Effect of weakening 1,3-β-glucan synthesis on sophorolipids biosynthesis in Komagataella phaffii

Abstract

Komagataella phaffii (formerly Pichia pastoris) is a widely used host for heterologous protein expression and biotransformation, and simplifying its cell wall polysaccharides is a promising strategy for designing advanced chassis strains. Previously, we constructed two superior chassis hosts by inactivating the β-glucan biosynthesis genes PAS_chr1-3_0225 and PAS_chr1-3_0661. In this study, PAS_chr2-1_0263 gene responsible for β-glucan synthase was inactivated to investigate the impact of β-glucan deficiency on sophorolipid (SL) biosynthesis. Furthermore, we systematically evaluated the combined effects of this mutation and faa1 inactivation on SL production and host performance. Firstly, the SL biosynthesis-related genes (comprising cyp52M1, ugtA1, ugtB1, sble, at, and mdr) were overexpressed for the first time, demonstrating that P_GAP-driven K. phaffii GS115 can synthesize seven structural types of SLs with a total titer of 4.09 g/L. Subsequently, to facilitate subsequent gene editing, the DNA repair-related genes ku70 and mph1 were deleted; this deletion did not depress SL production. PAS_chr2-1_0263 was then knocked out to obtain a novel chassis host, and the SL productivity, oil-to-SLs ratio, and precursor UDP-glucose level were systematically investigated. The results showed that knocking out PAS_chr2-1_0263 reduced glucan content by 24.21% while increasing total SLs to 7.69 g/L, a 43.8% increase, and improving the conversion ratios of both oil and glucose to SLs. Moreover, the combined deletion of PAS_chr2-1_0263 and faa1, which encodes fatty acid acyl-CoA synthase, further elevated the SL titer to 11.53 g/L and achieved even higher glucose-to-SLs and oil-to-SLs conversion rates. These findings indicate that weakening 1,3-β-glucan synthesis not only improves the utilization ratio of glucose but also increases intracellular UDP-glucose levels, both of which contribute to enhanced SL biosynthesis. This study demonstrates for the first time that attenuating 1,3-β-glucan synthesis in K. phaffii is an effective strategy to boost SL biosynthesis and improve host performance, and this novel chassis possesses excellent potential for the biotransformation of other glycolipids.

Keywords

Komagataella phaffii / Sophorolipids / 1,3-β-glucan synthase / Fatty acid / UDP-glucose

References

[1] Barone G, Emmerstorfer-Augustin A, Biundo A, Pisano I, Coccetti P, Mapelli V, et al.. Industrial production of proteins with Pichia pastoris-Komagataella phaffii. Biomolecules, 2023, 13(3): 441.

[2] Barrero J, Casler J, Valero F, Ferrer P, Glick B. An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microb Cell Fact. 2018;17. https://doi.org/10.1186/s12934-018-1009-5.

[3] Bhangale A, Wadekar S, Kale S, Bhowmick D, Pratap A. Production of sophorolipids synthesized on castor oil with glucose and glycerol by using Starmerella bombicola (ATCC 22214). Eur J Lipid Sci Technol, 2014, 116(3): 336-43.

[4] Cai P, Duan X, Wu X, Gao L, Ye M, Zhou Y. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Res, 2021, 49(13): 7791-805.

[5] Cai P, Wu X, Deng J, Gao L, Shen Y, Yao L, et al.. Methanol biotransformation toward high-level production of fatty acid derivatives by engineering the industrial yeast Pichia pastoris. Proc Natl Acad Sci USA, 2022, 119(29): 1-26.

[6] Carlos Fragoso-Jimenez J, Gutierrez-Rios M, Flores R, Martinez N, Lara A, Delvigne AR. Glucose consumption rate-dependent transcriptome profiling of Escherichia coli provides insight on performance as microbial factories. Microb Cell Fact. 2022;21(1). https://doi.org/10.1186/s12934-022-01909-y.

[7] Cheng B, Yu K, Weng X, Liu Z, Huang X, Jiang Y, et al.. Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications. Microb Cell Fact, 2024, 23(1): 55.

[8] Chen N, Yang S, You D, Shen J, Ruan B, Wu M, et al.. Systematic genetic modifications of cell wall biosynthesis enhanced the secretion and surface-display of polysaccharide degrading enzymes in Saccharomyces cerevisiae. Metab Eng, 2023, 77: 273-82.

[9] Ciesielska K, Van Bogaert I, Chevineau S, Li B, Groeneboer S, Soetaert W, et al.. Exoproteome analysis of Starmerella bombicola results in the discovery of an esterase required for lactonization of sophorolipids. J Proteom, 2014, 98: 159-74.

[10] Deshpande M, Daniels L. Evaluation of sophorolipid biosurfactant production by Candida bombicola using animal fat. Bioresour Technol, 1995, 54(2): 143-50.

