Non-conventional yeasts: Versatile platforms for the expression and secretion of eukaryotic proteins

Keywords

Bioprospecting / Genetic manipulation / Metabolic engineering / Recombinant proteins / Protein secretion / Yeast vectors

References

[1] Álvarez-Cao ME, Rico-Díaz A, Cerdán ME, Becerra M, González-Siso MI. Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase. Microb Cell Fact, 2018, 17: 137

[2] Bartkevičiute D, Sasnauskas K. Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae. FEMS Yeast Res, 2004, 4: 833-840

[3] Blobel G. Protein targeting (Nobel lecture). ChemBioChem, 2000, 1: 86-102

[4] Blondeau K, B O, Boze H, M G, G P. Influence of culture conditions on the production of heterologous interleukin 1β by Kluveromyces lactis. Biotechnol Tech, 1993, 7: 609-614

[5] Böer E, Steinborn G, Florschütz K, Körner M, Gellissen G, Kunze G. Arxula adeninivorans (Blastobotrys adeninivorans) - a dimorphic yeast of great biotechnological potential. Yeast Biotechnol: Divers Appl Springer, 2009, 6: 15-34

[6] Böer E, Breuer FS, Weniger M, Denter S, Piontek M, Kunze G. Large-scale production of tannase using the yeast Arxula adeninivorans. Appl Microbiol Biotechnol, 2011, 92: 105-114

[7] Bragança CRS, Colombo LT, Roberti AS, Alvim MCT, Cardoso SA, Reis KCP, et al. . Construction of recombinant Kluyveromyces marxianus UFV-3 to express dengue virus type 1 nonstructural protein 1 (NS1). Appl Microbiol Biotechnol, 2015, 99: 1191-1203

[8] Bredell H, Smith JJ, Görgens JF, van Zyl WH. Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1-L2 proteins in Pichia pastoris and Hansenula polymorpha. Yeast, 2018, 35: 519-529

[9] Breuer U, Harms H. Debaryomyces hansenii - an extremophilic yeast with biotechnological potential. Yeast, 2006, 23: 415-437

[10] Celińska E, Borkowska M, Białas W, Korpys P, Nicaud JM. Robust signal peptides for protein secretion in Yarrowia lipolytica: identification and characterization of novel secretory tags. Appl Microbiol Biotechnol, 2018, 102: 5221-5233

[11] Contesini FJ, Davanço MG, Borin GP, Vanegas KG, Cirino JPG, de Melo RR, et al. . Advances in recombinant lipases: production, engineering, immobilization and application in the pharmaceutical industry. Catalysts, 2020, 10: 1032

[12] Corredor M, Davila A-M, Casaregola S, Gaillardin G. Chromosomal polymorphism in the yeast species Debaryomyces hansenii. Antonie Van Leeuwenhoek, 2003, 83: 215-222

[13] Cruz A, Sterten H, Steinhoff FS, Mydland LT, Øverland M. Cyberlindnera jadinii yeast as a protein source for broiler chickens: effects on growth performance and digestive function from hatching to 30 days of age. Poult Sci, 2020, 99: 3168-3178

[14] Dekker WJC, Ortiz-Merino RA, Kaljouw A, Battjes J, Wiering FW, Mooiman C, et al. . Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth. Metab Eng, 2021, 67: 347-364

[15] Delic M, Valli M, Graf AB, Pfeffer M, Mattanovich D, Gasser B. The secretory pathway: exploring yeast diversity. FEMS Microbiol Rev, 2013, 37: 872-914

[16] dos Santos VC, Bragança CRS, Passos FJV, Passos FML. Kinetics of growth and ethanol formation from a mix of glucose/xylose substrate by Kluyveromyces marxianus UFV-3. Antonie Van Leeuwenhoek, 2013, 103: 53-161

[17] Du Preez JC, Bosch M, Prior BA. Xylose fermentation by Candida shehatae and Pichia stipitis: effects of pH, temperature and substrate concentration. Enzyme Microb Technol, 1986, 8: 360-364

[18] Dusny C, Schmid A. The MOX promoter in Hansenula polymorpha is ultrasensitive to glucose-mediated carbon catabolite repression. FEMS Yeast Res, 2016, 16: fow067

[19] Dyerberg ASB, Navarrete C, Martínez JL, 2022, High-throughput screening of a Debaryomyces hansenii library for potential candidates with improved stress tolerance and wider carbon utilisation capabilities. BioRxiv. 2022–03.

