Coupling optimization of cell growth cycle and key enzyme membrane localization for enhanced synthesis of high molecular weight heparosan by Corynebacterium glutamicum

Abstract

High-molecular weight heparosan (HMW-heparosan) is a member of the glycosaminoglycan family. It possesses various chemical and physical properties suitable for a range of high-quality tissue engineering biomaterials, gels, scaffolds, and drug delivery systems. In this study, the HMW-heparosan biosynthesis pathway was engineered in Corynebacterium glutamicum through the introduction of heparosan synthase PmHS2 from Pasteurella multocida combined with overexpression of the key genes ugdA and galU, resulting in the generation of a stable HMW-heparosan-producing strain. Subsequently, to address metabolic flux competition, endogenous glycosyltransferases were systematically deleted to minimize UDP-glucose consumption, leading to a significant increase in HMW-heparosan accumulation. Additionally, cell growth was optimized by overexpressing transcriptional regulators whcD and PnkB, which was found to improve cell growth while creating an improved intracellular environment for biosynthesis. Notably, the critical enzyme heparosan synthase PmHS2 was relocated to the cell membrane by cell membrane display motifs porB, with its stability and catalytic efficiency being significantly enhanced so that the titer of HMW-heparosan reached 1.40 g/L in shake-flasks. Ultimately, the engineered strain was demonstrated to achieve HMW-heparosan production at 7.02 g/L with an average molecular weight (Mw) of 801 kDa in 5 L fed-batch bioreactor. These results demonstrate combinatorial optimization of cell factories, especially cell morphology and membrane localization of key enzymes, is efficacious and likely applicable for the production of other biopolymers.

References

[1] Capila I, Linhardt RJ (2002) Heparin–protein interactions. Angew Chem Int Ed 41:390–412.

[2] CarlssonP, PrestoJ, SpillmannD, LindahlU, KjellénL. Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains. J Biol Chem, 2008, 283: 20008-20014

[3] ChavarocheAAE, SpringerJ, KooyF, BoeriuC, EgginkG. In vitro synthesis of heparosan using Recombinant Pasteurella multocida heparosan synthase PmHS2. Appl Microbiol Biotechnol, 2010, 85: 1881-1891

[4] ChenJ, JonesCL, LiuJ. Using an enzymatic combinatorial approach to identify anticoagulant Heparan sulfate structures. Chem Biol, 2007, 14: 986-993

[5] ChenYP, HwangIE, LinCJ, WangHJ, TsengCP. Enhancing the stability of Xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli. J Appl Microbiol, 2012, 112: 455-463

[6] ChenJX, LiuW, ZhangM, ChenJH. Heparosan-based negatively charged nanocarrier for rapid intracellular drug delivery. Int J Pharm, 2014, 473: 493-500

[7] ChenX, ChenR, YuX, TangD, YaoW, GaoX. Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella Multocida, PmHS1. Bioprocess Biosyst Eng, 2017, 40: 675-681

[8] CorbettD, RobertsI. Capsular polysaccharides in Escherichia coli. Adv Appl Microbiol, 2008, 65: 1-26

[9] CuthbertsonL, KosV, WhitfieldC. ABC transporters involved in export of cell surface glycoconjugates. Microbiol Mol Biol Rev, 2010, 74: 369-417

[10] CuthbertsonLK, KosVV, WhitfieldCH. ABC transporters involved in export of cell surface glycoconjugates. Microbiol Mol Biol Rev, 2010, 74: 341-362

[11] DeAngelisPL, WhiteCL. Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D. J Biol Chem, 2002, 277: 7209-7213

[12] DonotF, FontanaA, BaccouJC, Schorr-GalindoS. Microbial exopolysaccharides: main examples of synthesis, excretion, genetics and extraction. Carbohydr Polym, 2012, 87: 951-962

[13] FiuzaM, CanovaMJ, Zanella-CléonI, BecchiM, CozzoneAJ, MateosLM, KremerL, GilJA, MolleV. From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division. J Biol Chem, 2008, 283: 18099-18112

[14] GibsonDG, YoungL, ChuangRY, VenterJC, HutchisonCA, SmithHO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009, 6: 343-345

[15] HuS, ZhaoLL, HuLT, XiXT, ZhangYL, WangY, ChenJM, ChenJ, KangZ. Engineering the probiotic bacterium Escherichia coli Nissle 1917 as an efficient cell factory for heparosan biosynthesis. Enzyme Microb Technol, 2022, 158: 110038

[16] HuS, ZhouS, WangY, ChenW, YinG, ChenJ, DuGC, KangZ. Coordinated optimization of the polymerization and transportation processes to enhance the yield of exopolysaccharide heparosan. Carbohydr Polym, 2024, 333: 121983

[17] Jackson M (2025) The mycobacterial cell envelope—lipids. Cold Spring Harb Perspect Med

[18] JinP, ZhangLP, YuanPH, KangZ, DuGC, ChenJ. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis. Carbohydr Polym, 2016, 140: 424-432

[19] Jin C, Li J, Huang Z, Han XS, Bao J (2022) Engineering Corynebacterium glutamicum for synthesis of poly(3-hydroxybutyrate) from lignocellulose biomass. Biotechnol Bioeng 119:1598–1613. https://doi.org/10.1002/bit.28074

[20] LaneRS, AngeK, ZolghadrB, LiuX, SchäfferC, LinhardtRJ, DeAngelisPL. Expanding glycosaminoglycan chemical space: towards the creation of sulfated analogs, novel polymers and chimeric constructs. Glycobiology, 2017, 27(7): 646-656

[21] LeeDS, KimY, LeeHS. The WhcD gene of Corynebacterium glutamicum plays roles in cell division and envelope formation. Microbiology, 2017, 163: 131-143