[11] Elshafie A, Joshi S, Al-Wahaibi Y, Al-Bemani A, Al-Bahry S, Al-Maqbali D, et al.. Sophorolipids production by Candida bombicola ATCC 22214 and its potential application in microbial enhanced oil recovery. Front Microbiol, 2015, 6: 1324.

[12] Ferrari M, Rawal C, Lodovichi S, Vietri M, Pellicioli A. Rad9/53BP1 promotes DNA repair via crossover recombination by limiting the Sgs1 and Mph1 helicases. Nat Commun, 2020, 11(1): 1-11.

[13] García-Ortega X, Camara E, Ferrer P, Albiol J, Montesinos-Seguí J, Valero F. Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand?. New Biotechnol, 2019, 53: 24-34.

[14] Gonzalez-Ramos D, Gonzalez R. Genetic determinants of the release of mannoproteins of enological interest by Saccharomyces cerevisiae. J Agric Food Chem, 2006, 54(25): 9411-16.

[15] Gorin PAJ, Spencer JF, T.,Tulloch AP. Hydroxy fatty acid glycosides of sophorose from Torulopsis magnoliae. Can J Chem, 1961, 39(4): 846-55.

[16] Ichihara K, Fukubayashi Y. Preparation of fatty acid methyl esters for gas-liquid chromatography. J Lipid Res, 2010, 51(3): 635-40.

[17] Ingham B, Hollywood K, Wongsirichot P, Veitch A, Winterburn J. Uncovering the fragmentation and separation characteristics of sophorolipid biosurfactants with LC-MS-ESI. J Ind Microbiol Biotechnol, 2024, 51: kuae035.

[18] Jezierska S, Claus S, Ledesma-Amaro R, Van Bogaert I. Redirecting the lipid metabolism of the yeast Starmerella bombicola from glycolipid to fatty acid production. J Ind Microbiol Biotechnol, 2019, 46(12): 1697-706.

[19] Jiang SLH, Zhang L, et al.. Generic Diagramming Platform (GDP): a comprehensive database of high-quality biomedical graphics. Nucleic Acids Res, 2025, 53(D1): D1670-76.

[20] Johnson M, Edlind T. Topological and mutational analysis of Saccharomyces cerevisiae Fks1. Eukaryot Cell, 2012, 11(7): 952-60.

[21] Kumanski S, Forey R, Cazevieille C, Moriel-Carretero M. Nuclear lipid droplet birth during replicative stress. Cells. 2022;11(9). https://doi.org/10.3390/cells11091390.

[22] Lan C, Zhao B, Yang L, Zhou Y, Guo S, Zhang X, et al.. Determination of UDP-glucose and UDP-galactose in maize by hydrophilic interaction liquid chromatography and tandem mass spectrometry. J Anal Methods Chem, 2022, 2022: 7015311.

[23] Lin Cereghino GP, Sunga AJ, Lin Cereghino J, Cregg JM. Expression of foreign genes in the yeast Pichia pastoris. Genet Eng, 2001, 23: 157-69.

[24] Li S, Qian X, Xu L, Xu N, Liu S, Xu A, et al.. Biological tailoring of novel sophorolipid molecules and their derivatives. Biofuels Bioprod Biorefining-Biofpr, 2021, 15(6): 1938-49.

[25] Liu J, Zhang X, Liu G, Zhao G, Fang X, Song X. A Cumulative Effect by Multiple-Gene Knockout Strategy Leads to a Significant Increase in the Production of Sophorolipids in Starmerella bombicola CGMCC 1576. Front Bioeng Biotechnol, 2022, 10: 818445.

[26] Li X, Yu W, Lyu X, Liu Y, Li J, Du G, et al.. Metabolic engineering of Saccharomyces cerevisiae to synthesize p-glucan efficiently. Food Ferment Industries, 2022, 48: 1-7.

[27] Ma Y, Chen X, Shen W, Yang H, Zhou L, Cao Y, et al.. Metabolic engineering of Starmerella bombicola for the production of sophorolipids. Syst Microbiol Biomanufacturing, 2025, 5(4): 1634-42.

[28] Peña D, Gasser B, Zanghellini J, Steiger M, Mattanovich D. Metabolic engineering of Pichia pastoris. Metab Eng, 2018, 50: 2-15.

[29] Qazi M, Wang Q, Dai Z. Sophorolipids bioproduction in the yeast Starmerella bombicola: Current trends and perspectives. Bioresour Technol, 2022, 346: 126593.

[30] Ribeiro I, Bronze M, Castro M, Ribeiro M. Optimization and correlation of HPLC-ELSD and HPLC-MS/MS methods for identification and characterization of sophorolipids. J Chromatogr B-Analytical Technol Biomedical Life Sci, 2012, 899: 72-80.

