yeast cell nucleus, so as to increase the production of heparinogen.

[0099] The amino acid sequence corresponding to the nucleotide (nt) sequence SEQ ID NO:54 is SEQ ID NO:53.

[0100] In a preferred embodiment, the amino acid sequence of the Hsp70-type chaperone SSB1 includes the sequence SEQ ID NO: 56, preferably consists of the sequence SEQ ID NO: 56. The amino acid sequence is encoded by a nucleotide (nt) sequence including the nucleotide (nt) sequence SEQ ID NO: 55, or preferably is encoded by the nucleotide (nt) sequence SEQ ID NO: 55.

[0101] (14) A method for producing proheparin using the recombinant yeast cell described in item (11) or (13), wherein the preferred S. cerevisiae strain is ST_GNS_016.

[0102] (15) The recombinant yeast cell according to any one of items (11) to (13) or defined in item (14), characterized in that the recombinant yeast cell additionally comprises genes encoding N-deacetylase (ND) and / or N-deacetylase-N-sulfotransferase (NDST), and / or C5 isomerase (C5-epi), and / or 2-O-sulfotransferase (HS2ST), and / or 3-O-sulfotransferase (HS3ST), and / or 6-O-sulfotransferase (HS6ST), and / or 1-O-sulfotransferase (1-O-sulfotransferase) ... HS6ST), thereby being able to produce heparan sulfate; preferably, the recombinant yeast cell additionally expresses genes encoding N-deacetylase (ND), and / or N-deacetylase-N-sulfotransferase (NDST), and / or C5-isomerase (C5-epi), and / or 2-O-sulfotransferase (HS2ST), and / or 3-O-sulfotransferase (HS3ST), and / or 6-O-sulfotransferase (HS6ST), thereby being able to produce heparan sulfate.

[0103] In a preferred embodiment, the heparan sulfate produced by the recombinant S. cerevisiae strain is heparin.

[0104] (16) A recombinant yeast cell according to any one of items (11) to (13), characterized in that the recombinant yeast cell additionally comprises a gene encoding ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST, in which the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragment is truncated; preferably, the recombinant yeast cell expresses a gene encoding ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST, in which the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragment is truncated, so that the ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST and / or HS6ST enzymes are specifically expressed in the cytoplasm to increase the production of heparan sulfate and / or heparin with different types and clear sulfation patterns.

[0105] (17) The recombinant yeast cell according to item (15) or (16), characterized in that the N-deacetylase is ND1 and / or ND1-similar; the N-sulfotransferase is NDST1 and / or NDST1-similar; the 2-O-sulfotransferase is HS2ST1 and / or HS2ST1-similar; the 3-O-sulfotransferase is HS3ST1, HS3ST3 or HS3ST5 and / or HS3ST1-similar, HS3ST3-similar or HS3ST5-similar, respectively; and the 6-O-sulfotransferase is HS6ST1 and / or HS6ST1-similar.

[0106] (18) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (1).

[0107] (19) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, HS3ST5 and / or HS3ST5, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (2).

[0108] (20) The recombinant yeast cell according to item (15), characterized in that genes encoding NDST and HS6ST are additionally expressed to produce Figure 3 Heparan sulfate is represented by formula (3).

[0109] (21) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, HS2ST, and HS6ST, thereby producing Figure 3 Heparan sulfate as described by formula (4).

[0110] (22) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-similar, and HS6ST, thereby being capable of producing Figure 3 Heparan sulfate represented by formula (5).

[0111] (23) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (6).

[0112] (24) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding ND, HS3ST5 and / or HS3ST5-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (7).

[0113] (25) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, HS2ST, HS3ST5 and / or HS3ST5-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (8).

[0114] (26) The recombinant yeast cell according to item (15), characterized in that it additionally expresses genes encoding NDST, C5-epi, HS2ST, HS3ST3 and / or HS3ST3-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (9).

[0115] (27) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, HS2ST-ΔMD, HS3ST1-ΔSD and / or HS3ST1-ΔSD-similar, and HS6ST-ΔMD, thereby producing Figure 3 The intracellular concentration of heparan sulfate represented by formula (1) increases.

[0116] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-019 strain or ST_GNS-022 strain.

[0117] (28) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, HS3ST5-ΔMD and / or HS3ST5-ΔMD, and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (2) increases.

[0118] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-027 strain.

[0119] (29) The recombinant yeast cell according to item (16), characterized in that genes encoding NDST-ΔMD and HS6ST-ΔMD are additionally expressed so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (3) increases.

[0120] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-026 strain.

[0121] (30) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, HS2ST-ΔMD and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (4) increases.

[0122] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-018 strain or ST_GNS-021 strain.

[0123] (31) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, HS2ST-ΔMD, HS3ST1-ΔSD and / or HS3ST1-ΔSD, and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (5) increases.

[0124] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-019 strain or ST_GNS-022 strain.

[0125] (32) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, and HS6ST-ΔMD, thereby producing Figure 3 The intracellular concentration of heparan sulfate represented by formula (6) increases.

[0126] In a preferred embodiment, the yeast cell is a recombinant S. cerevisiae ST_GNS-028 strain.

[0127] (33) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding ND-ΔMD, HS3ST5-ΔMD and / or HS3ST5-ΔMD, and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (7) increases.

[0128] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-027 strain.

[0129] (34) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, HS2ST-ΔMD, HS3ST5-ΔMD and / or HS3ST5-ΔMD, and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (8) increases.

[0130] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-024 strain.

[0131] (35) The recombinant yeast cell according to item (16), characterized in that it additionally expresses genes encoding NDST-ΔMD, C5-epi-ΔMD, HS2ST-ΔMD, HS3ST3-ΔMD and / or HS3ST3-ΔMD, and HS6ST-ΔMD, so that it produces Figure 3 The intracellular concentration of heparan sulfate represented by formula (9) increases.

[0132] In a preferred embodiment, the recombinant yeast cell is S. cerevisiae ST_GNS-023 strain.

[0133] (36) Recombinant S. cerevisiae strains ST_GNS-001, ST_GNS-002, ST_GNS-003, ST_GNS-004, ST_GNS-005, ST_GNS-006, ST_GNS-007, S T_GNS-008, ST_GNS-009, ST_GNS-010, ST_GNS-011, ST_GNS-012, ST_GNS-013, ST_GNS-014, ST_GNS-015, ST_GNS-016, ST_GNS-017, ST_GNS-018, ST_GNS-019, ST_GNS-020, ST_GNS-021, ST_GNS-022, ST_GNS-023, ST_GNS-024, ST_GNS-025, ST_GNS-026, ST_GNS-027, ST_GNS-028 and ST_GNS-029.

[0134] ST_GNS-014 is deposited in DSMZ (German Collection of Microorganisms and Cell Cultures GmbH) with the deposit number DSM 34100.

[0135] ST_GNS-022 is deposited in DSMZ with the accession number DSM 34101.

[0136] (37) The recombinant yeast cell according to any one of items (18) to (35) or the recombinant S. cerevisiae strain according to item (36), characterized in that the heparan sulfate produced by the recombinant yeast cell or the recombinant yeast strain comprises sugar residues of 4 or more in length.

[0137] (38) A recombinant microorganism capable of producing heparan sulfate.

[0138] (39) A method for producing heparan sulfate using a recombinant microorganism, preferably the recombinant microorganism of the present invention.

[0139] In order to produce heparan sulfate, the production strain (i.e., recombinant microorganism or recombinant yeast strain) is cultured in a culture medium under appropriate conditions. The cultivation of the corresponding strain (production strain) and the purification of the product are carried out in a conventional manner well known to those skilled in the art.

[0140] For example, the production strain is cultured in a suitable culture vessel such as a flask or container containing a suitable culture solution for a suitable and determined period of time, such as 28 to 144 hours, preferably 48 to 96 hours.

[0141] The culture is centrifuged and the recovered supernatant is filtered. The filtered supernatant is then purified, for example, using a chromatography column: the supernatant is loaded into the chromatography column for washing, and the product is then eluted from the chromatography column with an appropriate buffer. The product is then precipitated and the pellet is washed to obtain a purified product.

[0142] The purified product thus obtained is then analyzed by LC-MS analysis, for example with regard to its sulfation pattern.

[0143] (40) A method for producing heparan sulfate using the recombinant yeast cell or the recombinant S. cerevisiae strain according to any one of items (18) to (37).

[0144] (40.1) A method for producing heparan sulfate, comprising the following steps

[0145] (i) culturing a recombinant yeast cell in a culture medium, wherein the recombinant yeast cell is characterized in that:

[0146] Compared with its wild-type cells, the recombinant yeast cells produce increased production of UDP-GlcA (UDP-D-glucuronic acid) and UDP-GlcNAc (UDP-N-acetyl-D-glucosamine);

[0147] Compared with wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcA (UDP-D-glucuronic acid) and PAPS (3′-phospho-adenosine sulfate);

[0148] Compared with its wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate);

[0149] The recombinant yeast cell has an increased production of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) compared to its wild-type cell; or

[0150] Compared with wild-type cells, the recombinant yeast cells have increased production of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate);

[0151] (ii) obtaining heparan sulfate produced by the recombinant yeast cell in step (i); and optionally

[0152] (iii) purifying the heparan sulfate obtained in step (ii).

[0153] (40.2) A method according to item (40.1), wherein the recombinant yeast cell is characterized in that, compared with its wild-type cell, the recombinant yeast cell expresses an increased amount of one or more enzymes selected from the following groups: UTP-glucose-1-phosphate-uridylyl transferase (UTP-glucose pyrophosphorylase), phosphoglucomutase, glutamine-fructose-6-phosphate transaminase, N-acetyl-glucosamine-phosphomutase, ATP-sulfurylase, adenylate sulfate kinase and bisphospho-3'-nucleotidase.

[0154] (40.3) The method according to item (40.1), wherein the recombinant yeast cell is characterized by

[0155] (i) overexpressing one or more genes involved in the synthesis of UDP-GlcA and one or more genes involved in the synthesis of UDP-GlcNAc; or

[0156] (ii) overexpressing one or more genes involved in the synthesis of UDP-GlcA and one or more genes involved in the synthesis of PAPS; or

[0157] (iii) overexpressing one or more genes involved in UDP-GlcNAc synthesis and one or more genes involved in PAPS synthesis; or

[0158] (iv) overexpression of one or more genes involved in UDP-GlcNAc synthesis; or

[0159] (v) overexpressing one or more genes involved in UDP-GlcA synthesis, one or more genes involved in UDP-GlcNAc synthesis, and one or more genes involved in PAPS synthesis;

[0160] and

[0161] Expression of a gene encoding a heterologous UDP-glucose dehydrogenase;

[0162] Thus, the recombinant yeast cell has increased production of UDP-GlcA, UDP-GlcNAc and PAPS compared with the expression of the corresponding genes in its wild-type cells.

[0163] (40.4) The method according to item (40.3), wherein the recombinant yeast cell is characterized by

[0164] The genes involved in (i) UDP-GlcA synthesis are genes encoding phosphoglucomutase, preferably phosphoglucomutase 1 / 2 (PGM1 / 2); and genes encoding UTP-glucose-1-phosphate-uridylyl transferase (UTP-glucose pyrophosphorylase), preferably UDP-glucose pyrophosphorylase 1 (UGP1);

[0165] The gene involved in (ii) UDP-GlcNAc synthesis is a gene encoding glutamine-fructose-6-phosphate aminotransferase, preferably glutamine-fructose-6-phosphate aminotransferase 1 (GFA1); and a gene encoding N-acetyl-glucosamine-phosphomutase, preferably phosphoacetylglucosamine mutase 1 (PCM1); and / or

[0166] Genes involved in (iii) PAPS synthesis are genes encoding sulfate adenylyltransferase (ATP-sulfurylase), preferably methionine-requiring protein 3 (MET3); adenylate sulfate kinase, preferably methionine-requiring protein 14 (MET14); and bisphospho-3'-nucleotidase, preferably methionine-requiring protein 22 (MET22).

[0167] (40.5) The method according to any one of items (40.1) to (40.4), wherein the recombinant yeast cell is characterized by

[0168] Down-regulating the gene encoding chitin synthase, preferably chitin synthase 2 / 3 (CHS2 / 3), so that the production of chitin synthase is reduced compared to its wild-type strain; and / or

[0169] Down-regulating a gene encoding a 3′-phospho-adenylate sulphate reductase, preferably MET16, such that the production of the corresponding enzyme encoded by the down-regulated gene is reduced compared to the wild-type strain.

[0170] (40.6) A method according to any one of items (40.1) to (40.5), wherein the recombinant yeast cell is characterized in that the recombinant yeast cell additionally expresses a gene encoding heparin synthetase, preferably heparin synthetase 1 and / or heparin synthetase 2, so that the recombinant yeast cell is capable of producing, preferably producing, heparin.

[0171] (40.7) A method according to any one of items (40.1) to (40.6), wherein the recombinant yeast cell is characterized in that the recombinant yeast cell additionally expresses genes encoding N-deacetylase (ND), and / or N-deacetylase-N-sulfotransferase (NDST), and / or C5-isomerase (C5-epi), and / or 2-O-sulfotransferase (HS2ST), and / or 3-O-sulfotransferase (HS3ST), and / or 6-O-sulfotransferase (HS6ST), thereby enabling it to produce heparan sulfate.

[0172] (40.8) A method according to any one of items (40.1) to (40.7), wherein the recombinant yeast cell is characterized in that the recombinant yeast cell additionally expresses a gene encoding ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST, in which the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragment is truncated, so that the ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST and / or HS6ST enzymes are specifically expressed in the cytoplasm to increase the production of heparan sulfate and / or heparin with different types and clear sulfation patterns.

[0173] (40.9) The method according to any one of items (40.1) to (40.8), wherein the recombinant yeast cell is characterized in that one or more heterologous genes are stably integrated into the genome.