[22] LetekM, OrdoñezE, VaqueraJ, MargolinW, FlärdhK, MateosLM, GilJA. DivIVA is required for Polar growth in the MreB-Lacking Rod-Shaped actinomycete Corynebacterium glutamicum. J Bacteriol, 2008, 190: 3283-3292

[23] LiZM, WangQT, LiuHU, WangYT, ZhengZY, ZhangYA, TanTW. Engineering Corynebacterium glutamicum for the efficient production of N-acetylglucosamine. Bioresour Technol, 2023, 390: 129865

[24] LyM, WangZ, LaremoreTN, ZhangF, ZhongW, PuD, ZagorevskiDV, DordickJS, LinhardtRJ. Analysis of E. coli K5 capsular polysaccharide heparosan. Anal Bioanal Chem, 2011, 399: 737-745

[25] MayBJ, ZhangQ, LiLL, PaustianML, WhittamTS, KapurV. Complete genomic sequence of Pasteurella multocida, Pm70. Proc Natl Acad Sci U S A, 2001, 98: 3460-3465

[26] OttoNJ, GreenDE, MasukoS, MayerA, TannerME, LinhardtRJ, DeAngelisPL. Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts. J Biol Chem, 2012, 287: 7203-7212

[27] PuechV, ChamiM, LemassuA, LaneelleM-A, SchifflerB, GounonP, BayanN, BenzR, DaffeM. Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiol, 2001, 147: 1365-1382

[28] PuechV, ChamiM, LemassuA, LanéelleMA, SchifflerB, GounonP, BayanN, BenzR, DafféM. Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane. Microbiology, 2001, 147(5): 1365-1372

[29] RamosA, LetekM, CampeloAB, VaqueraJ, MateosLM, GilJA. Altered morphology produced by FtsZ expression in Corynebacterium glutamicum ATCC13869. Microbiology, 2005, 151: 2563-2572

[30] RomanEL, RobertsIA, LidholtKE, Kusche-GullbergMK. Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide. Biochem J, 2003, 374(3): 767-772

[31] SchäferA, TauchA, JägerW, KalinowskiJ, ThierbachG, PühlerA. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene, 1994, 145: 69-73

[32] SiegerB, SchubertK, DonovanC, BramkampM. The lipid II flippase RodA determines morphology and growth in Corynebacterium glutamicum. Mol Microbiol, 2013, 90: 966-982

[33] Sismey-RagatzAE, GreenDE, OttoNJ, RejzekM, FieldRA, DeAngelisPL. Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures. J Biol Chem, 2007, 282(39): 28321-28327

[34] SuflitaM, FuL, HeW, KoffasM, LinhardtRJ. Heparin and related polysaccharides: synthesis using Recombinant enzymes and metabolic engineering. Appl Microbiol Biotechnol, 2015, 99: 7465-7479

[35] TangM, SunX, ZhangS, WanJ, LiL, NiH. Improved catalytic and antifungal activities of Bacillus thuringiensis cells with surface display of Chi9602∆SP. J Appl Microbiol, 2017, 122(1): 106-118

[36] TatenoT, HatadaK, TanakaT, FukudaH, KondoA. Development of novel cell surface display in Corynebacterium glutamicum using Porin. Appl Microbiol Biotechnol, 2009, 84: 733-739

[37] van den ChavarocheAAE, EgginkG. Production methods for heparosan, a precursor of heparin and Heparan sulfate. Carbohydr Polym, 2013, 93: 38-47

[38] WangZY, LyM, ZhangFM, ZhongWH, SuenA, HickeyAM, DordickJS, LinhardtRJ. E. coli K5 fermentation and the Preparation of heparosan, a bioengineered heparin precursor. Biotechnol Bioeng, 2010, 107: 964-973

[39] WangZY, DordickJS, LinhardtRJ. Escherichia coli K5 heparosan fermentation and improvement by genetic engineering. Bioeng Bugs, 2011, 2: 63-67

[40] WangWZ, ChenF, WangYQ, WangLN, FuHY, ZhengFP, BeecherL. Optimization of reactions between reducing sugars and 1-phenyl-3-methyl-5-pyrazolone (PMP) by response surface methodology. Food Chem, 2018, 254: 158-164

[41] WangY, HuL, HuangH, WangH, ZhangT, ChenJ, DuGC, KangZ. Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum. Nat Commun, 2020, 11: 3120

[42] WilliamsAW, GedeonKS, VaidyanathanD. Metabolic engineering of Bacillus megaterium for heparosan biosynthesis using Pasteurella multocida heparosan synthase, PmHS2. Microb Cell Fact, 2019, 18: 132

[43] YangF, MossL, PhillipsG. The molecular structure of green fluorescent protein. Nat Biotechnol, 1996, 14: 1246-1251

[44] YimSS, ChoiJW, LeeRJ, LeeYJ, LeeSH, KimSY, JeongKJ. Development of a new platform for secretory production of Recombinant proteins in Corynebacterium glutamicum. Biotechnol Bioeng, 2015, 113: 163-172

[45] YuYY, GongBX, WangHL, YangGX, ZhouXX. Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan. Biotechnol Bioeng, 2022, 120: 1081-1096

[46] ZhangLR, JiaHM, XuDQ. Construction of a novel twin-arginine translocation (Tat)-dependent type expression vector for secretory production of heterologous proteins in Corynebacterium glutamicum. Plasmid, 2015, 82: 50-55

[47] ZhangQ, YaoR, ChenXL, LiuLM, XuSQ, ChenJH, WuJ. Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors. Metab Eng, 2018, 47: 314-322

Funding

the National Key R&D Program of China (2021YFC2103100)

RIGHTS & PERMISSIONS

The Author(s)