[20] Egermeier M, Russmayer H, Sauer M, Marx H. Metabolic flexibility of Yarrowia lipolytica growing on glycerol. Front Microbiol, 2017, 8: 49

[21] Ernits K, Kjeldsen C, Persson K, Grigor E, Alamäe T, Visnapuu T. Structural insight into a yeast maltase—the BaAG2 from Blastobotrys adeninivorans with transglycosylating activity. J Fungi, 2021, 7: 816

[22] Estrada M, Navarrete C, Møller S, Procentese A, Martínez JL. Utilization of salt-rich by-products from the dairy industry as feedstock for recombinant protein production by Debaryomyces hansenii. Microb Biotechnol, 2023, 16: 404-407

[23] Fonseca GG, Heinzle E, Wittmann C, Gombert AK. The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol, 2008, 79: 339-354

[24] Gao J, Jiang L, Lian J. Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products. Synth Syst Biotechnol, 2021, 6: 110-119

[25] Gottardi D, Siroli L, Vannini L, Patrignani F, Lanciotti R. Recovery and valorization of agri-food wastes and by-products using the non-conventional yeast Yarrowia lipolytica. Trends Food Sci Technol, 2021, 115: 74-86

[26] Gu L, Zhang R, Fan X, Wang Y, Ma K, Jiang J, et al. . Development of CRISPR/Cas9-based genome editing tools for polyploid yeast Cyberlindnera jadinii and its application in engineering heterologous steroid-producing strains. ACS Synth Biol, 2023, 12: 2947-2960

[27] Haghighi Poodeh S, Ranaei Siadat SO, Arjmand S, Khalifeh Soltani M. Improving AOX1 promoter efficiency by overexpression of Mit1 transcription factor. Mol Biol Rep, 2022, 49: 9379-9386

[28] Hermansen C, Siao R, Chua GG, Lee MRX, Thong A, Weingarten M, Lindley N, et al. . Short-chain fatty acid utilization in Cyberlindnera jadinii for single-cell protein and odd-chain fatty acid production. Microorganisms, 2025, 13: 1558

[29] Hong YR, Chen YL, Farh L, Yang WJ, Liao CH, Shiuan D. Recombinant Candida utilis for the production of biotin. Appl Microbiol Biotechnol, 2006, 71: 211-221

[30] Hong J, Wang Y, Kumagai H, Tamaki H. Construction of thermotolerant yeast expressing thermostable cellulase genes. J Biotechnol, 2007, 130: 114-123

[31] Huang EL, Lefsrud MG. Fermentation monitoring of a co-culture process with Saccharomyces cerevisiae and Scheffersomyces stipitis using shotgun proteomics. J Bioprocess Biotechniq, 2014, 4: 144

[32] Huang M, Wang G, Qin J, Petranovic D, Nielsen J. Engineering the protein secretory pathway of Saccharomyces cerevisiae enables improved protein production. Proc Natl Acad Sci U S A, 2018, 115: E11025-E11032

[33] Ji L, Xu S, Zhang Y, Cheng H. Screening of broad-host expression promoters for shuttle expression vectors in non-conventional yeasts and bacteria. Microb Cell Fact, 2024, 23: 230

[34] Karbalaei M, Rezaee SA, Farsiani H. Pichia pastoris: a highly successful expression system for optimal synthesis of heterologous proteins. J Cell Physiol, 2020, 235: 5867-5881

[35] Karim A, Gerliani N, Aïder M. Kluyveromyces marxianus: an emerging yeast cell factory for applications in food and biotechnology. Int J Food Microbiol, 2020, 333: 108818

[36] Kobayashi Y, Inokuma K, Matsuda M, Kondo A, Hasunuma T. Resveratrol production from several types of saccharide sources by a recombinant Scheffersomyces stipitis strain. Metab Eng Commun, 2021, 13: 188

[37] Korpys-Woźniak P, Celińska E. Molecular background of HAC1-driven improvement in the secretion of recombinant protein in Yarrowia lipolytica based on comparative transcriptomics. Biotechnol Rep, 2023, 38: 801

[38] Krasovska OS, Stasyk OV, Sibirny AA. Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis. Appl Microbiol Biotechnol, 2013, 97: 9969-9979

[39] Kunigo M, Buerth C, Tielker D, Ernst JF. Heterologous protein secretion by Candida utilis. Appl Microbiol Biotechnol, 2013, 97: 7357-7368

[40] Kunze G, Gaillardin C, Czernicka M, Durrens P, Martin T, Böer E, et al. . The complete genome of Blastobotrys (Arxula) adeninivorans LS3-a yeast of biotechnological interest. Biotechnol Biofuels, 2014, 7: 66

[41] Kurtzman CP, Fell JW, Boekhout T. The yeasts: a taxonomic study. 5^th ed. Elsevier; 2011.