[0174] (40.10) The method according to any one of items (40.1) to (40.8), wherein the recombinant yeast cell is characterized in that:

[0175] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-like, and HS6ST, thereby producing Figure 3The heparan sulfate represented by formula (1), the preferred recombinant yeast strain is ST_GNS-022;

[0176] Additional expression of genes encoding NDST, HS3ST5 and / or HS3ST5-like, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (2);

[0177] · Additional expression of genes encoding NDST and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (3);

[0178] Additional expression of genes encoding NDST, C5-epi, HS2ST, and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (4);

[0179] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-like, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (5);

[0180] Additional expression of genes encoding NDST, C5-epi, and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (6);

[0181] Additional expression of genes encoding ND, HS3ST5 and / or HS3ST5-like and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (7);

[0182] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST5 and / or HS3ST5-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (8); and / or

[0183] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST3 and / or HS3ST3-similar, and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (9).

[0184] (41) Figure 3 The heparan sulfate represented by formula (1) to (9) is preferably Figure 3 Heparan sulfate represented by formula (2) to (9).

[0185] (42) Obtained by culturing (fermenting) the recombinant yeast strain defined in item (18) or item (27) Figure 3 Heparan sulfate represented by formula (1).

[0186] The obtained heparan sulfate contains one or more residue sequences as shown in formula (1) (heparin). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate having one or more residue sequences represented by formula (1) is capable of binding to antithrombin.

[0187] (43) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (19) or (28) Figure 3 Heparan sulfate represented by formula (2).

[0188] The obtained heparan sulfate contains one or more residue sequences as shown in formula (2). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (2) has enhanced binding affinity with Tau protein.

[0189] (44) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (20) or (29) Figure 3 Heparan sulfate represented by formula (3).

[0190] The obtained heparan sulfate contains one or more residue sequences as shown in formula (3). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (3) can optimally bind to human cytomegalovirus.

[0191] (45) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (21) or (30) Figure 3 Heparan sulfate represented by formula (4).

[0192] The obtained heparan sulfate contains one or more residue sequences as shown in formula (4). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (4) can bind to the growth factor FGF-2, thereby inhibiting the binding of FGF-2 to endothelial cells.

[0193] (46) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (22) or (31) Figure 3 Heparan sulfate as described by formula (5).

[0194] The obtained heparan sulfate contains one or more residue sequences as shown in formula (5). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (5) can optimally bind to the chemokine CCL2.

[0195] (47) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (23) or (32) Figure 3 Heparan sulfate represented by formula (6).

[0196] The obtained heparan sulfate contains one or more residue sequences as shown in formula (6). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (6) is capable of binding to chemokines CXCL1, CXCL2 and CCL2.

[0197] (48) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (24) or (33) Figure 3 Heparan sulfate as described by formula (7).

[0198] The obtained heparan sulfate contains one or more residue sequences as shown in formula (7). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 The heparan sulfate containing one or more residue sequences shown in formula (7) is capable of binding to A, B, C and D class chemokines.

[0199] (49) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (25) or (34) Figure 3 Heparan sulfate as described by formula (8).

[0200] The obtained heparan sulfate contains one or more residue sequences as shown in formula (8). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3 A heparan sulfate having one or more residue sequences as shown in formula (8).

[0201] (50) obtained by culturing (fermenting) a recombinant yeast strain as defined in item (26) or (35) Figure 3 Heparan sulfate represented by formula (9).

[0202] The obtained heparan sulfate contains one or more residue sequences as shown in formula (9). Therefore, in a preferred embodiment, the present invention also relates to a Figure 3Heparan sulfate containing one or more residue sequences represented by formula (9).

[0203] (51) The heparan sulfate according to any one of items (41) to (50), wherein the heparan sulfate includes a sugar residue having a length of 4 or more.

[0204] (52) Use of the recombinant yeast cell as defined in any one of items (1) to (37) in a method for producing heparan sulfate.

[0205] (53) Use of the recombinant microorganism defined in item (38) in a method for producing heparan sulfate.

[0206] (54) The recombinant yeast cell according to any of the preceding items, wherein the expression of the heterologous gene and / or the overexpression of the endogenous gene is achieved by stable gene integration.

[0207] In a preferred embodiment, stable integration does not rely on selection for a resistance marker. Thus, the strain obtained is marker-free.

[0208] Definitions of terms used in the meaning of the present invention

[0209] In the meaning of the present invention, the term "cell strain" refers to a cell derived from a single cell having one or more specific characteristics. Therefore, a "cell strain" includes a plurality of cells, all of which have the same one or more specific characteristics.

[0210] In the meaning of the present invention, the term "gene" refers to a nucleic acid sequence that encodes a polypeptide or RNA chain with a certain function in an organism. The "nucleic acid" sequence used herein includes DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), which can be in single-stranded or double-stranded form, or other forms.

[0211] Generally speaking, in the meaning of the present invention, the term "AtUGD1 gene" refers to a gene encoding UDP-glucose dehydrogenase 1, the gene comprising the nucleic acid sequence described by the sequence SEQ ID NO:2, preferably consisting of the nucleic acid sequence described by the sequence SEQ ID NO:2; the term "PmHS1 gene" refers to a gene encoding heparinogen synthetase 1, the gene comprising the nucleic acid sequence described by the sequence SEQ ID NO:4, preferably consisting of the nucleic acid sequence described by the sequence SEQ ID NO:4; the term "PmHS2 gene" refers to a gene encoding heparinogen synthetase 2, the gene comprising the nucleic acid sequence described by the sequence SEQ ID NO:6, preferably consisting of the nucleic acid sequence described by the sequence SEQ ID NO:6; the term "PmHS1_NES gene" refers to a gene encoding heparinogen synthetase 1, the C-terminus of which is fused to the nuclear export sequence (NES) of the Hsp70 chaperone SSB1 protein of S. cerevisiae, the gene comprising the nucleic acid sequence described by the sequence SEQ ID NO:8, preferably consisting of the nucleic acid sequence described by the sequence SEQ ID NO:9. NO:8; the term "PmHS2_NES gene" refers to a gene encoding heparinogen synthetase 2, and its C-terminus is fused to the nuclear export sequence (NES) of the Hsp70 chaperone SSB1 protein of S. cerevisiae, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:10, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:10; as described herein, the term "-NES" refers to a nuclear export signal, i.e., a short target peptide in a protein, which utilizes nuclear transport to target and locate the protein to be exported from the cell nucleus through the nuclear pore complex to the cytoplasm. In a preferred embodiment, NES is encoded by a nucleotide (nt) sequence comprising the sequence SEQ ID NO:54, preferably by a nucleotide (nt) sequence consisting only of the sequence SEQ ID NO:54. The corresponding amino acid sequence (protein sequence) is SEQ ID NO:53. In another preferred embodiment, the amino acid sequence of SSB1 includes SEQ ID NO:56, preferably consists of the amino acid sequence SEQ ID NO:56, and SSB1 is encoded by a nucleotide (nt) sequence including the sequence SEQ ID NO:55, preferably is encoded by a nucleotide (nt) sequence consisting only of the sequence SEQ ID NO:55.

[0212] The term "ND1-ΔMD gene" refers to a gene encoding N-deacetylase 1 but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:58, preferably consisting of the nucleic acid sequence described in SEQ ID NO:58; the term "NDST1-ΔMD gene" refers to a gene encoding N-deacetylase-N-sulfotransferase 1 but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:12, preferably consisting of the nucleic acid sequence described in SEQ ID NO:12; the term "C5-epi-ΔMD gene" refers to a gene encoding D-glucaldehyde C5-isomerase but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:14, preferably consisting of the nucleic acid sequence described in SEQ ID NO:14; the term "HS2ST1-ΔMD gene" refers to a gene encoding heparan sulfate 2-O-sulfotransferase 1 but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:16, preferably consisting of the nucleic acid sequence described in SEQ ID NO:17. NO:16; the term "HS3ST1-ΔSD gene" refers to a gene encoding heparan sulfate 3-O-sulfotransferase 1 but not encoding an N-terminal signal domain ( / N-terminal signal peptide), the gene comprising the nucleic acid sequence described in SEQ ID NO:18, preferably consisting of the nucleic acid sequence described in SEQ ID NO:18; the term "HS3ST3 A1-ΔMD gene" refers to a gene encoding heparan sulfate 3-O-sulfotransferase 3A1 but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:20, preferably consisting of the nucleic acid sequence described in SEQ ID NO:20; the term "HS3ST5-ΔMD gene" refers to a gene encoding heparan sulfate 3-O-sulfotransferase 5 but not encoding an N-terminal membrane domain, the gene comprising the nucleic acid sequence described in SEQ ID NO:22, preferably consisting of the nucleic acid sequence described in SEQ ID NO:23. NO:22 described nucleic acid sequence composition; the term "HS6ST1-ΔMD gene" refers to a gene encoding heparan sulfate 6-O-sulfotransferase 1, but not encoding the N-terminal membrane domain, the gene comprising the nucleic acid described in SEQ ID NO:24 sequence The term "-ΔMD" as used herein refers to the nucleic acid sequence described by SEQ ID NO: 24.The term "-ΔSD" refers to the gene encoding the N-terminal signal peptide removed. In the meaning of the present application, the term "domain" refers to a specific physical region or amino acid sequence in a protein that is associated with a specific function or a corresponding DNA fragment. In the meaning of the present invention, the term "N-terminal membrane domain" refers to the first domain of a protein, which is located at the N-terminus of the protein and is inserted into or otherwise attached to the lipid bilayer. In the meaning of the present invention, the term "N-terminal signal peptide" refers to a peptide sequence located at the N-terminus of a protein that directs protein transport to a specific organelle.

[0213] The term "PGM1 gene" refers to a gene encoding phosphoglucomutase 1, the gene comprising the nucleic acid sequence described in SEQ ID NO:26, preferably consisting of the nucleic acid sequence described in SEQ ID NO:26; the term "PGM2 gene" refers to a gene encoding phosphoglucomutase 2, the gene comprising the nucleic acid sequence described in SEQ ID NO:28, preferably consisting of the nucleic acid sequence described in SEQ ID NO:28; the term "UGP1 gene" refers to a gene encoding UDP-glucose pyrophosphorylase 1, the gene comprising the nucleic acid sequence described in SEQ ID NO:30, preferably consisting of the nucleic acid sequence described in SEQ ID NO:30; the term "GFA1 gene" refers to a gene encoding glutamine-fructose-6-phosphate aminotransferase 1, the gene comprising the nucleic acid sequence described in SEQ ID NO:32, preferably consisting of the nucleic acid sequence described in SEQ ID NO:32; the term "PCM1 gene" refers to a gene encoding phosphoglucoacetyl glucosamine mutase 1, the gene comprising the nucleic acid sequence described in SEQ ID NO:34, preferably consisting of the nucleic acid sequence described in SEQ ID NO:35. NO:34; the term "MET3 gene" refers to a gene encoding a protein 3 requiring methionine, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:36, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:36; the term "MET14 gene" refers to a gene encoding a protein 14 requiring methionine, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:38, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:38; the term "MET22 gene" refers to a gene encoding a protein 22 requiring methionine, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:40, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:40; the term "CHS2 gene" refers to a gene encoding a chitin synthase 2, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:42, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:42; the term "CHS3 gene" refers to a gene encoding a chitin synthase 3, the gene comprising the nucleic acid sequence described in the sequence SEQ ID NO:44, preferably consisting of the nucleic acid sequence described in the sequence SEQ ID NO:44. NO:44; the term "MET16 gene" refers to a gene encoding 3'-phospho-adenylate sulfate reductase, the gene comprising the nucleic acid sequence described in SEQ ID NO:46, preferably consisting of the nucleic acid sequence described in SEQ ID NO:46;The term "gRNA carrying the natMX6 nourseoin resistance marker (auxiliary gRNA vector)" refers to the artificially synthesized nucleotide sequence described in the sequence SEQ ID NO: 59. ;

[0214] In the meaning of the present invention, the term "UDP-glucose dehydrogenase 1" refers to a protein comprising the amino acid sequence described in SEQ ID NO: 1, preferably consisting of the amino acid sequence described in SEQ ID NO: 1; the term "heparinogen synthetase 1" refers to a protein comprising the amino acid sequence described in SEQ ID NO: 3, preferably consisting of the amino acid sequence described in SEQ ID NO: 3; the term "heparinogen synthetase 2" refers to a protein comprising the amino acid sequence described in SEQ ID NO: 5, preferably consisting of the amino acid sequence described in SEQ ID NO: 5. Protein; term " The "heparinogen synthetase 1" encoded by the PmHS1_NES gene refers to the heparinogen synthetase 1 described in SEQ ID NO:7The term "heparinogen synthetase 2 encoded by the PmHS2_NES gene" refers to a protein comprising the amino acid sequence described in SEQ ID NO:9, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:9; the term "N-deacetylase 1 not encoding an N-terminal membrane domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:57, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:57; the term "N-deacetylase-N-sulfotransferase 1 not encoding an N-terminal membrane domain" refers to a protein comprising (preferably comprising) the amino acid sequence described in SEQ ID NO:11, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:11; the term "D-glucuronic acid C5-isomerase not encoding an N-terminal membrane domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:13, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:14. NO:13; the term "heparan sulfate 2-O-sulfotransferase 1 not encoding an N-terminal membrane domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:15, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:15; the term "heparan sulfate 3-O-sulfotransferase 1 not encoding an N-terminal signaling domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:17, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:17; the term "heparan sulfate 3-O-sulfotransferase 3A1 not encoding an N-terminal membrane domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:19, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:19; the term "heparan sulfate 3-O-sulfotransferase 5 not encoding an N-terminal membrane domain" refers to a protein comprising the amino acid sequence described in SEQ ID NO:21, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:22. The term "heparan sulfate 6-O-sulfotransferase 1 not encoding an N-terminal membrane domain" refers to a protein consisting of the amino acid sequence described in SEQ ID NO:23, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:23; the term "phosphoglucomutase 1" refers to a protein consisting of the amino acid sequence described in SEQ ID NO:25, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:25;The term "phosphoglucomutase 2" refers to a protein comprising the amino acid sequence described in SEQ ID NO:27, preferably consisting of the amino acid sequence described in SEQ ID NO:27; the term "UDP-glucose pyrophosphorylase 1" refers to a protein comprising the amino acid sequence described in SEQ ID NO:29, preferably consisting of the amino acid sequence described in SEQ ID NO:29; the term "glutamine-fructose-6-phosphate aminotransferase 1" refers to a protein comprising the amino acid sequence described in SEQ ID NO:31, preferably consisting of the amino acid sequence described in SEQ ID NO:31; the term "phosphoglucoacetylglucosamine mutase 1" refers to a protein comprising the amino acid sequence described in SEQ ID NO:33, preferably consisting of the amino acid sequence described in SEQ ID NO:33; the term "methionine-requiring protein 3" refers to a protein comprising the amino acid sequence described in SEQ ID NO:35, preferably consisting of the amino acid sequence described in SEQ ID NO:35; the term "methionine-requiring protein 14" refers to a protein comprising the amino acid sequence described in SEQ ID NO:37, preferably consisting of the amino acid sequence described in SEQ ID NO:38. ID NO:37; the term "methionine-requiring protein 22" refers to a protein comprising the amino acid sequence described in SEQ ID NO:39, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:39; the term "chitin synthase 2" refers to; A protein comprising the amino acid sequence described in SEQ ID NO:41, preferably a protein comprising the amino acid sequence described in SEQ ID NO:41 describe The term "chitin synthase 3" refers to a protein comprising the amino acid sequence described in SEQ ID NO:43, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:43; the term "3'-phospho-adenylate sulfate reductase" refers to a protein comprising the amino acid sequence described in SEQ ID NO:4, preferably a protein consisting of the amino acid sequence described in SEQ ID NO:4.

