Heterologous expression and functional characterization of two alginate lyases in corynebacterium glutamicum

Keywords

Alginate lyases / Corynebacterium glutamicum / Uronates / Alginate depolymerization / Enzyme synergy

References

[1] Enquist-Newman M, Faust AM, Bravo DD, Santos CN, Raisner RM, Hanel A, et al.. Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform. Nature, 2014, 505(7482): 239-243

[2] Chades T, Scully SM, Ingvadottir EM, Orlygsson J. Fermentation of mannitol extracts from brown macro algae by thermophilic clostridia. Front Microbiol, 2018

[3] Motone K, Takagi T, Sasaki Y, Kuroda K, Ueda M. Direct ethanol fermentation of the algal storage polysaccharide laminarin with an optimized combination of engineered yeasts. J Biotechnol, 2016, 231: 129-135

[4] Bilan MI, Grachev AA, Ustuzhanina NE, Shashkov AS, Nifantiev NE, Usov AI. Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag. Carbohydr Res, 2002, 337(8): 719-730

[5] Kawai S, Murata K. Biofuel production based on carbohydrates from both brown and red macroalgae: recent developments in key biotechnologies. Int J Mol Sci, 2016, 17(2): 145

[6] Doi H, Tokura Y, Mori Y, Mori K, Asakura Y, Usuda Y, et al.. Identification of enzymes responsible for extracellular alginate depolymerization and alginate metabolism in Vibrio algivorus. Appl Microbiol Biotechnol, 2017, 101(41581-1592

[7] Takeda H, Yoneyama F, Kawai S, Hashimoto W, Murata K. Bioethanol production from marine biomass alginate by metabolically engineered bacteria. Energy Environ Sci, 2011, 4(7): 2575-2581

[8] Haug A, Larsen B, Smidsrød O, Møller J, Brunvoll J, Bunnenberg E, et al.. A Study of the Constitution of Alginic Acid by Partial Acid Hydrolysis. Acta Chem Scand, 1966, 20: 183-190

[9] Atkins ED, Nieduszynski IA, Mackie W, Parker KD, Smolko EE. Structural components of alginic acid. I. The crystalline structure of poly-beta-D-mannuronic acid. Results of x-ray diffraction and polarized infrared studies. Biopolymers, 1973, 12(8): 1865-1878

[10] Garron ML, Cygler M. Structural and mechanistic classification of uronic acid-containing polysaccharide lyases. Glycobiology, 2010, 20(12): 1547-1573

[11] Hobbs JK, Lee SM, Robb M, Hof F, Barr C, Abe KT, et al.. KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate. Proc Natl Acad Sci U S A, 2016, 113(22): 6188-6193

[12] Rønne ME, Dybdahl Andersen C, Teze D, Petersen AB, Fredslund F, Stender EGP, et al.. Action and cooperation in alginate degradation by three enzymes from the human gut bacterium Bacteroides eggerthii DSM 20697. J Biol Chem, 2024, 300(9): 107596

[13] Drula E, Garron ML, Dogan S, Lombard V, Henrissat B, Terrapon N. The carbohydrate-active enzyme database: functions and literature. Nucleic Acids Res, 2022, 50(D1D571-D577

[14] Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat B. A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J, 2010, 432(3): 437-444

[15] Xu F, Wang P, Zhang YZ, Chen XL. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases. Appl Environ Microbiol, 2018

[16] Aarstad OA, Tøndervik A, Sletta H, Skjåk-Bræk G. Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes. Biomacromol, 2012, 13(1): 106-116

[17] Rønne ME, Tandrup T, Madsen M, Hunt CJ, Myers PN, Moll JM, et al.. Three alginate lyases provide a new gut Bacteroides ovatus isolate with the ability to grow on alginate. Appl Environ Microbiol, 2023, 89(10): e0118523