[42] Kurylenko OO, Ruchala J, Vasylyshyn RV, Stasyk OV, Dmytruk OV, Dmytruk KV, et al. . Peroxisomes and peroxisomal transketolase and transaldolase enzymes are essential for xylose alcoholic fermentation by the methylotrophic thermotolerant yeast, Ogataea (Hansenula) polymorpha. Biotechnol Biofuels, 2018, 11: 197

[43] Lacerda MP, Oh EJ, Eckert C. The model system Saccharomyces cerevisiae versus emerging non-model yeasts for the production of biofuels. Life, 2020, 10: 299

[44] Lane MM, Morrissey JP. Kluyveromyces marxianus: a yeast emerging from its sister’s shadow. Fungal Biol Rev, 2010, 24: 17-26

[45] Lapeña D, Kosa G, Hansen LD, Mydland LT, Passoth V, Horn SJ, et al. . Production and characterization of yeasts grown on media composed of spruce-derived sugars and protein hydrolysates from chicken by-products. Microb Cell Fact, 2020, 19: 19

[46] Laukens B, De Visscher C, Callewaert N. Engineering yeast for producing human glycoproteins: where are we now?. Future Microbiol, 2015, 10: 21-34

[47] Lee YJ, Kim CS, Oh DK. Lactulose production by β-galactosidase in permeabilized cells of Kluyveromyces lactis. Appl Microbiol Biotechnol, 2004, 64: 787-793

[48] Lee MH, Hsu TL, Lin JJ, Lin YJ, Kao YY, Chang JJ, et al. . Constructing a human complex type N-linked glycosylation pathway in Kluyveromyces marxianus. PLoS ONE, 2020, 15: 233492

[49] Li H, Ma W, Lyv Y, Gao S, Zhou J. Glycosylation modification enhances (2S)-naringenin production in Saccharomyces cerevisiae. ACS Synth Biol, 2022, 11: 2339-2347

[50] Li M, Gao S, Yang P, Li H. Improvement of ribonucleic acid production in Cyberlindnera jadinii and optimization of fermentation medium. AMB Express, 2024, 14: 24

[51] Liang Z, Deng M, Zhang Z, Li M, Zhou SJ, Zhao ZG, et al. . One-step construction of a food-grade expression system based on the URA3 gene in Kluyveromyces lactis. Plasmid, 2021, 116: 102577

[52] Lin X, Ding W, Zheng S, Wu L, Chen X, Xie C, et al. . Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins. Microb Cell Fact, 2024, 23: 206

[53] Liping X, Zhang R, Wen Q, Fan X, Lishan G, Ma K, et al. . J Agri Food Chem, 2025, 73: 31511-31522

[54] Litwińska K, Bischoff F, Matthes F, Bode R, Rutten T, Kunze G. Characterization of recombinant laccase from trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals. AMB Express, 2019, 9(1): 102

[55] Liu B, Gong X, Chang S, Yang Y, Song M, Duan D, et al. . Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis. J Biotechnol, 2009, 143: 95-102

[56] Liu C, Yao Y, Yang X, Bai H, Huang W, Xia Y, et al. . Production of recombinant human papillomavirus type 52 L1 protein in Hansenula polymorpha formed virus-like particles. J Microbiol Biotechnol, 2015, 25: 936-940

[57] Liu Y, Tu X, Xu Q, Bai C, Kong C, Liu Q, et al. . Engineered monoculture and co-culture of methylotrophic yeast for de novo production of monacolin J and lovastatin from methanol. Metab Eng, 2018, 45: 189-199

[58] Lv Y, Edwards H, Zhou J, Xu P. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth Biol, 2019, 8: 568-576

[59] Madzak C. Yarrowia lipolytica strains and their biotechnological applications: how natural biodiversity and metabolic engineering could contribute to cell factories improvement. J Fungi, 2021, 7: 548