[0215] Nucleic acid sequences include regulatory regions (also referred to herein as regulatory polynucleotide sequences or regulatory sequences), transcribed regions (such as regions encoding proteins, and also regions that are transcribed but do not encode proteins, such as introns) and / or other functional sequence regions. Examples of regulatory polynucleotide sequences include promoters, enhancers, terminators, silencers, IRES sequences, ribosome binding sites, and sequences that stabilize or destroy mRNA stability through secondary structures. In the present invention, the term "promoter" refers to a DNA sequence that directs transcription of a (structural) gene. Typically, the promoter is located in the 5'-region of a gene, close to the transcription start site of the (structural) gene. The promoter sequence can be constitutive, inducible or repressive. In the meaning of the present invention, the term "native promoter" refers to a single DNA endogenous sequence downstream of an endogenous gene that can direct the transcription of the gene. In a specific embodiment of the present invention, the native promoter is the native promoter CHS3 (pCH3), directing the transcription of chitin synthase 2 / 3, and the promoter CHS3 is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 47, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 47. In another specific embodiment of the present invention, the native promoter is the native promoter MET16 (pMET16), directing the transcription of 3'-phospho-adenylate sulfate reductase, and the promoter MET16 is encoded by a nucleotide (nt) sequence, and the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 48, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 48.

[0216] In the present invention, "strong promoter" refers to a gene regulation promoter with a relatively high transcription level, and the level of the corresponding protein transcribed by it is also relatively high, while a gene regulation promoter with a low transcription level resulting in a low level of the corresponding protein is called a "weak promoter". In a preferred embodiment, the strong promoter is promoter TDH3 (pTDH3), encoded by a nucleotide (nt) sequence, the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 50, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 50; promoter TEF1 (pTEF1) is encoded by a nucleotide (nt) sequence, the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 51, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 51; or promoter PGK1 (pPGK1), encoded by a nucleotide (nt) sequence, the nucleotide (nt) sequence includes the nucleotide (nt) sequence SEQ ID NO: 52, preferably consisting of the nucleotide (nt) sequence SEQ ID NO: 52. In a preferred embodiment, the weaker promoter is promoter KEX2 (pKEX2), encoded by a nucleotide (nt) sequence comprising the nucleotide (nt) sequence SEQ ID NO:49, preferably consisting of the nucleotide (nt) sequence SEQ ID NO:49.

[0217] In the meaning of the present invention, the term "recombinant (microorganism / yeast / strain)" refers to a cell / strain containing nucleic acids, which are the result of different gene combinations and ultimately form unique gametes with chromosomes different from the parental chromosomes. In particular, the recombinant cell or recombinant strain may contain nucleic acids that are not present in the corresponding wild-type cell or wild-type strain, and this nucleic acid is introduced into the strain (cell) using DNA recombinant technology. DNA recombinant technology has been described elsewhere in this article. Specifically, the recombinant strain can produce a targeted substance that the wild-type strain cannot produce. For example, the recombinant S.cerevisiae comprises a heterologous AtUGD1 gene, which can express UD-glucose dehydrogenase; the recombinant S.cerevisiae comprises a heterologous PmHS1 and / or PmHS2 gene, which can express proheparin synthetase 1 and / or proheparin synthetase 2; the recombinant S.cerevisiae comprises a heterologous PmHS1_NES and / or PmHS2_NES gene, which can express proheparin synthetase 1 and / or proheparin synthetase 2 specifically in the cytoplasm instead of being localized in the yeast nucleus; the recombinant S.cerevisiae capable of producing proheparin comprises a heterologous encoding NDST1-ΔMD and / or C5-epi-ΔMD, and / or HS2ST1-ΔMD, and / or HS3ST1-ΔSD, and / or HS3ST3 A1-ΔMD, and / or HS3ST5-ΔMD, and / or HS6ST1-ΔMD and / or their corresponding similar forms of genes, which can respectively express N-deacetylase 1 and / or N-sulfotransferase 1 and / or C5-isomerase, and / or heparan sulfate 2-O-sulfotransferase 1, and / or heparan sulfate 3-O-sulfotransferase 1, and / or heparan sulfate 3-O-sulfotransferase 3A1, and / or heparan sulfate 3-O-sulfotransferase 5, and / or heparan sulfate 6-O-sulfotransferase 1 specifically in the cytoplasm instead of being localized in the endoplasmic reticulum or Golgi apparatus.

[0218] In the meaning of the present invention, the term "wild type" refers to a substance in its naturally occurring state, thereby exerting its natural function and / or activity. As used herein, "a substance" can be an organism, such as a microorganism, a cell, or, preferably, a yeast strain, such as S. cerevisiae. The wild type state or wild type form of a substance can be distinguished from the recombinant form of the substance.

[0219] In the meaning of the present invention, "increased production / amount" (e.g. UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) or PAPS (3'-phospho-adenosine sulfate)) means that the content of the corresponding substance (e.g. precursor) is at least 103% by weight of the dry matter of the substance, preferably at least 105% by weight of the dry matter of the substance, more preferably at least 110% by weight of the dry matter of the substance, even more preferably at least 120% by weight of the dry matter of the substance, even more preferably at least 130% by weight of the dry matter of the substance, and most preferably at least 140% by weight of the dry matter of the substance, compared to the amount produced by the corresponding wild-type cell / cell strain (wherein the amount produced by the wild-type cell / cell strain is 100% by weight of the dry matter of the substance).

[0220] In the meaning of the present invention, the term "heterologous", such as a heterologous protein or gene, refers to a protein or gene from a different organism. Specifically, a protein encoded by a heterologous gene means that the protein is originally cloned or derived from a different cell type or species from the recipient, preferably a strain different from the wild-type strain. For example

[0221] AtUGD gene, PmHS1 gene, PmHS2 gene, PmHS1_NES gene, PmHS2_NES gene, NDST gene, C5-epi gene, HS2ST gene, HS3ST1 gene, HS3ST3 gene, HS3ST5 gene and HS6ST gene and their respective encoded proteins are heterologous genes and proteins to wild-type S.cerevisiae. In the meaning of the present invention, the term "homologous" means "same type" or similar, having corresponding parts, similar structures or the same anatomical position, and may refer to a common origin. For example, chitin synthase CHS2 and CHS3 are homologous enzymes; phosphoglucomutase PGM 1 and PGM 2 are homologous enzymes; heparanogen synthase PmHS1 and PmHS2 are homologous enzymes; heparan sulfate 3-O-sulfotransferase 1, heparan sulfate 3-O-sulfotransferase 3A1 and heparan sulfate 3-O-sulfotransferase 5 are homologous enzymes.

[0222] The introduction (or integration) of the heterologous gene into the genome can be performed by using DNA recombination techniques known to those skilled in the art according to any suitable method, such as using marker-free CRISPR-Cas9 assisted gene integration technology ( Figure 4 ).

[0223] In the meaning of the present invention, the term "stable integration" / "stable gene integration" refers to the integration of a DNA fragment into the host genome (ie into one of the host's chromosomes), thereby ensuring that the integrated DNA fragment is maintained for many generations.

[0224] In the meaning of the present invention, the term "marker-free", for example marker-free gene integration, means that there is no selection marker (such as kanamycin). Marker-free gene integration is advantageous for industrial strains.

[0225] In the meaning of the present invention, the term "endogenous", such as endogenous proteins or genes, refers to proteins or genes that originate from the inside of a system such as an organism, tissue or cell, such as an organism, tissue or cell. In particular, proteins encoded by endogenous genes refer to proteins that were originally cloned or derived from wild-type strains. For example, HXT1 / 2 / 4 / 5 / 7 genes, GLK1 genes, PGM1 / 2 genes, UGP1 genes, PGI1 genes, GFA1 genes, GNA1 genes, PCM1 genes, QRI1 genes, CHS2 / 3 genes, Sul1 / 2 genes, MET3 genes, MET14 genes, MET16 genes and MET22 genes and the proteins encoded by them are all endogenous genes and proteins of wild-type S.cerevisiae.

[0226] In the meaning of the present invention, DNA recombinant technologies known to those skilled in the art include molecular cloning methods (such as preparation of plasmid vectors, digestion of plasmid vectors, preparation of DNA by polymerase chain reaction, assembly of recombinant DNA by a combination of Gibson and T5 exonucleases, Phusion polymerase, and Taq ligase), methods for integrating recombinant DNA into the host genome (such as CRISPR / Cas9-mediated homology-directed repair), and methods for screening whether an organism has the correct recombinant DNA (e.g., screening using antibiotic resistance, colony PCR, and Sanger sequencing).

[0227] In the meaning of the present invention, the term "recombinant DNA" refers to a DNA molecule formed by combining DNA from different sources by DNA recombination technology.

[0228] In the meaning of the present invention, the term "chassis" refers to a platform that serves as a framework and support for biological components and ingredients, preferably recombinant yeast, preferably S. cerevisiae, as a framework and support for the production and / or modification of targeted substances. In a preferred embodiment of the present invention, the recombinant yeast chassis is a recombinant S. cerevisiae strain that is capable of providing high cytoplasmic concentrations of the precursors UDP-GlcA and UDP-GlcNAc for the synthesis of heparinogen, and optionally providing high cytoplasmic concentrations of the precursor PAPS for the synthesis of heparan sulfate. In the meaning of the present invention, the term "precursor" refers to a substance, cell or cell component from which another substance, cell or cell component is formed.

[0229] In the meaning of the present invention, the term "increased", e.g., "increased" transcription means that the total transcription amount of a gene encoding, e.g., GFA1, PCM1, PMG1 / 2, UGP1, MET3, MET14, or MET22 protein in a recombinant strain is at least 3% more than the total transcription amount of a gene encoding GFA1, PCM1, PMG1 / 2, UGP1, MET3, MET14, or MET22 protein in a wild-type strain, preferably at least 5% more, preferably at least 10% more, more preferably at least 15% more, even more preferably at least 20% more. By "increased" (cytoplasmic) concentration or "increased" amount is meant that the total concentration or total production of a substance (e.g., D-glucosamine-6-P, N-acetyl-D-glucosamine-1-P, D-glucose-1-P, UDP-D-glucose, UDP-GlcA, UDP-GlcNAc, APS, PAPS, AMP, proheparin, heparan sulfate or heparin) is at least 5% greater, preferably at least 10% greater, more preferably at least 15% greater, and even more preferably at least 20% greater than the total concentration or total production of the substance (e.g., D-glucosamine-6-P, N-acetyl-D-glucosamine-1-P, D-glucose-1-P, UDP-D-glucose, UDP-GlcA, UDP-GlcNAc, APS, PAPS, AMP, proheparin, heparan sulfate or heparin) produced by a wild-type strain.

[0230] In the meaning of the present invention, the term "reduced", for example, "reduced" transcription means that the total transcription of genes encoding proteins such as CHS2 / 3 or MET16 in the recombinant strain is at least 5% less than the total transcription of genes encoding proteins such as CHS2 / 3 or MET16 in its wild-type strain, preferably at least 10% less, more preferably at least 15% less, and even more preferably at least 20% less. "Reduced" (cytoplasmic) concentration or "reduced" amount means that the total (cytoplasmic) concentration or production of a substance (such as chitin or PAP) is at least 5% less than the total concentration or production of the substance (such as chitin or PAP) produced by its wild-type strain, preferably at least 10% less, more preferably at least 15% less, and even more preferably at least 20% less.

[0231] In the meaning of the present invention, the term "transcription", such as "transcription of a gene", refers to the process of copying the information in a DNA strand into a new messenger RNA (mRNA) molecule.

[0232] In the meaning of the present invention, the term "expression", such as "expression of a gene", refers to the process of copying the DNA code into RNA (transcription) and translating the information carried by the nucleic acid into the amino acid sequence constituting the protein.

[0233] In the meaning of the present invention, the term "overexpression" refers to (eg, artificially) overexpressing a gene to produce an abnormally large amount of protein or other substances compared to the expression of the gene in a corresponding wild-type cell.