[31] Rocha T, Marcelino P, Munoz S, Ruiz E, Balbino T, Moraes E, et al.. Utilization of renewable feedstocks for the production of sophorolipids by native yeasts from brazilian cerrado biome. Bioenergy Res, 2023, 16(4): 1956-72.

[32] Roelants S, Ciesielska K, De Maeseneire S, Moens H, Everaert B, Verweire S, et al.. Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola. Biotechnol Bioeng, 2016, 113(3): 550-59.

[33] Romanauska A, Koehler A. The inner nuclear membrane is a metabolically active territory that generates nuclear lipid droplets. Cell, 2018, 174(3): 700.

[34] Saerens K, Roelants S, Van Bogaert I, Soetaert W. Identification of the UDP-glucosyltransferase gene UGTA1, responsible for the first glucosylation step in the sophorolipid biosynthetic pathway of Candida bombicola ATCC 22214. FEMS Yeast Res, 2011, 11(1): 123-32.

[35] Saerens K, Saey L, Soetaert W. One-Step production of unacetylated sophorolipids by an acetyltransferase negative Candida bombicola. Biotechnol Bioeng, 2011, 108(12): 2923-31.

[36] Saerens K, Zhang J, Saey L, Van Bogaert I, Soetaert W. Cloning and functional characterization of the UDP-glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid-producing yeast strain. Yeast, 2011, 28(4): 279-92.

[37] Schimoler-O’Rourke R, Renault S, Mo W, Selitrennikoff C. Neurospora crassa FKS protein binds to the (1,3)β-glucan synthase substrate, UDP-glucose. Curr Microbiol, 2003, 46(6): 408-12.

[38] SHAW HONGZMANNP, K.,DIDOMENICO B. Analysis of β-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography. Yeast, 1994, 10(8): 1083-92.

[39] Takahashi F, Igarashi K, Hagihara H. Identification of the fatty alcohol oxidase FAO1 from Starmerella bombicola and improved novel glycolipids production in an FAO1 knockout mutant. Appl Microbiol Biotechnol, 2016, 100(22): 9519-28.

[40] Tamm T, Kristjuhan A. Protocol for rapid and cost-effective extraction of genomic DNA from a wide range of wild yeast species for use in PCR-based applications. Star Protocols. 2024;5(3). https://doi.org/10.1016/j.xpro.2024.103282.

[41] Van Bogaert I, Buyst D, Martins J, Roelants S, Soetaert W. Synthesis of bolaform biosurfactants by an engineered Starmerella bombicola yeast. Biotechnol Bioeng, 2016, 113(12): 2644-51.

[42] Van Bogaert I, De Mey M, Develter D, Soetaert W, Vandamme E. Importance of the cytochrome P450 monooxygenase CYP52 family for the sophorolipid-producing yeast Candida bombicola. FEMS Yeast Res, 2009, 9(1): 87-94.

[43] Van Bogaert I, Holvoet K, Roelants S, Li B, Lin Y, Van de Peer Y, et al.. The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by Starmerella bombicola. Mol Microbiol, 2013, 88(3): 501-09.

[44] Van Bogaert I, Zhang J, Soetaert W. Microbial synthesis of sophorolipids. Process Biochem, 2011, 46(4): 821-33.

[45] Wang J, Mao J, Yang G, Zheng F, Niu C, Li Y, et al.. The FKS family genes cause changes in cell wall morphology resulted in regulation of anti-autolytic ability in Saccharomyces cerevisiae. Bioresour Technol, 2018, 249: 49-56.

[46] Weninger A, Fischer J, Raschmanová H, Kniely C, Vogl T, Glieder A. Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers. J Cell Biochem, 2018, 119(4): 3183-98.

[47] Weninger A, Hatzl A, Schmid C, Vogl T, Glieder A. Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J Biotechnol, 2016, 235: 139-49.

[48] Xia Y, Shi Y, Chu J, Zhu S, Luo X, Shen W, et al.. Efficient biosynthesis of acidic/lactonic sophorolipids and their application in the remediation of cyanobacterial harmful algal blooms. Int J Mol Sci, 2023, 24(15): 12389.

[49] Zhou YJ, Buijs NA, Zhu Z, Qin J, Siewers V, Nielsen J. Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories. Nat Commun, 2016, 7: 11709.

[50] Zhu T, Sun H, Wang M, Li Y. Pichia pastoris as a versatile cell factory for the production of industrial enzymes and chemicals: current status and future perspectives. Biotechnol J, 2019, 14(6): 1800694.

[51] Zulkifli W, Razak N, Yatim A, Hayes D. Acid precipitation versus solvent extraction: Two techniques leading to different lactone/acidic sophorolipid ratios. J Surfactants Deterg, 2019, 22(2): 365-71.

Funding

National Key Research and Development Program of China(2023YFE0104400)

RIGHTS & PERMISSIONS

Jiangnan University