[60] Manfrão-Netto JHC, Queiroz EB, Rodrigues KA, Coelho CM, Paes HC, Rech EL, et al. . Evaluation of Ogataea (Hansenula) polymorpha for hyaluronic acid production. Microorganisms, 2021, 9: 312

[61] Matanović A, Marđetko N, Slišković A, Didak B, Hanousek Čiča K, Žunar B, et al. . Production of lactate by metabolically engineered Scheffersomyces stipitis. J Fungi, 2025, 11: 413

[62] Mei M, Li J, Wang S, Lee KB, Iverson BL, Zhang G, et al. . Prompting fab yeast surface display efficiency by ER retention and molecular chaperon co-expression. Front Bioeng Biotechnol, 2019, 7: 362

[63] Moon SY, An NY, Lee JY. Transforming non-conventional yeasts into key players in biotechnology: advances in synthetic biology applications. Front Microbiol, 2025, 16: 1600187

[64] Morin N, Cescut J, Beopoulos A, Lelandais G, Le Berre V, Uribelarrea JL, et al. . Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica. PLoS ONE, 2011, 6: 27966

[65] Moussa M, Ibrahim M, El GM, Rohde J, Gnoth S, Anton A, et al. . Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha. BMC Biotechnol, 2012, 12: 96

[66] Mueller F, Moussa M, El Ghazaly M, Rohde J, Bartsch N, Parthier A, et al. . Efficient production of recombinant parathyroid hormone (rPTH) fragment 1–34 in the methylotrophic yeast Hansenula polymorpha. GaBI J, 2013, 2: 114-122

[67] Murphy A, Kavanagh K. Emergence of Saccharomyces cerevisiae as a human pathogen: implications for biotechnology. Enzyme Microb Technol, 1999, 25(7): 551-557

[68] Nambu-Nishida Y, Nishida K, Hasunuma T, Kondo A. Genetic and physiological basis for antibody production by Kluyveromyces marxianus. AMB Express, 2018, 8: 56

[69] Naveira-Pazos C, Robles-Iglesias R, Fernández-Blanco C, Veiga MC, Kennes C. State-of-the-art in the accumulation of lipids and other bioproducts from sustainable sources by Yarrowia lipolytica. Rev Environ Sci Biotechnol, 2023, 22: 1131-1158

[70] Neiers F, Belloir C, Poirier N, Naumer C, Krohn M, Briand L. Comparison of different signal peptides for the efficient secretion of the sweet-tasting plant protein brazzein in Pichia pastoris. Life, 2021, 11: 46

[71] Nicaud J-M, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, et al. . Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res, 2002, 2: 371-379

[72] Nurdiani D, Putro EW, Septisetyani EP, Utami N, Hariyatun H, Prasetyaningrum PW, et al. . Characterization of functional human insulin produced using the Pichia pastoris expression system. J Appl Pharm Sci, 2024, 14: 216-226

[73] Ochoa-Gutiérrez D, Reyes-Torres AM, de la Fuente-Colmenares I, Escobar-Sánchez V, González J, Ortiz-Hernández R, et al. . Alternative CUG codon usage in the halotolerant yeast Debaryomyces hansenii: gene expression profiles provide new insights into ambiguous translation. J Fungi, 2022, 8: 970

[74] Onésime D, Lebrun E, Petrovic GS, Celińska E, Nicaud JM. New Yarrowia lipolytica chassis strains for industrial enzyme production. Microb Cell Fact, 2025, 24: 164

[75] Papini M, Nookaew I, Uhlén M, Nielsen J. Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae. Microb Cell Fact, 2012, 11 136

[76] Parapouli M, Vasileiadis A, Afendra AS, Hatziloukas E. Saccharomyces cerevisiae and its industrial applications. AIMS Microbiol, 2020, 6: 1-31

[77] Park YK, Vandermies M, Soudier P, Telek S, Thomas S, Nicaud JM, et al. . Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions. Microb Cell Fact, 2019, 18: 167

[78] Passoth V, Hahn-Hägerdal B. Production of a heterologous endo-1,4-xylanase in the yeast Pichia stipitis with an O₂-regulated promoter. Enzyme Microb Technol, 2000, 26: 781-784

[79] Patra P, Das M, Kundu P, Ghosh A. Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts. Biotechnol Adv, 2021, 47 107695

[80] Pereira I, Madeira A, Prista C, Loureiro-Dias MC, Leandro MJ. Characterization of new polyol/H+ symporters in Debaryomyces hansenii. PLoS ONE, 2014, 9: 88180