[0234] In the meaning of the present invention, the term "downregulation" refers to a process in which a cell reduces the amount of a cellular component (such as RNA, protein or metabolite) compared to a corresponding wild-type cell when subjected to external stimulation, and the process may be a natural process.

[0235] "Overexpression" or "downregulation" can be achieved by any appropriate method known to those skilled in the art. For example, it can be achieved by modifying the nucleic acid encoding a specific (desired) gene product. For example, such modification can be the introduction of point mutations, such as substitution, insertion or deletion of single or multiple nucleotides; partial or complete deletion; and / or replacement with a different nucleotide sequence (such as a heterologous nucleotide sequence); or, in the case of "overexpression", the introduction of one or more additional gene copies of the gene to be overexpressed; or, in the case of "downregulation", the reduction of the copy number of the gene to be "downregulated".

[0236] "Overexpression" or "downregulation" may also be achieved by introducing point mutations, such as replacing, inserting or deleting a single or multiple nucleotides in the regulatory polynucleotide sequence of the affected gene; partially deleting or completely deleting or replacing the regulatory polynucleotide sequence of the affected gene; and / or replacing the regulatory polynucleotide sequence of the affected gene with a different nucleotide sequence, such as a heterologous nucleotide sequence.

[0237] Regulatory sequences are sequences that control the expression of certain genes, such as promoters, enhancers, terminators, silencers, IRES sequences, ribosome binding sites, and sequences that stabilize or destabilize mRNA through secondary structures.

[0238] In a preferred embodiment, regarding "overexpression", endogenous transaminases GFA1 and P-acetyl-glucosamine mutase PCM1 are overexpressed by integrating an additional gene copy with a strong promoter upstream of the gene in the chromosome. In another preferred embodiment, sulfate adenylate transferase (ATP-sulfatase) MET3, sulfate adenylate kinase MET14 and 3',5'-diphosphoadenosine nucleotidase MET22 are overexpressed by integrating an additional gene copy with a strong promoter upstream of the gene in the chromosome. Strong promoters include promoter TDH3 (pTDH3), promoter TEF1 (pTEF1) or promoter PGK1 (pPGK1). Overexpression can also be achieved by transcriptional programming mediated by inactivated Cas9 (dCas9). Here, dCas9 can be modified as a transcription factor for specific genes, thereby increasing the expression of these genes.

[0239] In a preferred embodiment, regarding "downregulation", a gene encoding a chitin synthase, preferably a gene encoding CHS2 / 3, is downregulated by a weaker promoter, preferably, the native promoter of CHS2 / 3 is replaced by a KEX2 promoter, the effect of which is that the transcription of the CHS2 / 3 gene is reduced, and thus the amount of chitin synthase is reduced; in another preferred embodiment, a gene encoding a 3'-phospho-adenylate sulfate reductase, preferably a gene encoding MET16, is downregulated by a weaker promoter, preferably, the native promoter of CHS2 / 3 is replaced by a weaker KEX2 promoter (pKEX2), so that the transcription of the MET16 gene is reduced, and thus the amount of 3'-phospho-adenylate sulfate reductase is reduced. Downregulation can also be achieved by dCas9-mediated transcriptional programming. Here, dCas9 can be modified as a transcription factor for specific genes, thereby reducing the expression of these genes. In addition, downregulation can also be achieved by knocking out / deleting genes such as CHS2 / 3 from the yeast genome, as long as the deleted genes do not cause yeast death.

[0240] In the meaning of the present invention, the term "upregulation" refers to a process in which a cell increases the amount of a cell component (such as RNA, protein or metabolite) under external stimulation, and the process may be a natural process. For example, treating a culture of S. cerevisiae with mating pheromone will upregulate the production of UDP-GlcNAc in S. cerevisiae.

[0241] In the meaning of the present invention, the term "modification" refers to chemical or structural changes in the structure of a molecule by enzymatic means, resulting in a change in the properties of said molecule.

[0242] In the present invention, the term "polymerization" refers to the act or process of monomer molecules joining together to form a polymer or a three-dimensional network. As used herein, polymerization occurs through a chemical reaction, and ultimately the monomers are linked by chemical bonds to form a polymer. Polymers produced by organisms are called biopolymers. They include proteins and polypeptides, polynucleotides and polysaccharides. Polysaccharides are biopolymers formed by monosaccharide units linked together by glycosidic bonds.

[0243] In the present invention, the term "titer" refers to the production concentration of a compound.

[0244] In the meaning of the present invention, the term "-similar" or "similar form", such as "similar protein / gene", means that the protein / gene exhibits substantially the same functions and activities as the protein / gene itself. For example, HS3TS1-similar protein / gene exhibits substantially the same functions and activities as HS3TS1 protein / gene. As used herein, "substantially the same" means that the deviation does not exceed 20% of a given value, preferably does not exceed 10%, more preferably does not exceed 7%, and even more preferably does not exceed 3%.

[0245] In the meaning of the present invention, the term "isoform", such as "protein isoform", refers to a member of a group of highly similar proteins that are derived from a single gene or gene family and are the result of genetic differences. Many isoforms have the same or similar biological effects, but some have unique functions. For example, heparin synthetase isoform 2 (PmHS2) is less dependent on polysaccharide primers, while PmHS1 elongates the heparin prosugar chain three times faster than PmHS2, thereby producing polymers with larger molecular weights [3].

[0246] For example, HS3ST isoform 1 (HS3ST1) catalyzes sulfate transfer only to the 3-OH of glucosamine linked to GlcA or IdoA. Unlike HS3ST1, HS3ST3 requires an adjacent 2-O-thiolated IdoA (IdoA2S) residue for sulfate transfer. The activity of HS3ST5 is independent of the attached uric acid residue [4].

[0247] In the meaning of the present invention, the term "microorganism" refers to an organism that can only be seen under a microscope. Microorganisms include bacteria, protozoa, algae and fungi.

[0248] In the meaning of the present invention, the term "yeast" or "yeast cell" refers to a diverse group of unicellular fungi in biological systems, most of which belong to the Ascomycetes and Basidiomycetes. Budding yeast ("true yeast") is classified in the class Saccharomyces, of which the most famous species is Saccharomyces cerevisiae. In the present invention, yeast or yeast cell can be selected from the genus of yeast cell and is selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporium, Cryptococcus, Spore yeast and Lipomyces, such as Saccharomyces cerevisiae, Saccharomyces boulardi, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica. In a more preferred embodiment, yeast cell is selected from Saccharomyces. In a particularly preferred embodiment, the yeast cell is S. cerevisiae.

[0249] In the meaning of the present invention, the term "glycosaminoglycan" refers to a linear polysaccharide composed of repeating disaccharide (i.e., disaccharide) units of uric acid and amino sugars or uric acid and galactose. Heparinogen, carrageenan, chondroitin, dermatan, hyaluronic acid and its sulfated derivatives are examples of glycosaminoglycans. In the meaning of the present invention, the term "heparinogen" refers to a glycosaminoglycan having the following chemical structure: [→4) β-D-glucuronic acid (GlcA) (1→4) N-acetyl-α-D-glucosamine (GlcNAc) (1→)] n .

[0250] In the meaning of the present invention, the term "heparinogen synthetase" refers to an enzyme having UDP-GlcA glucosyltransferase and / or UDP-GlcNAc glucosyltransferase activity, which enzyme itself or together with other enzymes is capable of synthesizing heparinogen. In the meaning of the present invention, the term "hyaluronic acid synthetase" refers to an enzyme having UDP-GlcA glucosyltransferase and / or UDP-GlcNAc glucosyltransferase activity, which enzyme itself or together with other enzymes is capable of synthesizing hyaluronic acid.

[0251] In the meaning of the present invention, the term "heparan sulfate" refers to a glycosaminoglycan similar to heparinogen, which is sulfated on the 3-O and / or 6-O and / or nitrogen of the glucosamine residues and / or sulfated on the 2-O of the glucuronic acid residues. In addition, the glucuronic acid residues may be partially exo-embedded as iduronic acid. In the meaning of the present invention, the term "heparin" refers to heparan sulfate that inhibits blood coagulation. In the meaning of the present invention, the term "sulfation pattern" refers to a specific sequence modification (such as deacetylation, N-sulfation, O-sulfation) of the uric and amino sugar residues of the glycosaminoglycan.

[0252] Detailed description of the invention

[0253] The present invention will now be described in more detail by way of preferred embodiments and examples, but these embodiments and examples are for illustration only and are not to be construed as limiting the scope of the present invention in any way.

[0254] Traditional production of heparin and its analogs relies on extraction from animal tissues (especially porcine intestines), which can lead to drug contamination and cause shortages during animal disease outbreaks. Although bacterial enzymes can be used to synthesize proheparin (a precursor of heparin sulfate and heparin), all enzymes required for the conversion of proheparin to heparin sulfate and heparin biosynthesis are derived entirely from vertebrates. Since enzymes derived from eukaryotic cells are generally more stable and more functional when expressed in eukaryotic hosts, yeast cells are effective candidate cell factories and are more suitable for the biosynthesis of heparin sulfate and heparin than bacterial systems. In addition, yeast cell factories can precisely control the type and degree of enzymatic modification of proheparin through a strictly controlled enzyme expression process, thereby providing a solution for the industrial-scale production of homogeneous, well-defined heparan sulfate.

[0255] The present invention utilizes eukaryotic cell factories, particularly yeast cell factories, to provide an excellent production platform for the production of glycosaminoglycans (GAGs), particularly heparinogen, its sulfated derivatives (such as heparan sulfate and heparin), and for the industrial production of heparan sulfate with a defined sulfation pattern that has a wide range of medical and / or research application value. In a preferred embodiment, the yeast cell is a S. cerevisiae cell.

[0256] The present invention is based on three units, including unit (1), which refers to an engineered optimized yeast chassis, which has a high substrate utilization rate and can produce high concentrations of precursors for the biosynthesis of proheparin; unit (2), which refers to an engineered recombinant yeast strain, which can produce high titers of proheparin; unit (3), which refers to an engineered recombinant yeast strain, which can produce high titers of heparan sulfate, heparin and derivatives with a defined sulfation pattern. These three units will be described in detail below.

[0257] The present invention is described using S. cerevisiae yeast as an example, but as described elsewhere herein, any type of yeast cell may be used.

[0258] Unit (1) Optimizing chassis engineering

[0259] In order to build a yeast cell factory into an economically viable production platform for proheparin and heparan sulfate, the present invention provides a recombinant yeast chassis that can efficiently utilize relatively cheap substrates (such as D-glucose, (NH4)SO4), and provide high cytoplasmic concentration / increased cytoplasmic concentration of precursors UDP-D-glucuronic acid (UDP-GlcA) and UDP-acetyl-D-glucosamine (UDP-GlcNAc) for synthesizing proheparin, and high cytoplasmic concentration / increased cytoplasmic concentration of 3'-phospho-adenosine sulfate (PAPS) for modifying proheparin into heparan sulfate, such as heparin. Since the biosynthetic pathways of UDP-sugars are not related to each other, they can be optimized separately through modular pathway engineering methods (see Figure 1).

[0260] Specifically, the scope of the first unit (1) is to design a modular pathway to obtain high / increased cytoplasmic concentrations of UDP-GlcA and / or UDP-GlcNAc in a yeast chassis, preferably, to obtain high / increased cytoplasmic concentrations of UDP-GlcA and UDP-GlcNAc. As is well known, UDP-GlcA is not endogenously produced by yeast, and therefore a heterologous gene encoding UDP-glucose dehydrogenase is designed to be integrated into the mother yeast, which can directly convert UDP-glucose (the direct precursor of UDP-GlcA) into UDP-GlcA. Therefore, in a preferred embodiment of the present invention, a heterologous gene encoding UDP-glucose dehydrogenase is integrated into the mother yeast, and the mother yeast is preferably S. cerevisiae S288C, wherein the heterologous gene encoding UDP-glucose dehydrogenase is AtUGD1 [SEQ ID NO: 2] from Arabidopsis thaliana, thereby obtaining a recombinant strain ST_GNS-005 capable of producing UDP-GlcA. Strain ST_GNS-005 can also be further modified to produce proheparin and / or heparan sulfate.

[0261] Although the UDP-glucose concentration in the cytoplasm is inherently high in S.cerevisiae, in order to further increase the cytoplasmic concentration of UDP-GlcA, the overexpression of endogenous phosphoglucomutase and UTP-glucose-1-phosphate-uridyltransferase (UDP-glucose pyrophosphorylase) in yeast cells that can directly convert D-glucose-6-P into UDP-glucose was designed in subsequent strains that can produce UDP-GlcA. Overexpression can be achieved by any suitable method known to those skilled in the art. In one embodiment of the invention, overexpression of phosphoglucomutase in yeast cells can be achieved by integrating an extra gene copy encoding phosphoglucomutase and a strong promoter (e.g., pTEF1 upstream of the gene) in the chromosome.

[0262] In another embodiment of the present invention, overexpression of UTP-glucose-1-phosphate-uridyl transferase (UTP-glucose pyrophosphorylase) in yeast cells can be achieved by integrating a gene encoding UTP-glucose-1-phosphate-uridine This can be achieved by including an additional gene copy of the acyltransferase and a strong promoter such as pTDH3 upstream of the gene.

[0263] As disclosed herein, in a preferred embodiment, PGM1 / 2 and UGP1 are overexpressed in a subsequent recombinant strain capable of producing UDP-GlcA by simultaneously integrating an additional gene copy of PGM2 with a strong promoter pTEF1 and an additional gene copy of UGP1 with a strong promoter pTDH3 into the chromosome of a parent strain expressing AtUGD1, preferably strain ST_GNS-005, thereby obtaining a recombinant yeast strain ST_GNS-009 capable of producing a higher UDP-GlcA cytoplasmic concentration. Strain ST_GNS-009 can also be further modified to produce proheparin and / heparan sulfate.