[18] Thomas F, Lundqvist LC, Jam M, Jeudy A, Barbeyron T, Sandström C, et al.. Comparative characterization of two marine alginate lyases from Zobellia galactanivorans reveals distinct modes of action and exquisite adaptation to their natural substrate. J Biol Chem, 2013, 288(3223021-23037

[19] Pilgaard B, Vuillemin M, Holck J, Wilkens C, Meyer AS. Specificities and synergistic actions of novel PL8 and PL7 alginate lyases from the marine fungus Paradendryphiella salina. J Fungi (Basel), 2021

[20] Doi H, Chinen A, Fukuda H, Usuda Y. Vibrio algivorus sp. nov., an alginate- and agarose-assimilating bacterium isolated from the gut flora of a turban shell marine snail. Int J Syst Evol Microbiol, 2016, 66(8): 3164-3169

[21] Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res, 2014, 42: D490-D495

[22] Zahoor A, Lindner SN, Wendisch VF. Metabolic engineering of aimed at alternative carbon sources and new products. Comput Struct Biotechnol J, 2012, 3(4): e201210004

[23] Brito LF, Frøystad MG, Vandenhove AH, Brautaset T, Pérez-García F. Red seaweed-based bioprocesses with Corynebacterium glutamicum. Bioresour Technol Rep, 2025, 31 102203

[24] Hoffmann SL, Kohlstedt M, Jungmann L, Hutter M, Poblete-Castro I, Becker J, et al.. Cascaded valorization of brown seaweed to produce l-lysine and value-added products using Corynebacterium glutamicum streamlined by systems metabolic engineering. Metab Eng, 2021, 67: 293-307

[25] Schwardmann LS, Wu T, Dransfeld AK, Lindner SN, Wendisch VF. Formamide-based production of amines by metabolically engineering Corynebacterium glutamicum. Appl Microbiol Biotechnol, 2023, 107(134245-4260

[26] Schneider J, Wendisch VF. Putrescine production by engineered Corynebacterium glutamicum. Appl Microbiol Biotechnol, 2010, 88(4): 859-868

[27] Kind S, Kreye S, Wittmann C. Metabolic engineering of cellular transport for overproduction of the platform chemical 1,5-diaminopentane in Corynebacterium glutamicum. Metab Eng, 2011, 13(5): 617-627

[28] Inui M, Kawaguchi H, Murakami S, Vertès AA, Yukawa H. Metabolic engineering of Corynebacterium glutamicum for fuel ethanol production under oxygen-deprivation conditions. J Mol Microbiol Biotechnol, 2004, 8(4): 243-254

[29] Blombach B, Riester T, Wieschalka S, Ziert C, Youn JW, Wendisch VF, et al.. Corynebacterium glutamicum tailored for efficient isobutanol production. Appl Environ Microbiol, 2011, 77(10): 3300-3310

[30] Henke NA, Wiebe D, Pérez-García F, Peters-Wendisch P, Wendisch VF. Coproduction of cell-bound and secreted value-added compounds: simultaneous production of carotenoids and amino acids by Corynebacterium glutamicum. Bioresour Technol, 2018, 247: 744-752

[31] Pérez-García F, Brito LF, Bakken TI, Brautaset T. Riboflavin overproduction from diverse feedstocks with engineered Corynebacterium glutamicum. Biofabrication, 2024

[32] Freudl R. Beyond amino acids: use of the Corynebacterium glutamicum cell factory for the secretion of heterologous proteins. J Biotechnol, 2017, 258: 101-109

[33] Gimmestad M, Sletta H, Ertesvåg H, Bakkevig K, Jain S, Suh SJ, et al.. The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation. J Bacteriol, 2003, 185(12): 3515-3523

[34] Ballance S, Aarstad OA, Aachmann FL, Skjåk-Braek G, Christensen BE. Preparation of high purity monodisperse oligosaccharides derived from mannuronan by size-exclusion chromatography followed by semi-preparative high-performance anion-exchange chromatography with pulsed amperometric detection. Carbohydr Res, 2009, 344(2): 255-259