[81] Pratama A, Putranto WS, Suradi K, Adawiyah R, Risanawati R, Ninda C. The effect of Kluyveromyces lactis starter concentration and fermentation time to the physicochemical and functional properties of egg white powder. J Ilmu Teknol Hasil Ternak, 2019, 14: 117-125

[82] Purushothaman K, Crawford AD, Rocha SDC, Göksu AB, Lange BM, Mydland LT, et al. . Cyberlindnera jadinii yeast as a functional protein source: modulation of immunoregulatory pathways in the intestinal proteome of zebrafish (Danio rerio). Heliyon, 2024, 10: 26547

[83] Puseenam A, Kocharin K, Tanapongpipat S, Eurwilaichitr L, Ingsriswang S, Roongsawang N. A novel sucrose-based expression system for heterologous proteins expression in thermotolerant methylotrophic yeast Ogataea thermomethanolica. FEMS Microbiol Lett, 2018, 365 238

[84] Qiao K, Wasylenko TM, Zhou K, Xu P, Stephanopoulos G. Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat Biotechnol, 2017, 35: 173-177

[85] Rajoka MI, Khan S, Shahid R. Kinetics and regulation studies of the production of β-Galactosidase from Kluyveromyces marxianus grown on different substrates. Food Technol Biotechnol, 2003, 41: 315-320

[86] Rakicka-Pustułka M, Mirończuk AM, Celińska E, Białas W, Rymowicz W. Scale-up of the erythritol production technology–process simulation and techno-economic analysis. J Clean Prod, 2020, 257: 120533

[87] Rico-Díaz A, Álvarez-Cao ME, Escuder-Rodríguez JJ, González-Siso MI, Cerdán ME, Becerra M. Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications. Sci Rep, 2017, 7: 45535

[88] Rodicio R, Heinisch JJ. Yeast on the Milky Way: genetics, physiology and biotechnology of Kluyveromyces lactis. Yeast, 2013, 30: 165-177

[89] Rodríguez ÁP, Leiro RF, Cristina MC, Cerdán ME, González Siso MI, Becerra M. Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase. Microb Cell Fact, 2006, 5: 41

[90] Sakhtah H, Behler J, Ali-Reynolds A, Causey TB, Vainauskas S, Taron CH. A novel regulated hybrid promoter that permits autoinduction of heterologous protein expression in Kluyveromyces lactis. Appl Environ Microbiol, 2019, 85 19

[91] Seike T, Narazaki Y, Kaneko Y, Shimizu H, Matsuda F. Random transfer of Ogataea polymorpha genes into Saccharomyces cerevisiae reveals a complex background of heat tolerance. J Fungi, 2021, 7: 302

[92] Shi T, Zhou J, Xue A, Lu H, He Y, Yu Y. Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins. Biotechnol Biofuels, 2021, 14: 236

[93] Sibirny AA. Yeast peroxisomes: structure, functions and biotechnological opportunities. FEMS Yeast Res, 2016, 16: fow038

[94] Sinha J, Plantz BA, Inan M, Meagher MM. Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-τ. Biotechnol Bioeng, 2005, 89: 102-112

[95] Slininger PJ, Shea-Andersh MA, Thompson SR, Dien BS, Kurtzman CP, Balan V, Sousa CL, et al. . Evolved strains of Scheffersomyces stipitis achieving high ethanol productivity on acid-and base-pretreated biomass hydrolyzate at high solids loading. Biotechnol Biofuels, 2015, 8: 60

[96] Sousa-Silva M, Vieira D, Soares P, Casal M, Soares-Silva I. Expanding the knowledge on the skillful yeast Cyberlindnera jadinii. J Fungi, 2021, 7: 36

[97] de Souza Varize C, Maria Christofoleti-Furlan R, de Souza Miranda Muynarsk E, Vinícius de Melo Pereira G, Dantas Lopes L, Carlos Basso L. Biotechnological applications of non-conventional yeasts. Yeasts in Biotechnol. IntechOpen; 2019.