[0264] As the main precursor of chitin, UDP-GlcNAc is synthesized from fructose-6-P in S.cerevisiae through four steps: fructose-6-P is first induced to transaminate into glucosamine-6-P by the transaminase encoded by the GFA1 gene using L-glutamine as a nitrogen donor. Glucosamine-6-P is then acetylated to N-acetyl-glucosamine-6-P by the acetylase encoded by the GNA1 gene, and then N-acetyl-glucosamine-6-P is converted to N-acetyl-glucosamine-1-P by the P-acetylglucosamine mutase encoded by the PCM1 gene. In the last synthesis step, N-acetyl-glucosamine-1-P is converted to UDP-GlcNAc by the UDP-GlcNAc pyrophosphorylase encoded by the QRI1 gene. Studies have shown that treatment of S.cerevisiae cultures with mating pheromones upregulates the production of UDP-GlcNAc, which in turn upregulates the production of chitin, thereby increasing the transcription of the GFA1 and PCM1 genes. This dynamic change indicates that glutamine-fructose-6-phosphate aminotransferase 1 (GFA1) and glucosamine-phosphomutase 1 (PCM1) are bottlenecks of the UDP-GlcNAc pathway. Therefore, it is designed to overexpress endogenous GFA1 and PCM1 genes to increase the cytoplasmic concentration of UDP-GlcNAc. In one embodiment of the present invention, overexpression of GFA1 in yeast cells can be achieved by integrating an additional gene copy of GFA1 and a strong promoter pTDH3 in the chromosome. In one embodiment of the present invention, overexpression of PCM1 in yeast cells can be achieved by integrating an additional gene copy of PCM1 and a strong promoter pTEF1 in the chromosome.

[0265] As disclosed herein, in certain embodiments of the present invention, by simultaneously integrating GFA1 and an additional gene copy of the strong promoter pTDH3 and PCM1 and an additional gene copy of the strong promoter pTEF1 into the chromosome of any recombinant yeast strain previously designed to produce UDP-GlcA, a recombinant yeast strain capable of simultaneously producing UDP-GlcA and UDP-GlcNAc is obtained. In a preferred embodiment, GFA1 and PCM1 are overexpressed in the recombinant strain ST_GNS-005 by simultaneously integrating an additional gene copy of GFA1 and a strong promoter pTDH3 and an additional gene copy of PCM1 and a strong promoter pTEF1 on its chromosome, thereby obtaining a recombinant yeast strain ST_GNS-008, which is capable of producing higher cytoplasmic concentrations of UDP-GlcA and UDP-GlcNAc. The strain ST_GNS-008 can also be further modified to produce heparinogen and / or heparan sulfate. In a more preferred embodiment, GFA1 and PCM1 are overexpressed in the recombinant strain ST_GNS-009 by simultaneously integrating an additional gene copy of GFA1 and a strong promoter pTDH3 and an additional gene copy of PCM1 and a strong promoter pTEF1 on its chromosome, thereby obtaining a recombinant yeast strain ST_GNS-010, which is capable of producing higher cytoplasmic concentrations of UDP-GlcA and UDP-GlcNAc. The strain ST_GNS-010 can also be further modified to produce proheparin and / or heparan sulfate.

[0266] In addition, in order to prevent the consumption of UDP-GlcNAc in the cytoplasm, the transcription of the CHS3 gene encoding chitin synthase III is also designed to be down-regulated, because chitin synthase III synthesizes about 90% of chitin in yeast cells, especially in S.cerevisiae. Down-regulation of CHS3 gene transcription can be achieved by replacing the native CHS3 promoter downstream of the CHS3 gene in S.cerevisiae with a weaker promoter. As disclosed herein, in some embodiments of the present invention, in any subsequent bacterial recombinant strain previously designed to produce a higher UDP-GlcNAc cytoplasmic concentration compared to its wild-type strain, the native CHS3 promoter is replaced with a weaker promoter, and the weaker promoter can be selected from the group consisting of pKEX2 and pHXT1, preferably pKEX2.

[0267] In a preferred embodiment, the gene of the native promoter CHS3 in the recombinant strain ST_GNS-010 is replaced by the weaker promoter KEX2, thereby obtaining the subsequent recombinant strain ST_GNS-011, which further improves the cytoplasmic concentration of UDP-GlcA and UDP-GlcNAc produced by the strain. The strain ST_GNS-011 can also be further modified to produce proheparin and / or heparan sulfate.

[0268] In addition to the previous improvement (increase) of the concentration of UDP-sugar, the second aspect of unit (1) is to increase the cell membrane concentration of 3′-phospho-adenylic acid sulfate (PAPS) by a modular pathway engineering approach of recombinant yeast strains. To this end, overexpression of sulfate adenylate transferase (ATP-sulfatase) MET3, adenylate sulfate kinase MET14 and adenosine 3',5'-diphosphonucleotide nucleotidase MET22 in any of the previously engineered recombinant yeast strains was designed. Overexpression of the MET3 gene can be achieved by integrating an additional gene copy of MET3 and a strong promoter (such as pTDH3) in the chromosome. Overexpression of the MET14 gene can be achieved by integrating an additional gene copy of MET14 and a strong promoter (such as pTEF1) in the chromosome. Overexpression of the MET22 gene can be achieved by integrating an additional gene copy of MET22 and a strong promoter (such as pPGK1) in the chromosome.

[0269] As disclosed herein, in certain embodiments of the present invention, MET3, MET14 and / or MET22 genes are overexpressed in any of the previously designed subsequent strains, thereby generating a group of recombinant yeast strains capable of producing UDP-GlcA, UDP-GlcNAc and PAPS. In a preferred embodiment, MET3 and MET14 genes are overexpressed in the recombinant strain ST_GNS-011 by simultaneously integrating an additional gene copy of MET3 and a strong promoter pTDH3 and an additional gene copy of MET14 and a strong promoter pTEF1 in its chromosome, thereby obtaining a subsequent recombinant yeast strain ST_GNS-012, which is capable of producing higher cytoplasmic concentrations of UDP-GlcA, UDP-GlcNAc and PAPS. The strain ST_GNS-012 can also be further modified to produce heparinogen and / or heparan sulfate. In a more preferred embodiment, the MET3, MET14 and MET22 genes are overexpressed in the recombinant strain ST_GNS-0011 by simultaneously integrating an additional gene copy of MET3 and a strong promoter pTDH3, an additional gene copy of MET14 and a strong promoter pTEF1, and an additional gene copy of MET22 and a strong promoter pPGK1 into its chromosome, thereby obtaining a recombinant yeast strain ST_GNS-013, which is capable of producing optimal (increased) cytoplasmic concentrations of UDP-GlcA, UDP-GlcNAc and PAPS. The strain ST_GNS-013 can also be further modified to produce proheparin and / or heparan sulfate.

[0270] In addition, to prevent the depletion of PAPS in the cytoplasm, a method was designed to downregulate the transcription of the gene encoding the endogenous 3′-phospho-adenylate sulfate reductase MET16. Downregulation of the transcription of the MET16 gene can be achieved by replacing the native promoter of MET16 in the yeast genome with a relatively weak promoter, or by dCas9-mediated transcriptional programming, or by completely knocking out the gene.

[0271] As disclosed herein, in some embodiments of the invention, by in any of the previously designed subsequent bacteria The relatively weak KEX2 promoter (pKEX2) was used to replace the native promoter of MET16 in the yeast genome.(pMET16) is used to down-regulate the expression of the MET16 gene, thereby obtaining a group of subsequent recombinant yeast strains capable of producing UDP-GlcA, UDP-GlcNAc and PAPS. In a more preferred embodiment, in the recombinant strain ST_GNS-013, the expression of the MET16 gene is down-regulated by replacing the native promoter (pMET16) of MET16 in the yeast genome with a relatively weak KEX2 promoter (pKEX2), thereby obtaining a subsequent recombinant yeast strain ST_GNS-014, which is capable of producing UDP-GlcA, UDP-GlcNAc and PAPS at optimal cytoplasmic concentrations. The strain ST_GNS-014 can be used as an optimal yeast chassis for further engineering to produce heparinogen and / or heparan sulfate.

[0272] Unit (2) Engineering of Heparinogenic Yeast Strain

[0273] Heparinogen is a glycosaminoglycan composed of repeating disaccharide units (-GlcA-β1,4-GlcNAc-ɑ1,4-) and is the precursor of unmodified GAGs of heparin and other heparan sulfates. In mast cells, the biosynthesis of heparinogen is initiated by the formation of a tetrasaccharide linker (-GlcA-β1,3-Gal-β1,3-Gal-β1,4-Xyl-O-Ser) on specific Ser residues of the sericin core protein. The formation of this linker is catalyzed by four enzymes, each of which elongates the linker by one residue from its respective UDP-sugar. The tetrasaccharide linker serves as a primer for the subsequent synthesis of heparinogen ([-GlcA-β1,4-GlcNAc-ɑ1,4-]n) by alternating addition of glucuronic acid residues and N-acetylglucosamine residues from their UDP-sugars. This polymerization is performed by isomeric complexes of the exonuclease family of GlcA / GlcNAc transferase II EXT1 and EXT2. This complex eukaryotic heparan synthesis pathway, which requires multiple different enzymes and UDP-sugars to synthesize heparanogen, can be replaced by a bacterial heparanogen synthetase that requires only UDP-GlcA and UDP-GlcNAcd. Escherichia coli K5 expresses two genes, KfiC and KfiA, encoding UDP-GlcA glucosyltransferase and UDP-GlcNAc glucosyltransferase, respectively, while Pasteurella multocida (P. multocida) expresses two highly homologous dual-acting heparanogen synthases, PmHS1 and PmHS2. Although both enzymes of P. multocida have glucuronidation and hexasaccharide transfer activities (dual action), their different catalytic preferences suggest that they play different roles in heparanogen synthesis: PmHS2 is less dependent on polysaccharide primers, while PmHS1 elongates heparanogen sugar chains three times faster than PmHS2, thereby producing polymers with larger molecular weights. When PmHS2 was heterologously expressed in Bacillus megaterium, the proheparin titer in shake flasks was approximately 250 mg / L and the proheparin titer in the bioreactor was 2.74 g / L.

[0274] Therefore, the gene encoding the proheparin synthetase PmHS1 and / or PmHS2 is designed to be integrated into a recombinant yeast strain capable of producing UDP-GlcA and UDP-GlcNAc, thereby obtaining a recombinant yeast strain capable of producing proheparin. Therefore, in certain embodiments of the present invention, the gene encoding the proheparin synthetase PmHS1 and / or PmHS2 (preferably PmHS1 and PmHS2) is integrated into the genome of any of the previous engineered yeast strains, thereby obtaining a group of subsequent recombinant yeast strains capable of producing proheparin. In a preferred embodiment, the PmHS1 gene is integrated into the recombinant strain ST_GNS-005, thereby producing a subsequent recombinant yeast strain ST_GNS-001 capable of producing proheparin. The strain ST_GNS-001 can also be further modified to produce heparan sulfate. In another preferred embodiment, the PmHS2 gene is integrated into the recombinant strain ST_GNS-005, thereby obtaining a subsequent recombinant yeast strain ST_GNS-02 capable of producing proheparin. Strain ST_GNS-002 can be further modified to produce heparan sulfate.

[0275] However, it has been observed that bacterial PmHS1 and PmHS2 enzymes tend to localize in the yeast nucleus, thus hindering heparin To counteract this nuclear localization, the nuclear export sequence of Hsp70 from S. cerevisiae could be The NES was fused to the C-terminus of the proheparin synthetases PmHS1 and PmHS2 to obtain proheparin synthetases with high cytoplasmic concentrations. When PmHS1_NES and PmHS2_NES were expressed in S.cerevisiae, the production of proheparin was improved, especially in the strain expressing PmHS1_NES. Co-expression of PmHS1_NES and PmHS2_NES further increased the titer of proheparin, which may be due to the different properties of the two isoforms: PmHS2 is better at synthesizing proheparin primers, and PmHS2 is a super proheparin synthetase that is good at elongating proheparin sugar chains.

[0276] Therefore, in certain embodiments of the present invention, PmHS1_NES and / or PmHS2_NES genes, preferably PmHS1_NES and PmHS2_NES genes, are integrated into the genome of any of the previously engineered yeast strains, thereby obtaining a group of subsequent recombinant yeast strains, which can specifically express heparinogen synthase PmHS1 and / or PmHS2, preferably PmHS1 and PmHS2, in the cell sap rather than in the yeast nucleus, thereby increasing the yield of heparinogen. In a preferred embodiment, the PmHS1_NES gene is integrated into the genome of the recombinant strain ST_GNS-005, and the recombinant yeast strain ST_GNS-003 obtained subsequently can produce heparinogen with a higher cytoplasmic concentration. The strain ST_GNS-003 can also be further modified to produce heparan sulfate. In another preferred embodiment, the PmHS2_NES gene is integrated into the genome of the recombinant strain (ST_GNS-005), and the recombinant yeast strain ST_GNS-004 obtained subsequently can produce the heparinogen with a higher cytoplasmic concentration. The strain ST_GNS-004 can also be further modified to produce heparan sulfate. In another preferred embodiment, the PmHS1_NES gene and the PmHS2_NES gene are integrated into the genome of the recombinant strain ST_GNS-005, and the recombinant yeast strain ST_GNS-007 obtained subsequently can produce heparinogen, and its heparinogen cytoplasmic concentration is further improved. The strain ST_GNS-007 can also be further modified to produce heparan sulfate. In a more preferred embodiment, the PmHS2_NES gene is integrated into the genome of the recombinant strain ST_GNS-014, and the recombinant yeast strain ST_GNS-015 obtained subsequently can produce heparinogen, and its heparinogen cytoplasmic concentration is further improved. Bacterial strain ST_GNS-015 can also be further modified to produce heparan sulfate. In a more preferred embodiment, PmHS1_NES gene and PmHS2_NES gene are integrated into the genome of recombinant strain (ST_GNS-014), and the recombinant yeast strain ST_GNS-016 obtained subsequently can produce heparin with optimal (increase) cytoplasmic concentration. Bacterial strain ST_GNS-016 can also be further modified to produce heparan sulfate.