[35] Ertesvåg H, Skjåk-Bræk G. Bucke C. Modification of alginate using mannuronan C-5-epimerases. Carbohydrate biotechnology protocols, 1999, Totowa, NJ, Humana Press7178

[36] Eggeling L, Bott M. Handbook of Corynebacterium glutamicum, 2005, Boca Raton, Taylor & Francis

[37] Miller JH. Experiments in molecular genetics, 1972, New York, Cold Spring Harbor Laboratory

[38] Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA3rd, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 2009, 6(5): 343-345

[39] Green MR, Sambrook J. Molecular cloning : a laboratory manual, 20124Cold Spring Harbor, N.Y, Cold Spring Harbor Laboratory Press

[40] Nonaka K, Osamura T, Takahashi F. A 4-hydroxybenzoate 3-hydroxylase mutant enables 4-amino-3-hydroxybenzoic acid production from glucose in Corynebacterium glutamicum. Microb Cell Fact, 2023, 22(1): 168

[41] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(1248-254

[42] Peters-Wendisch PG, Schiel B, Wendisch VF, Katsoulidis E, Möckel B, Sahm H, et al.. Pyruvate carboxylase is a major bottleneck for glutamate and lysine production by Corynebacterium glutamicum. J Mol Microbiol Biotechnol, 2001, 3(2): 295-300

[43] Pérez-García F, Klein VJ, Brito LF, Brautaset T. From brown seaweed to a sustainable microbial feedstock for the production of riboflavin. Front Bioeng Biotechnol, 2022

[44] Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol, 1983, 166(4): 557-580

[45] Blattner FR, Plunkett G3rd, Bloch CA, Perna NT, Burland V, Riley M, et al.. The complete genome sequence of Escherichia coli K-12. Science, 1997, 277(5331): 1453-1462

[46] Abe S, Takayama K-I, Kinoshita S. Taxonomical studies on glutamic acid-producing bacteria. J Gen Appl Microbiol, 1967, 13(3): 279-301

[47] Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD. ¹H, ¹³C and ¹⁵N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR, 1995, 5(1): 67-81

[48] Ballance S, Holtan S, Aarstad OA, Sikorski P, Skjåk-Braek G, Christensen BE. undefined. J Chromatogr A, 2005, 1093(1-2): 59-68

[49] Hoffmann F, Rinas U. Stress induced by recombinant protein production in Escherichia coli. Adv Biochem Eng Biotechnol, 2004, 89: 73-92

[50] Snoeck S, Guidi C, De Mey M. “Metabolic burden” explained: stress symptoms and its related responses induced by (over)expression of (heterologous) proteins in Escherichia coli. Microb Cell Fact, 2024, 23(1): 96

[51] Li Z, Rinas U. Recombinant protein production associated growth inhibition results mainly from transcription and not from translation. Microb Cell Fact, 2020, 19(183

[52] Chander D, Koul D, Tickoo A, Chaubey A. Advancing recombinant protein production by bacteria: strategies and challenges in heterologous systems. Cur Res Biotechnol, 2025, 10: 100342

[53] Preiss J, Ashwell G. Alginic acid metabolism in bacteria: I. enzymatic formation of unsaturated oligosaccharides and 4-deoxy-l-erythro-5-hexoseulose uronic acid. J Biol Chem, 1962, 237(2309-316

[54] Labourel F, Rajon E. Resource uptake and the evolution of moderately efficient enzymes. Mol Biol Evol, 2021, 38(93938-3952

[55] Green ER, Mecsas J. Bacterial secretion systems: an overview. Microbiol Spectr, 2016

[56] Vertès AA. Yukawa H, Inui M. Protein secretion systems of corynebacterium glutamicum. : biology and biotechnologyCorynebacterium glutamicum, 2013, Heidelberg, Springer, Berlin Heidelberg, Berlin351389