[98] Spohner SC, Schaum V, Quitmann H, Czermak P. Kluyveromyces lactis: an emerging tool in biotechnology. J Biotechnol, 2016, 222: 104-116

[99] Stöckmann C, Scheidle M, Dittrich B, Merckelbach A, Hehmann G, Melmer G, et al. . Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment. Microb Cell Fact, 2009, 8: 22

[100] Streck AF, Canal CW, Truyen U. Molecular epidemiology evolution of porcine parvoviruses. Infect Genet Evol, 2015, 36: 300-306

[101] Su C, Li L, Jin Z, Han X, Zhao P, Wang L, et al. . Fermentation, purification and immunogenicity evaluation of hepatitis E virus-like particles expressed in Hansenula polymorpha. Chin J Biotechnol, 2017, 33: 653-663

[102] Suppi S, Michelson T, Viigand K, Alamäe T. Repression vs. activation of MOX, FMD, MPP1 and MAL1 promoters by sugars in Hansenula polymorpha: the outcome depends on cell’s ability to phosphorylate sugar. FEMS Yeast Res, 2013, 13: 219-232

[103] Swinkels BW, van Ooyen AJ, Bonekamp FJ. The yeast Kluyveromyces lactis as an efficient host for heterologous expression. Antonie Van Leeuwenhoek, 1993, 64: 187-201

[104] Theron CW, Vandermies M, Telek S, Steels S, Fickers P. Comprehensive comparison of Yarrowia lipolytica and Pichia pastoris for production of Candida antarctica lipase B. Sci Rep, 2020, 10: 1741

[105] Thomas KC, Hynes SH, Ingledew WM. Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids. Appl Environ Microbiol, 2002, 68: 1616-1623

[106] Tong L, Huang H, Zheng J, Wang X, Bai Y, Wang X, et al. . Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris. Microb Cell Fact, 2022, 21: 95

[107] Tsuda M, Nonaka K. Recent progress on heterologous protein production in methylotrophic yeast systems. World J Microbiol Biotechnol, 2024, 40: 200

[108] Unrean P, Nguyen NHA. Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments. Biotechnol Prog, 2012, 28: 1119-1125

[109] Vai M, Brambilla L, Orlandi I, Rota N, Ranzi BM, Alberghina L, Porro D. Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells. Appl Environ Microbiol, 2000, 66: 5477-5479

[110] Van den Berg JA, van der Laken KJ, van Ooyen AJ, Renniers TC, Rietveld K, Schaap A, Brake AJ, Bishop RJ, Schultz K, Moyer D, Richman M. Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/technol, 1990, 8(2): 135-139

[111] Van Ooyen AJJ, Dekker P, Huang M, Olsthoorn MMA, Jacobs DI, Colussi PA, et al. . Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res, 2006, 6: 381-392

[112] van Zutphen T, Baerends RJS, Susanna KA, de Jong A, Kuipers OP, Veenhuis M, et al. . Adaptation of Hansenula polymorpha to methanol: a transcriptome analysis. BMC Genomics, 2010, 11: 1

[113] Vanderwaeren L, Dok R, Voordeckers K, Nuyts S, Verstrepen KJ. Saccharomyces cerevisiae as a model system for eukaryotic cell biology, from cell cycle control to DNA damage response. Int J Mol Sci, 2022, 23: 11665

[114] Vasylyshyn R, Dmytruk O, Sybirnyy A, Ruchała J. Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose. FEMS Yeast Res, 2024, 24: foae07

[115] Vivier D, Ratomahenina R, Moulin G, Galzy P. Study of physicochemical factors limiting the growth of Kluyveromyces marxianus. J Ind Microbiol, 1993, 11: 157-161

[116] Wang W, Blenner MA. Engineering heterologous enzyme secretion in Yarrowia lipolytica. Microb Cell Fact, 2022, 21: 134

[117] Wang H, Song HL, Wang Q, Qiu BS. Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha. World J Microbiol Biotechnol, 2013, 29: 447-458

[118] Wang DH, Zhang JL, Dong YY, Wei GY, Qi B. Glutathione is involved in physiological response of Candida utilis to acid stress. Appl Microbiol Biotechnol, 2015, 99: 10669-10679

[119] Wang W, Wang X, Tan Y, Zhao S, Zhao L, Zhu Z. CRISPR-Cas9 mediated genome editing of Kluyveromyces marxianus for iterative, multiplexed gene disruption and pathway integration. Biotechnol Bioeng, 2024, 121: 3269-3282

[120] Wefelmeier K, Schmitz S, Haut AM, Otten J, Jülich T, Blank LM. Engineering the methylotrophic yeast Ogataea polymorpha for lactate production from methanol. Front Bioeng Biotechnol, 2023, 11: 1223726