[0277] (3) Engineered yeast strains producing heparan sulfate and heparin

[0278] In the biosynthesis of heparinogen, polysaccharides are modified by various enzymes to produce various heparan sulfates with different biological functions ( Figure 2a). The first two heparinogen modification steps, N-deacetylation and PAPS-dependent N-sulfation of N-acetylglucosamine, are carried out by dual-acting enzymes called N-deacetylase-N-sulfotransferases (NDSTs). Although four highly homologous vertebrate NDST isoforms are known, only NDST1 and NDST2 have high N-deacetylation and N-sulfation activities. In contrast, the NDST3 isoform has high N-deacetylation activity but low N-sulfation activity. N-sulfated glucosamine (GlcNS) is required for the subsequent C5-epimerization of the adjacent D-glucuronic acid (GlcA) residue to L-iduronic acid (IdoA) by C5-isomerase (C5-epi). The epimerized L-iduronic acid increases the affinity of the residue for heparan sulfate 2-O-sulfotransferase (HS2ST) by 5-fold, facilitating the transfer of sulfate from PAPS to C2 of the L-iduronic residue. In addition to the modification of proheparin by N-deacetylation, N-sulfation, C5-epimeterization, and 2-O-sulfation, three isoforms of heparan sulfate 6-O-sulfotransferase (HS6ST) have been reported to catalyze the transfer of sulfate from PAPS to the glucosamine residue C6 of proheparin. All three isoforms have a clear preference for GlcNS / GlcNAc residues immediately downstream of the IdoA residue. The final modification of heparan sulfate, the transfer of sulfate to the 3-OH of the glucosamine unit, is rare in nature but is critical to many of the biological functions of the resulting GAGs. The seven known heparan sulfate 3-O-sulfotransferase (HS3ST) isoforms can be classified by their ability to transfer a sulfate group to the non-reducing end of a glucosamine bond attached to GlcA, IdoA, IdoA2S, or a combination thereof ( Figure 2b ). For example, HS3ST isoform 1 (HS3ST1) only catalyzes sulfate transfer to the 3-OH of glucosamine linked to GlcA or IdoA. Unlike HS3ST1, HS3ST3 requires an adjacent 2-O-thio IdoA (IdoA2S) residue for sulfate transfer. The activity of HS3ST5 is independent of the attached uric acid residue [4].

[0279] The large number of possible proheparin modifications, namely N-deacetylation, N-sulfation, C5-episomerization, 2-O-sulfation, 6-O-sulfation, 3-O-sulfation and combinations of modifications, has led to a large number of different proheparin derivatives. The possible modifications of proheparin under enzyme catalysis and the modifications produced by their combination are further complicated by the different substrate preferences of the isoforms. This is particularly true for the isoforms of HS3ST, which mediate a variety of different sulfation patterns (SPs) with different substrate preferences. This wealth of structural information translates into a large number of biological functions, which is an area that has yet to be clarified.

[0280] Therefore, in certain embodiments of the present invention, genes encoding N-deacetylase and / or N-deacetylase-N-sulfotransferase and / or C5-isomerase and / or 2-O-sulfotransferase and / or 3-O-sulfotransferase and / or 6-O-sulfotransferase are additionally integrated into the genome of any previously designed recombinant yeast strain capable of producing proheparin, thereby generating a set of subsequent recombinant yeast strains capable of producing different types of heparan sulfate with defined sulfation patterns. In a preferred embodiment, the N-deacetylase is ND1 and / or ND1-analogous; the N-deacetylase-N-sulfotransferase is NDST1 and / or NDST1-analogous; the 3-O-sulfotransferase is HS3ST1, HS3ST3 A1 or HS3ST5, and / or an analogous form of HS3ST1, HS3ST3 A1 or HS3ST5, respectively; the 2-O-sulfotransferase is HS2ST1 and / or HS2ST1, and / or the 6-O-sulfotransferase is HS6ST1 and / or HS6ST1-analogous.

[0281] Since heparin is used as an anticoagulant, the sulfation pattern (SP) that leads to the binding of antithrombin to heparan sulfate (GlcNS6S-GlcA-GlcnS3S6s-IdoA2S-GlcNS6S) has been well characterized. However, the biological functions of heparan sulfates with different sulfation patterns and their mechanisms of action have only recently begun to be investigated. Initial studies on the subject have shown that heparan sulfates with specific modifications and sulfation patterns can mediate a variety of biological functions, enabling their therapeutic use as anticancer, anti-inflammatory, antimicrobial, antidiabetic or anti-neurodegenerative agents ( Figure 3 ). Research into the biological functions and therapeutic applications of structurally defined heparan sulfate has been hampered by a lack of production capacity.

[0282] Therefore, the yeast cell factory provided by the present invention can produce heparan sulfate with different modifications and sulfation patterns on an industrial scale. To this end, the present invention is designed to integrate the heterologous genes required for the modification of heparinogen into the recombinant yeast strain capable of producing heparinogen. Since many required enzymes are originally located in the endoplasmic reticulum, the N-terminal membrane domain (MD) or N-terminal signal peptide (SD) gene encoding the corresponding enzyme is cut off. In this way, the heterologous enzyme can be localized in the cytoplasm.

[0283] Therefore, in some embodiments of the present invention, the N-terminal membrane domain (MD) gene fragment encoding N-deacetylase and / or N-deacetylase-N-sulfotransferase and / or C5-isomerase and / or 2-O-sulfotransferase and / or 3-O-sulfotransferase and / or 6-O-sulfotransferase is truncated, wherein the N-terminal signal domain / signal peptide (SD) gene fragment of HS3ST1 is truncated, thereby obtaining a group of subsequent recombinant yeast strains that are capable of specifically expressing N-deacetylase-N-sulfotransferase and / or C5-isomerase and / or 2-O-sulfotransferase and / or 3-O-sulfotransferase and / or 6-O-sulfotransferase in the cytoplasm, rather than expressing them in the yeast endoplasmic reticulum or Golgi apparatus, thereby increasing the production of different types of heparan sulfates with clear sulfation patterns. In a preferred embodiment, the N-deacetylase with the N-terminal membrane domain (MD) deleted is ND1-ΔMD and / or a similar form of ND1-ΔMD; the N-deacetylase-N-sulfotransferase with the N-terminal membrane domain (MD) deleted is NDST1-ΔMD and / or a similar form of NDST1-ΔMD; the 3-O-sulfotransferase with the N-terminal membrane domain (MD) deleted is HS3ST3 A1-ΔMD, or HS3ST5-ΔMD, and / or HS3ST3 A1-ΔMD, or a similar form of HS3ST5-ΔMD; the 3-O-sulfotransferase with the truncated terminal signal domain / signal peptide (SD) is HS3ST1-ΔSD and / or a similar form of HS3ST1-ΔSD; the 2-O-sulfotransferase with the truncated N-terminal membrane domain (MD) is HS2ST1-ΔMD and / or a similar form of HS2ST1-ΔMD; the 6-O-sulfotransferase with the truncated N-terminal membrane domain (MD) is HS6ST1-ΔMD and / or a similar form of HS6ST1-ΔMD.

[0284] In addition, in a preferred embodiment, the NDST1-ΔMD gene and the C5-epi-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-002, thereby obtaining a recombinant yeast strain ST_GNS-006, which is capable of producing heparan-N-sulfate with iduronic acid residues. The strain ST_GNS-006 can also be further modified to transform heparan-N-sulfate with iduronic acid residues into different types of heparan sulfates with clear sulfation patterns.

[0285] In a more preferred embodiment, the NDST1-ΔMD gene and the C5-epi-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-007, thereby obtaining the recombinant yeast strain ST_GNS-017, which is capable of producing heparan-N-sulfate with iduronic acid residues. The strain ST_GNS-017 can also be further modified so as to transform the heparan-N-sulfate with iduronic acid residues into different types of heparan sulfates with a clear sulfation pattern.

[0286] In a more preferred embodiment, the NDST1-ΔMD gene and the C5-epi-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-016, thereby obtaining a recombinant yeast strain ST_GNS-020, which is capable of producing an optimal (higher) cell membrane concentration of heparan-N-sulfuric acid with iduronic acid residues. The strain ST_GNS-020 can also be further modified so that the heparan-N-sulfuric acid with iduronic acid residues is transformed into different types of heparan sulfates with a clear sulfation pattern.

[0287] In another more preferred embodiment, the NDST1-ΔMD gene is additionally integrated into the genome of the recombinant strain ST_GNS-016, thereby obtaining a recombinant yeast strain ST_GNS-025, which is capable of producing heparan-N-sulfate with an optimal (increased) cell membrane concentration. The strain ST_GNS-025 can also be further modified to transform heparan-N-sulfate into different types of heparan sulfate with a clear sulfation pattern.

[0288] To achieve the correct sulfation pattern, especially 2-O-sulfation, 3-O-sulfation, and 5-O-sulfation, the integration of genes encoding HS2ST, HS6ST, HS3ST1 / HS3ST1-like, HS3ST3 / HS3ST3-like, and HS3ST5 / HS3ST5-like sulfotransferases is essential.

[0289] In a preferred embodiment, the HS2ST1-ΔMD gene and the HS6ST1-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-017, thereby obtaining the recombinant yeast strain ST_GNS-018, which is capable of producing heparan sulfates and / or heparan sulfates sulfated at the N-, IdoA2-O, and 6-O positions as shown in formula (4), which contain one or more residue sequences as shown in formula (4) ( Figure 3 ), the heparan sulfate can bind to the growth factor FGF-2, thereby inhibiting the binding of FGF-2 to endothelial cells.

[0290] In another preferred embodiment, the HS3ST1-ΔSD gene is additionally integrated into the genome of the recombinant strain ST_GNS-018 to obtain the recombinant yeast strain ST_GNS-019, which is capable of producing heparan sulfate and / or heparan sulfate sulfated at the N-, IdoA2-O, 3-O, and 6-O positions as shown in formula (1) (heparin) and / or formula (5), which contains one or more residue sequences as shown in formula (1) (heparin) and / or formula (5), and the heparan sulfate is capable of binding to antithrombin.

[0291] In another more preferred embodiment, the HS2ST1-ΔMD gene and the HS6ST1-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-020, thereby obtaining the recombinant yeast strain ST_GNS-021, which is capable of producing optimal (increased) cytoplasmic concentrations of N-, IdoA2-O,6-O-sulfated heparan sulfate and / or heparan sulfate containing one or more residue sequences as shown in formula (4) ( Figure 3 ), the heparan sulfate can bind to the growth factor FGF-2, thereby inhibiting the binding of FGF-2 to endothelial cells.

[0292] In another preferred embodiment, the HS3ST1-ΔSD gene is additionally integrated into the genome of the recombinant strain ST_GNS-021 to obtain the recombinant yeast strain ST_GNS-022, which is capable of producing optimal (increased) cytoplasmic concentrations of N-, IdoA2-O, 3-O, 6-O sulfated heparan sulfate and / or heparan sulfate as shown in formula (1) (heparin) and / or formula (5), which contains one or more residue sequences as shown in formula (1) (heparin) and / or formula (5), and the heparan sulfate is capable of binding to antithrombin.

[0293] In another preferred embodiment, the HS3ST3 A1-ΔMD gene is additionally integrated into the genome of the recombinant strain ST_GNS-021, thereby obtaining the recombinant yeast strain ST_GNS-023, which is capable of producing optimal (increased) cytoplasmic concentrations of heparan sulfates and / or heparan sulfates sulfated at the N-, IdoA2-O, 3-O, 6-O positions as described in formula (9), which contain one or more Figure 3 The residue sequence shown in formula (9).

[0294] In another preferred embodiment, the HS3ST5-ΔMD gene is additionally integrated into the genome of the recombinant strain ST_GNS-021, thereby obtaining the recombinant yeast strain ST_GNS-024, which is capable of producing optimal (increased) cytoplasmic concentrations of heparan sulfates and / or heparan sulfates sulfated at the N-, IdoA2-O, 3-O, 6-O positions as shown in formula (8), which contain one or more Figure 3 The residue sequence shown in formula (8).

[0295] In another preferred embodiment, the HS6ST1-ΔMD gene is additionally integrated into the genome of the recombinant strain ST_GNS-025, thereby obtaining the recombinant yeast strain ST_GNS-026, which is capable of producing an optimal (increased) cytoplasmic concentration of N-, 6-O-sulfated heparan sulfate and / or heparan sulfate as shown in formula (3), which contains one or more Figure 3 The residue sequence shown in formula (3) can optimally bind to human cytomegalovirus.

[0296] In another preferred embodiment, the HS3ST5-ΔMD gene is additionally integrated into the recombinant strain ST_GNS- 026, thereby obtaining a recombinant yeast strain ST_GNS-027, which is capable of producing optimal (increased) Cytoplasmic concentration of heparan sulfate and / or heparan sulfate sulfated at the N-, 3-O, and 6-O positions as shown in formula (2), which contains one or more Figure 3 The residue sequence shown in formula (2), the heparan sulfate has enhanced binding affinity with Tau protein.

[0297] In another preferred embodiment, the HS6ST1-ΔMD gene is additionally integrated into the genome of the recombinant strain ST_GNS-020, thereby obtaining the recombinant yeast strain ST_GNS-028, which is capable of producing an optimal (increased) cytoplasmic concentration of N-, 6-O-sulfated heparan sulfate and / or heparan sulfate as shown in formula (6), which contains one or more Figure 3 The residue sequence shown in formula (6), the heparan sulfate can bind to the chemokines CXCL1, CXCL2, and CCL2.