[57] Braunstein M, Bensing BA, Sullam PM. The two distinct types of SecA2-dependent export systems. Microbiol Spectr, 2019

[58] Tsirigotaki A, De Geyter J, Šoštaric´ N, Economou A, Karamanou S. Protein export through the bacterial Sec pathway. Nat Rev Microbiol, 2017, 15(1): 21-36

[59] Lokireddy SR, Kunchala SR, Vadde R. Advancements in Escherichia coli secretion systems for enhanced recombinant protein production. World J Microbiol Biotechnol, 2025, 41(3): 90

[60] Palmer T, Berks BC. The twin-arginine translocation (Tat) protein export pathway. Nat Rev Microbiol, 2012, 10(7483-496

[61] Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, et al.. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol, 2022, 40(7): 1023-1025

[62] Emani SS, Kan A, Storms T, Bonanno S, Law J, Ray S, et al.. Periplasmic stress contributes to a trade-off between protein secretion and cell growth in Escherichia coli Nissle 1917. Synth Biol, 2023

[63] Gao SK, Yin R, Wang XC, Jiang HN, Liu XX, Lv W, et al.. Structure characteristics, biochemical properties, and pharmaceutical applications of alginate lyases. Mar Drugs, 2021

[64] Junker N, Sariyar Akbulut B, Wendisch VF. Utilization of orange peel waste for sustainable amino acid production by Corynebacterium glutamicum. Front Bioeng Biotechnol, 2024

[65] Arntzen M, Pedersen B, Klau LJ, Stokke R, Oftebro M, Antonsen SG, et al.. Alginate degradation: insights obtained through characterization of a thermophilic exolytic alginate lyase. Appl Environ Microbiol, 2021

[66] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al.. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25(17): 3389-3402

[67] Wang Z-P, Cao M, Li B, Ji X-F, Zhang X-Y, Zhang Y-Q, et al.. Cloning, secretory expression and characterization of a unique pH-stable and cold-adapted alginate lyase. Mar Drugs, 2020, 18(4): 189

[68] Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al.. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 2024, 630(8016493-500

[69] Tandrup T, Rivas-Fernández JP, Madsen M, Rønne ME, Peterson AB, Klau LJ, et al.. The swiss army knife of alginate metabolism: mechanistic analysis of a mixed-function polysaccharide lyase/epimerase of the human gut microbiota. J Am Chem Soc, 2025, 147(27): 23594-23607

[70] Paalme T, Elken R, Kahru A, Vanatalu K, Vilu R. The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach. Antonie Van Leeuwenhoek, 1997, 71(3): 217-230

[71] Mandrand-Berthelot M-A, Condemine G, Hugouvieux-Cotte-Pattat N (2004). Catabolism of Hexuronides, Hexuronates, Aldonates, Aldarates. EcoSal Plus 1(1). https://doi.org/10.1128/ecosalplus.3.4.2

[72] Sharp PM, Bailes E, Grocock RJ, Peden JF, Sockett RE. Variation in the strength of selected codon usage bias among bacteria. Nucleic Acids Res, 2005, 33(4): 1141-1153

[73] Hadiati A, Krahn I, Lindner SN, Wendisch VF. Engineering of Corynebacterium glutamicum for growth and production of L-ornithine, L-lysine, and lycopene from hexuronic acids. Bioresour Bioprocess, 2014, 1(125

[74] Park SH, Kim HU, Kim TY, Park JS, Kim SS, Lee SY. Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat Commun, 2014, 5: 4618

[75] Liu J, Xu J-Z, Rao Z-M, Zhang W-G. Industrial production of L-lysine in Corynebacterium glutamicum: progress and prospects. Microbiol Res, 2022, 262: 127101

Funding

NTNU Norwegian University of Science and Technology (incl St. Olavs Hospital - Trondheim University Hospital)

RIGHTS & PERMISSIONS

The Author(s)