[121] Wu L, Wang M, Zha G, Zhou J, Yu Y, Lu H. Improving the expression of a heterologous protein by genome shuffling in Kluyveromyces marxianus. J Biotechnol, 2020, 320: 11-16

[122] Wu P, Mo W, Tian T, Song K, Lyu Y, Ren H, Zhou J, Yu Y, Lu H. Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus. Mlife, 2024, 3(1): 129-142

[123] Wu J, Ohnuki S, Teyssonnière É, Yasukawa T, Masuo N, Schacherer J, et al. . Genetic and phenotypic insights into Cyberlindnera jadinii as a promising yeast for industrial biotechnology. G3: Genes, Genomes, Genet, 2025, 15: 145

[124] Yan J, Han B, Gui X, Wang G, Xu L, Yan Y, et al. . Engineering Yarrowia lipolytica to simultaneously produce lipase and single-cell protein from agro-industrial wastes for feed. Sci Rep, 2018, 8: 758

[125] Yan C, Yu W, Zhai X, Yao L, Guo X, Gao J, et al. . Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha. Synth Syst Biotechnol, 2022, 7: 498-505

[126] Yang D, Chen L, Duan J, Yu Y, Zhou J, Lu H. Investigation of Kluyveromyces marxianus as a novel host for large-scale production of porcine parvovirus virus-like particles. Microb Cell Fact, 2021, 20: 24

[127] Yarimizu T, Nakamura M, Hoshida H, Akada R. Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus. Microb Cell Fact, 2015, 14 20

[128] Ye M, Gao J, Zhou YJ. Global metabolic rewiring of the non-conventional yeast Ogataea polymorpha for biosynthesis of the sesquiterpenoid β-elemene. Metab Eng, 2023, 76: 225-231

[129] Youn JK, Shang L, Kim MI, Jeong CM, Chang HN, Hahm MS, Rhee SK, Kang HA. Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen. J Microb Biotechnol, 2010, 20(11): 1534-1538

[130] Yu W, Gao J, Zhai X, Zhou YJ. Screening neutral sites for metabolic engineering of methylotrophic yeast Ogataea polymorpha. Synth Syst Biotechnol, 2021, 6: 63-68

[131] Zhang Y, Li YF, Chi Z, Liu GL, Jiang H, Hu Z, et al. . Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis. Ann Microbiol, 2019, 69: 647-657

[132] Zhang J, Xu T, Wang X, Jing X, Zhang J, Hong J, et al. . Lignocellulosic xylitol production from corncob using engineered Kluyveromyces marxianus. Front Bioeng Biotechnol, 2022, 10: 1029203

[133] Zhao Y, Liu S, Lu Z, Zhao B, Wang S, Zhang C, et al. . Hybrid promoter engineering strategies in Yarrowia lipolytica: Isoamyl alcohol production as a test study. Biotechnol Biofuels, 2021, 14: 149

[134] Zhao M, Ma J, Zhang L, Qi H. Engineering strategies for enhanced heterologous protein production by Saccharomyces cerevisiae. Microb Cell Fact, 2024, 23 32

[135] Zhong Y, Yang L, Guo Y, Fang F, Wang D, Li R, Jiang M, et al. . High-temperature cultivation of recombinant Pichia pastoris increases endoplasmic reticulum stress and decreases production of human interleukin-10. Microb Cell Fact, 2014, 13: 163

[136] Zhou WJ, Yang JK, Mao L, Miao LH. Codon optimization, promoter and expression system selection that achieved high-level production of Yarrowia lipolytica lipase in Pichia pastoris. Enzyme Microb Technol, 2015, 71: 66-72

[137] Zhou J, Zhu P, Hu X, Lu H, Yu Y. Improved secretory expression of lignocellulolytic enzymes in Kluyveromyces marxianus by promoter and signal sequence engineering. Biotechnol Biofuels, 2018, 11: 235

[138] Zhou S, Wu P, Ren H, Zhou J, Yu Y, Lu H. Characterization and optimization of mnn11Δ-mediated enhancement in heterologous protein production in Kluyveromyces marxianus. Microb Cell Fact, 2025, 24 50

[139] Zhu X, Li M, Zhu R, Xin Y, Guo Z, Gu Z, et al. . Up front unfolded protein response combined with early protein secretion pathway engineering in Yarrowia lipolytica to attenuate ER stress caused by enzyme overproduction. Int J Mol Sci, 2023, 24: 16426

Funding

Symbiosis International (Deemed University)

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