[0298] In another preferred embodiment, the ND1-ΔMD gene, the HS3ST5-ΔMD gene and the HS6ST1-ΔMD gene are additionally integrated into the genome of the recombinant strain ST_GNS-016, thereby obtaining the recombinant yeast strain ST_GNS-029, which is capable of producing an optimal (increased) cytoplasmic concentration of N-, 6-O-sulfated heparan sulfate and / or heparan sulfate as shown in formula (7), which contains one or more Figure 3The residue sequence shown in formula (7) is capable of binding to A, B, C and D class chemokines. BRIEF DESCRIPTION OF THE DRAWINGS

[0299] Figure 1a : Achieving high cytoplasmic concentrations of UDP-D-glucuronic acid (UDP-GlcA), UDP-acetyl-D-glucosamine (UDP-GlcNAc), and 3'-phospho-adenosine sulfate (PAPS) through a modular pathway engineering approach. (e) indicates the location of D-glucose and sulfate outside the cell, and (c) indicates the location of D-glucose and sulfate inside the cell.

[0300] Figure 1b : Chassis optimization of yeast production of UDP-D-glucuronic acid and UDP-N-acetyl-D-glucosamine is essential for efficient production of glycosaminoglycans.

[0301] Figure 2a : Biosynthesis of heparan sulfate from proheparin.

[0302] Figure 2b : Substrate-specific HS3ST isoforms 1, 3, and 5 mediate heparan sulfate 3-O-sulfate transfer. Figure 2a .

[0303] Figure 3 :Heparan sulfates defined by different modification and sulfation patterns have different biological functions. In terms of modification, NS represents N-sulfated heparan sulfate, NH2 represents deacetylated heparan sulfate, 2S represents 2-O sulfated heparan sulfate, 3S represents 3-O sulfated heparan sulfate, 6S ​​represents 6-O sulfated heparan sulfate, and C5-epi represents the epiphase isomerization of glucuronic acid residues to iduronic acid residues.

[0304] Figure 4 : A cloning method for stable integration of genes into the yeast genome. The linearized integration vector and a helper vector containing the gRNA of the target site are co-transformed into yeast containing a Cas9 expression vector, and stable gene integration is achieved through Cas9-mediated double-stranded DNA breaks and subsequent homology-directed repair. DETAILED DESCRIPTION

[0305] Materials and Methods

[0306] Strains and culture media

[0307] Escherichia coli strain DH5α was used for cloning, propagation and storage of plasmids. E. coli was cultured in lysate broth (LB) liquid medium containing 100 mg / L ampicillin, with shaking at 250 rpm and a culture temperature of 37°C. For solid LB medium, 20 g / L agar was added to the medium.

[0308] When constructing yeast strains, Saccharomyces cerevisiae S288C containing an episomal Cas9 vector with a kanamycin resistance marker was used as the parent strain. Yeast peptone dextrose (YPD) medium consists of 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose, and 200 mg / L G418 (to ensure the maintenance of Cas9 vector expression), which is used to culture yeast, with shaking at 250 rpm and a culture temperature of 37°C. YPD agar medium can be prepared by adding 20 g / L agar.

[0309] Plasmid construction

[0310] Integration vectors and gRNA auxiliary vectors are listed in the plasmid list, which is disclosed elsewhere in this article. These vectors are constructed from PCR products using the Gibson assembly method or the AQUA cloning method. The Gibson assembly method is to incubate 100ng of the backbone (fragment carrying the replication origin) and the insert fragment at a molar ratio of 1:2-3 in a 20μl reaction volume containing 2X NEBGibson assembly master solution at 50°C for 60 minutes.

[0311] Similarly, the AQUA cloning method is to incubate 100 ng of backbone and insert at a molar ratio of 1:2-3 in a 10 μl aqueous reaction volume at room temperature for 60 minutes.

[0312] Chemical transformation was achieved by adding 2 μl of Gibson assembly solution or 5 μl of AQUA cloning mix to 50 μl of chemically competent E. coli cells and then incubating on ice for 30 minutes. The obtained cells were then heat shocked at 42°C for 30 seconds and mixed with 950 μl of LB medium. The transformed cells were cultured on LB-agar medium containing ampicillin. After culturing overnight at 37°C, colony PCR was performed using Promega's GoTaq G2 DNA polymerase according to the manufacturer's protocol to determine whether the plasmid construction was correct.

[0313] The fragments used to construct the plasmids ("bio-bricks") were obtained by PCR. PCR reactions with plasmids, genomic DNA or synthetic gene fragments as templates were performed using Q5 High-Fidelity DNA Polymerase from NEB according to the manufacturer's protocol.

[0314] All heterologous genes were codon-optimized for expression in S. cerevisiae and synthesized by Twist Bioscience in the form of synthetic gene fragments.

[0315] To ensure gene expression in yeast, the promoter needs to be cloned before the codon-optimized gene. Preferably, a strong promoter is cloned at its 5' end and a terminator is cloned at its 3' end ( Figure 4 The expression cassette (promoter, gene, terminator) or a number of linked expression cassettes are cloned into a structure sandwiched between homology arms to ensure stable chromosome integration in yeast. For example, a construct expressing two genes may include ( Figure 4 ):

[0316] (1) Upstream homology arm (i.e. 5'-homology arm) (about 500 bp)

[0317] (2) ADH1-terminator (tADH1)

[0318] (3) Gene 1

[0319] (4) TDH3-promoter (pTDH3)

[0320] (5)TEF1-promoter (pTEF1)

[0321] (6) Gene 2

[0322] (7) CYC1-terminator (tCYC1)

[0323] (8) Downstream homology arm (i.e. 3'-homology arm) (about 500 bp)

[0324] The construct (5'-homology arm, expression cassette, 3'-homology arm) was designed on a plasmid that simultaneously expresses an ampicillin resistance marker and an origin of replication (ori) to ensure the maintenance of the plasmid in E. coli. To facilitate the linearization of the construct (5'-homology arm, expression cassette, 3'-homology arm), the plasmid was designed to contain Not1 restriction sites on both sides.

[0325] Construction of strains

[0326] Stable integration of chromosomal genes into yeast can be achieved by using an integration vector and an auxiliary vector kit containing the corresponding gRNA. Before transformation, the integration vector for a certain chromosomal site containing a construct of a heterologous gene sandwiched between upstream and downstream homologous arms (such as a promoter, gene, terminator) is linearized with a restriction enzyme (NotI). Digestion is performed according to the method of the digestion enzyme manufacturer. Subsequently, 1.5-3μg of the linearized expression cassette is transformed into the parent strain expressing Cas9 together with 200-500ng of the gRNA auxiliary vector expressing the corresponding target gene and the nourseoin resistance marker ( Figure 4 ). Transformation was performed according to the standard lithium acetate transformation method. The transformed cells were cultured on YPD medium containing 200 mg / L G418 and 100 mg / L nourseoin. The transformants were verified by colony PCR using Promega's GoTaq G2 DNA polymerase according to its manufacturer's operating specifications, and plasmid solidification was performed to remove the gRNA vector. The gRNA auxiliary vector was solidified by culturing the strain in liquid YPD supplemented with 200 mg / L G418 but without nourseoin for 12 hours. After solidification, the strain is ready for the next round of stable chromosomal gene integration. Stable gene integration, especially marker-free stable gene integration (unlike plasmid integration of gene expression), does not require a co-expressed marker (such as an antibiotic resistance gene) to maintain the gene.

[0327] Stable integration technology in strain construction

[0328] As described below, stable and marker-free integration of recombinant DNA into yeast genes can be achieved by utilizing CRISPR / Cas9-induced homology-directed repair technology, as described below: the recombinant DNA to be integrated (usually an expression cassette consisting of a promoter, a gene, and a terminator) is cloned into a structure sandwiched by homology arms (the upstream homology arm is represented by UP and is the homology arm at the 5' end of the construct; the downstream homology arm is represented by DOWN and is the homology arm at the 3' end of the construct) ( Figure 4 The homology arms (each approximately 500 bp long) consist of DNA sequences that are identical to the DNA sequences downstream and upstream of the desired integration site ( Figure 4). The linearized DNA fragment consists of a 5' homology arm, the recombinant DNA to be integrated, and a 3' homology arm (in this order), and is then transformed into the mother strain together with a plasmid expressing a guide RNA (gRNA) corresponding to the target site of the homology arm of the linearized DNA fragment. The gRNA-expressing plasmid (auxiliary gRNA vector) carries a natMX6 nourseoin resistance marker to ensure that the plasmid is maintained in yeast. The expressed gRNA consists of 20 variable base pairs at the 5'-end (depending on the target gene site), and a gRNA scaffold sequence at the 3'-end (5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCUGUAU

[0329] CAACUUGAAAAAGUGGCACCGAGUCGGUG-3'). The parent yeast strain into which the linearized DNA fragment and the auxiliary gRNA vector (plasmid) are simultaneously introduced by transformation also carries a plasmid expressing the endonuclease Cas9 and a KanMX kanamycin resistance marker to ensure the maintenance of the plasmid. The expressed Cas9 is guided by the expressed gRNA (from the auxiliary gRNA vector) to the target chromosomal site, where the endonuclease induces a double-strand break. The double-strand break induces the homology-directed repair mechanism of the yeast cell, and the linearized DNA fragment (because its homology arms match the upstream and downstream regions of the induced double-strand break) is used as a repair template to integrate the recombinant DNA fragment that needs to be integrated.

[0330] The resulting series of yeast strains are disclosed elsewhere herein.

[0331] Production and purification

[0332] The production strain was cultured in a shake flask at 250 rpm and 30°C for 96 hours. The culture was then spun at 12,000g for 30 minutes at 4°C. Subsequently, the recovered supernatant was adjusted to pH 4 with acetic acid and then filtered using a filter funnel with a pore size of 60 microns. For purification, a chromatographic column loaded with Sepharose fast flow resin was first equilibrated with buffer A. Buffer A consisted of 50mM sodium chloride and 20mM sodium acetate with a pH of 4. After equilibration, the filtered culture supernatant was loaded into the chromatographic column and rinsed with three times buffer A. The product was then eluted from the chromatographic column with buffer B consisting of 1M sodium chloride and 20mM sodium acetate (pH 4). Three volumes of ethanol were added to the eluted product and spun at 12,000g for 30 minutes to obtain a precipitate. The precipitate was washed with 75% ethanol and spun again at 12,000g for 30 minutes to obtain a purified product.

[0333] Product analysis

[0334] The product was dissolved in 200 mM sodium phosphate buffer at pH 7 and then digested with 2 units of heparinase I and III from Flavobacterium heparinum obtained from Sigma-Aldrich for 12 hours at 30° C. The resulting disaccharides were sent to Eurofins Scientific SE for LC-MS analysis.

[0335] Analysis of precursors

[0336] To quantify precursor production (i.e., production of UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine, and 3'-phospho-adenylic acid sulfate), the fermentation broth was centrifuged at 10,000 g for 5 min. If cellular metabolite concentrations need to be determined, ultrasonic disruption can be used before the centrifugation step. After centrifugation, the supernatant was transferred to an amber vial for HPLC analysis. The samples were then analyzed by HPLC using a reverse phase column. Standards for HPLC quantification of metabolites can be obtained from Sigma-Aldrich (Product No. U6751-500MG for UDP-glucuronic acid, Product No. U4375-500MG for UDP-N-acetyl-D-glucosamine, Product No. U4375-500MG for UDP-N-acetyl-D-glucosamine, Product No. 94455-25MG for 3'-phospho-adenylic acid sulfate).

[0337] Transcriptional analysis

[0338] To measure mRNA levels, the following method can be used: About 10^7 cells need to be selected for RNA extraction using a Coulter counter. RNA for transcription analysis is then extracted using a commercial kit (such as NEB Monarch Total RNA Miniprep Kit) according to its manufacturer's operating procedures. Then, real-time quantitative PCR (RT-qPCR) is performed in a 96-well plate using a BioradICycler and SYBR-Green according to the manufacturer's operating procedures.

[0339] References

[0340] 1.Oka T.,Jigami Y.,Reconstruction of de novo pathway for synthesis ofUDP-glucuronicacid and UDP-xylose fromintrinsic UDP-glucose in Saccharomycescerevisiae.doi:10.1111 / j.1742-4658.2006.05281.x.The FEBS Journal 2006Jun;273(12):2645-57.

[0341] 2.Badri,A.,Williams,A.,Xia,K.,Linhardt,R.,Koffas,M.Increased 3'-phosphoadenosine- 5'-phosphosulfate levels in engineered Escherichia coli cells lysate facilitates in vitro synthesis o fChondroitin Sulfate A.BiotechnologyJournal 2019Sep;14(9):e1800436.doi:10.1002 / biot.201800436.

[0342] 3.Williams,A.,Gedeon,K.,Vaidyanathan,D.,Yu,Y.,Collins,C.,Dordick,J.,Linhardt,R.,Koffas.M.Metabolic engineering of Bacillus megaterium forheparosan biosynthesis usingPasteurella multocida heparosan synthase,PmHS2.Microbial Cell Factories 2019Aug12;18(1):132.doi:10.1186 / s12934-019-1187-9.

[0343] 4.Xu,D.,Moon,A.,Song,D.,Pedersen,L.,Liu,J.EngineeringSulfotransferases to ModifyHeparan Sulfate.Doi:10.1038 / nchembio.66.Epub2008Jan 27.

[0344] 5. Mah, D., Zhao, J., Liu,

[0345] Sequence Listing:

[0346]

[0347]

[0348]

[0349] Plasmid list

[0350]

[0351]

[0352] List of yeast strains

[0353]

[0354]

[0355] The present invention also relates to the following embodiments, wherein "claims" refer to "embodiments":

[0356] 1. A recombinant yeast cell, characterized in that the recombinant yeast cell produces an increased amount of UDP-GlcA (UDP-D-glucuronic acid) and / or UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and optionally PAPS (3'-phospho-adenosine sulfate) compared to its wild-type cell.

[0357] 2. The recombinant yeast cell according to claim 1, characterized in that compared with its wild-type cell, the recombinant The yeast cells express an increased amount of one or more enzymes selected from the group consisting of: UTP-glucose-1-phosphate-uridyl transferase (UDP-glucose pyrophosphorylase), phosphoglucomutase, glutamine-fructose-6-phosphate aminotransferase, N-acetyl-glucosamine-phosphomutase; and optionally ATP-sulfatase, adenosine sulfate kinase and bisphospho-3'-nucleotidase.

[0358] 3. The recombinant yeast cell according to claim 1, characterized in that

[0359] Overexpression

[0360] (i) one or more genes involved in the synthesis of UDP-GlcA; and / or

[0361] (ii) one or more genes involved in the synthesis of UDP-GlcNAc; and optionally

[0362] (iii) one or more genes involved in PAPS synthesis; and

[0363] Expression of a gene encoding a heterologous UDP-glucose dehydrogenase;

[0364] Thus, the recombinant yeast cell has increased production of UDP-GlcA, UDP-GlcNAc and PAPS compared with the expression levels of the corresponding genes in its wild-type cells.

[0365] 4. The recombinant yeast cell according to claim 3, characterized in that

[0366] The genes involved in (i) UDP-GlcA synthesis are genes encoding phosphoglucomutase, preferably phosphoglucomutase 1 / 2 (PGM1 / 2); and genes encoding UTP-glucose-1-phospholipid-uridyltransferase (UDP-glucose pyrophosphorylase), preferably UDP-glucose pyrophosphorylase 1 (UGP1);

[0367] The gene involved in (ii) UDP-GlcNAc synthesis is a gene encoding glutamine-fructose-6-phosphate aminotransferase, preferably glutamine-fructose-6-phosphate aminotransferase 1 (GFA1); and a gene encoding N-acetyl-glucosamine-phosphomutase, preferably phosphoacetylglucosamine mutase 1 (PCM1); and / or

[0368] The genes involved in (iii) PAPS synthesis are genes encoding sulfate adenylate transferase (ATP-sulfatase), preferably methionine requiring protein 3 (MET3); genes encoding adenosine sulfate kinase, preferably methionine requiring protein 14 (MET14); and genes encoding adenosine sulfate kinase and bisphospho-3'-nucleotidase, preferably methionine requiring protein 22 (MET22).

[0369] 5. The recombinant yeast cell according to any one of claims 1 to 4, characterized in that

[0370] Downward adjustment

[0371] a gene encoding a chitin synthase, preferably chitin synthase (CHS2 / 3), such that the production of chitin synthase is reduced compared to wild-type cells; and / or

[0372] A gene encoding 3'-phosphoadenylate sulphate reductase, preferably MET16, so that the production of the corresponding enzyme encoded by the down-regulated gene is reduced compared to wild-type cells.

[0373] 6. The recombinant yeast cell according to any one of claims 1 to 5, characterized in that the recombinant yeast cell additionally expresses a gene encoding a proheparin synthetase, preferably proheparin synthetase 1 and / or proheparin synthetase 2, so that the recombinant yeast cell can produce, preferably produce, proheparin.

[0374] 7. The recombinant yeast cell according to claim 6, characterized in that the recombinant yeast cell additionally expresses genes encoding N-deacetylase (ND), and / or N-deacetylase-N-sulfotransferase (NDST), and / or C5-isomerase (C5-epi), and / or 2-O-sulfotransferase (HS2ST), and / or 3-O-sulfotransferase (HS3ST), and / or 6-O-sulfotransferase (HS6ST), thereby enabling it to produce heparan sulfate.

[0375] 8. The recombinant yeast cell according to claim 6, characterized in that the recombinant yeast cell additionally expresses a gene encoding ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST, in which the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragment is truncated, so that the ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST and / or HS6ST enzymes are specifically expressed in the cytoplasm to increase the production of heparan sulfate and / or heparin with different types and clear sulfation patterns.

[0376] 9. The recombinant yeast cell according to claim 7 or 8, characterized in that

[0377] In addition, genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-like, and HS6ST are expressed, thereby producing Figure 3 The heparan sulfate represented by formula (1), the preferred recombinant yeast strain is ST_GNS-022;

[0378] Additional expression of genes encoding NDST, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing Figure 3 The heparan thioacetyl shown in formula (2);

[0379] ·Extra expression of genes encoding NDST and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (3);

[0380] Additional expression of genes encoding NDST, C5-epi, HS2ST, and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (4);

[0381] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-like and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (5);

[0382] Additional expression of genes encoding NDST, C5-epi, and HS6ST, resulting in Figure 3 Heparan sulfate represented by formula (6);

[0383] Additional expression of genes encoding ND, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (7); and / or

[0384] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (8); and / or

[0385] Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST3 and / or HS3ST3-like and HS6ST, thereby producing Figure 3 Heparan sulfate represented by formula (9).

[0386] 10. A method for producing heparan sulfate, comprising the following steps

[0387] (i) culturing the recombinant yeast cell as defined in any one of claims 1 to 9 in a culture medium;

[0388] (ii) obtaining heparan sulfate produced by the recombinant yeast cell in (i); and optionally

[0389] (iii) purifying the heparan sulfate obtained in step (ii).

[0390] 11. Use of a recombinant yeast cell as defined in any one of claims 1 to 9 for producing heparan sulfate.

[0391] 12. Heparan sulfate, characterized in that it exhibits a sulfation pattern produced by the process defined in claim 10.

[0392] PCT

[0393]

[0394]

[0395]

[0396] To be filled in by the receiving office

[0397]

[0398] To be filled in by the International Bureau

[0399]

Claims

1. A recombinant yeast cell, characterized in that Compared with its wild-type cells, the recombinant yeast cells produce increased production of UDP-GlcA (UDP-D-glucuronic acid) and UDP-GlcNAc (UDP-N-acetyl-D-glucosamine); Compared with its wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcA (UDP-D-glucuronic acid) and PAPS (3′-phospho-adenosine sulfate); Compared with its wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate); The recombinant yeast cell has an increased production of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) compared to its wild-type cell; or Compared with its wild-type cells, the recombinant yeast cells have increased production of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate), And it is further characterized in that the recombinant yeast cell additionally expresses a gene encoding proheparin synthase, so that the recombinant yeast cell can produce proheparin.

2. The recombinant yeast cell according to claim 1, characterized in that The recombinant yeast cell expresses an increased amount of one or more enzymes selected from the group consisting of: UTP-glucose-1-phosphate-uridyltransferase (UDP-glucose pyrophosphorylase), phosphoglucomutase, glutamine-fructose-6-phosphate transaminase, N-acetyl-glucosamine-phosphomutase; ATP-sulfatase, adenosine sulfate kinase and bisphospho-3'-nucleotidase compared to its wild-type cells.

3. The recombinant yeast cell according to claim 1, characterized in that Overexpression (i) one or more genes involved in the synthesis of UDP-GlcA and one or more genes involved in the synthesis of UDP-GlcNAc; or (ii) one or more genes involved in the synthesis of UDP-GlcA and one or more genes involved in the synthesis of PAPS; or (iii) one or more genes involved in the synthesis of UDP-GlcNAc and one or more genes involved in the synthesis of PAPS; or (iv) one or more genes involved in the synthesis of UDP-GlcNAc; or (v) one or more genes involved in the synthesis of UDP-GlcA and one or more genes involved in the synthesis of UDP-GlcNAc and one or more genes involved in the synthesis of PAPS; and Expression of a gene encoding a heterologous UDP-glucose dehydrogenase; Thus, the recombinant yeast cell has increased production of UDP-GlcA, UDP-GlcNAc and PAPS compared with the expression levels of the corresponding genes in its wild-type cells.

4. The recombinant yeast cell according to claim 3, characterized in that The genes involved in (i) UDP-GlcA synthesis are genes encoding phosphoglucomutase, preferably phosphoglucomutase 1 / 2 (PGM1 / 2); and genes encoding UTP-glucose-1-phospholipid-uridyltransferase (UDP-glucose pyrophosphorylase), preferably UDP-glucose pyrophosphorylase 1 (UGP1); The gene involved in (ii) UDP-GlcNAc synthesis is a gene encoding glutamine-fructose-6-phosphate aminotransferase, preferably glutamine-fructose-6-phosphate aminotransferase 1 (GFA1); and a gene encoding N-acetyl-glucosamine-phosphomutase, preferably phosphoacetylglucosamine mutase 1 (PCM1); and / or The genes involved in (iii) PAPS synthesis are genes encoding sulfate adenylate transferase (ATP-sulfatase), preferably methionine requiring protein 3 (MET3); genes encoding adenosine sulfate kinase, preferably methionine requiring protein 14 (MET14); and genes encoding adenosine sulfate kinase and bisphospho-3'-nucleotidase, preferably methionine requiring protein 22 (MET22).

5. The recombinant yeast cell according to any one of claims 1 to 4, characterized in that Downward adjustment a gene encoding a chitin synthase, preferably chitin synthase (CHS2 / 3), such that the production of chitin synthase is reduced compared to wild-type cells; and / or A gene encoding 3'-phosphoadenylate sulphate reductase, preferably MET16, so that the production of the corresponding enzyme encoded by the down-regulated gene is reduced compared to wild-type cells.

6. The recombinant yeast cell according to any one of claims 1 to 5, characterized in that The gene encoding proheparin synthetase encodes proheparin synthetase 1 and / or proheparin synthetase 2, so that the recombinant yeast cell produces proheparin.

7. The recombinant yeast cell according to any one of claims 1 to 6, characterized in that The recombinant yeast cell additionally expresses genes encoding N-deacetylase (ND), and / or N-deacetylase-N-sulfotransferase (NDST), and / or C5-isomerase (C5-epi), and / or 2-O-sulfotransferase (HS2ST), and / or 3-O-sulfotransferase (HS3ST), and / or 6-O-sulfotransferase (HS6ST), thereby enabling it to produce heparan sulfate.

8. The recombinant yeast cell according to any one of claims 1 to 6, characterized in that The recombinant yeast cell additionally expresses a gene encoding ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST in which the N-terminal membrane domain (MD) or N-terminal signal peptide / signal domain (SD) gene fragment is truncated, so that the ND, and / or NDST, and / or C5-epi, and / or HS2ST, and / or HS3ST, and / or HS6ST enzymes are specifically expressed in the cytoplasm to increase the production of heparan sulfate and / or heparin with different types and clear sulfation patterns.

9. The recombinant yeast cell according to any one of claims 1 to 8, characterized in that One or more heterologous genes are stably integrated into the genome.

10. The recombinant yeast cell according to any one of claims 7 to 9, characterized in that · additionally expressing genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-similar, and HS6ST, thereby producing heparan sulfate as shown in formula (1) in FIG. 3 , wherein the preferred recombinant yeast strain is ST_GNS-022; · additionally expressing genes encoding NDST, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing heparan thioacetyl as shown in formula (2) in FIG. 3 ; · additionally expressing genes encoding NDST and HS6ST, thereby producing heparan sulfate as shown in formula (3) in FIG. 3 ; · additionally expressing genes encoding NDST, C5-epi, HS2ST and HS6ST, thereby producing heparan sulfate as shown in formula (4) in FIG. 3 ; · additionally expressing genes encoding NDST, C5-epi, HS2ST, HS3ST1 and / or HS3ST1-like and HS6ST, thereby producing heparan sulfate as shown in formula (5) in Figure 3; · additionally expressing genes encoding NDST, C5-epi and HS6ST, thereby producing heparan sulfate as shown in formula (6) in FIG. 3 ; · additionally expressing genes encoding ND, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing heparan sulfate as shown in formula (7) in Figure 3; · additionally expressing genes encoding NDST, C5-epi, HS2ST, HS3ST5 and / or HS3ST5-like and HS6ST, thereby producing heparan sulfate as shown in formula (8) in Figure 3; and / or · Additional expression of genes encoding NDST, C5-epi, HS2ST, HS3ST3 and / or HS3ST3-like and HS6ST, thereby producing heparan sulfate as shown in formula (9) in Figure 3.

11. A method for producing heparan sulfate, comprising the following steps (i) culturing a recombinant yeast cell in a culture medium, wherein the recombinant yeast cell is characterized in that: Compared with its wild-type cells, the recombinant yeast cells produce increased production of UDP-GlcA (UDP-D-glucuronic acid) and UDP-GlcNAc (UDP-N-acetyl-D-glucosamine); Compared with wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcA (UDP-D-glucuronic acid) and PAPS (3′-phospho-adenosine sulfate); Compared with its wild-type cells, the recombinant yeast cells produce increased amounts of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate); The recombinant yeast cell has an increased production of UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) compared to its wild-type cell; or Compared with wild-type cells, the recombinant yeast cells have increased production of UDP-GlcA (UDP-D-glucuronic acid), UDP-GlcNAc (UDP-N-acetyl-D-glucosamine) and PAPS (3′-phospho-adenosine sulfate); (ii) obtaining heparan sulfate produced by the recombinant yeast cell in step (i); and optionally (iii) purifying the heparan sulfate obtained in step (ii).

12. The method according to claim 11, wherein the recombinant yeast cell is characterized in that the recombinant yeast cell additionally expresses a gene encoding proheparin synthase, so that the recombinant yeast cell produces proheparin; preferably, the recombinant yeast cell is a recombinant yeast cell as defined in claims 1 to 10.

13. Use of a recombinant yeast cell as defined in any one of claims 1 to 10 for producing heparan sulfate.

14. Heparan sulfate, characterized in that The heparan sulfate exhibits a sulfation pattern produced by a process as defined in claim 11 or 12.

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