Metabolic engineering of Starmerella bombicola for the production of sophorolipids

Abstract

Sophorolipids (SLs), a class of glycolipid biosurfactants, are naturally synthesized by Starmerella bombicola. Composed of sophorose and fatty acids, SLs exhibit excellent emulsification, reduced surface tension, low toxicity, and high biodegradability, making them promising for applications in food, detergent, and agricultural industries. In this study, we screened a S. bombicola capable of efficiently and stably synthesizing SLs through multiple rounds of rejuvenation as the wild-type strain. First, we knocked out PXA1, a gene critical for fatty acid β-oxidation (a competing pathway for SLs production). This weakened β-oxidation and generated the P1 strain. The SLs titer of the P1 strain reached 66.96 ± 4.29 g/L, representing a 50.5% increase compared to the wild-type strain (48.11 ± 3.50 g/L). Subsequently, by enhancing the expression of CYP52M1, a key enzyme in the fatty acid ω-oxidation pathway, we constructed the PC1 strain, which achieved an SLs titer of 88.50 ± 4.91 g/L, a 98.8% improvement over the wild-type strain. Finally, we scaled up the fermentation of the PC1 strain in a 5 L fermenter, and through fed-batch fermentation, the SLs titer reached 232.27 ± 13.83 g/L on the 7th day. This study shows that engineered S. bombicola strains can efficiently produce high SLs titers, making large-scale biosynthesis feasible.

Keywords

Sophorolipids / Starmerella bombicola / Metabolic engineering / β-oxidation / Uridine glucose diphosphate

References

[1] Thundiparambil Venu A, Vijayan J, Ammanamveetil MHA, Kottekkattu Padinchati K. An insightful overview of microbial biosurfactant: A promising Next-Generation biomolecule for sustainable future. J Basic Microbiol, 2024, 64: e2300757.

[2] Pal S, Chatterjee N, Das AK, McClements DJ, Dhar P. Sophorolipids: A comprehensive review on properties and applications. Adv Colloid Interface Sci, 2023, 313: 102856.

[3] De Clercq V, Roelants S, Castelein MG, De Maeseneire SL, Soetaert WK. Elucidation of the natural function of sophorolipids produced by Starmerella Bombicola. J Fungi, 2021, 7: 917.

[4] Dierickx S, Castelein M, Remmery J, De Clercq V, Lodens S. et al.. From bumblebee to bioeconomy: recent developments and perspectives for sophorolipid biosynthesis. Biotechnol Adv, 2022, 54: 107788.

[5] De Graeve M, De Maeseneire SL, Roelants S, Soetaert W. Starmerella Bombicola, an industrially relevant, yet fundamentally underexplored yeast. FEMS Yeast Res, 2018, 18: foy072.

[6] Qazi MA, Wang Q, Dai Z. Sophorolipids bioproduction in the yeast Starmerella Bombicola: current trends and perspectives. Bioresour Technol, 2022, 346: 126593.

[7] Huang FC, Peter A, Schwab W. Expression and characterization of CYP52 genes involved in the biosynthesis of sophorolipid and alkane metabolism from Starmerella Bombicola. Appl Environ Microbiol, 2014, 80: 766-76.

[8] Saerens KM, Roelants SL, Van Bogaert IN, 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: 123-32.

[9] Saerens KM, Zhang J, Saey L, Van Bogaert IN, 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: 279-92.

[10] Saerens KM, Saey L, Soetaert W. One-step production of unacetylated sophorolipids by an acetyltransferase negative Candida Bombicola. Biotechnol Bioeng, 2011, 108: 2923-31.

[11] Van Bogaert IN, Holvoet K, Roelants SL, Li B, Lin YC. et al.. The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by Starmerella Bombicola. Mol Microbiol, 2013, 88: 501-9.

[12] Ciesielska K, Roelants SL, Van Bogaert IN, De Waele S, Vandenberghe I. et al.. Characterization of a novel enzyme-Starmerella Bombicola lactone esterase (SBLE)-responsible for sophorolipid lactonization. Appl Microbiol Biotechnol, 2016, 100: 9529-41.

[13] Hirata Y, Igarashi K, Ueda A, Quan GL. Enhanced sophorolipid production and effective conversion of waste frying oil using dual lipophilic substrates. Biosci Biotechnol Biochem, 2021, 85: 1763-71.

[14] Kim YB, Yun HS, Kim EK. Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture. Bioresour Technol, 2009, 100: 6028-32.

[15] Zhou G, Tian X, Lin Y, Zhang S, Chu J. Rational high-throughput system for screening of high sophorolipids-producing strains of Candida Bombicola. Bioprocess Biosyst Eng, 2019, 42: 575-82.

[16] Zhang Y, Jia D, Sun W, Yang X, Zhang C. et al.. Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve-plates coupled with a novel separation system. Microb Biotechnol, 2018, 11: 455-64.

[17] Chen Y, Tang X, Li Y, Liu C, Zhuang Y. et al.. Ultrasound assisted in situ separation of sophorolipids in multi-phase fermentation system to achieve efficient production by Candida Bombicola. Biotechnol J, 2022, 17: e2100478.

[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: 1697-706.

[19] Zhang J, Zhang L, Shi Y, Shen W, Fan Y, Chen X. Metabolic engineering of Starmerella Bombicola for enhanced acid-type sophorolipid production. Acta Microbiol Sinica, 2019, 59: 2094-106

[20] Van Bogaert IN, Sabirova J, Develter D, Soetaert W, Vandamme EJ. Knocking out the MFE-2 gene of Candida Bombicola leads to improved medium-chain sophorolipid production. FEMS Yeast Res, 2009, 9: 610-7.

[21] Shi Y, Zhang L, Zhang M, Chu J, Xia Y. et al.. A CRISPR-Cas9 System-Mediated genetic disruption and Multi-fragment assembly in Starmerella Bombicola. ACS Synth Biol, 2022, 11: 1497-509.

[22] Yang L, Li Y, Zhang X, Liu T, Chen J. et al.. Metabolic profiling and flux distributions reveal a key role of acetyl-CoA in sophorolipid synthesis by Candida Bombicola. Biochem Eng J, 2019, 145: 74-82.

[23] Biggs BW, Lim CG, Sagliani K, Shankar S, Stephanopoulos G. et al.. Overcoming heterologous protein interdependency to optimize P450-mediated taxol precursor synthesis in Escherichia coli. Proc Natl Acad Sci U S A, 2016, 113: 3209-14.

[24] Sellner M, Fischer A, Don CG, Smieško M. Conformational landscape of cytochrome P450 reductase interactions. Int J Mol Sci, 2021, 22: 1023.

[25] Wang JH, Wang D, Li WX, Huang Y, Dai ZB, Zhang XL. [Optimization of UDP-glucose supply module and production of ginsenoside F(1) in Saccharomyces cerevisiae]. Zhongguo Zhong Yao Za Zhi, 2019, 44: 4596-604

[26] National Center for Biotechnology Information. PubChem Patent Summary for US-2018208935-A1, Sophorolipid Highly-Productive Mutant Strain. Retrieved April 15, 2025 from https://pubchem.ncbi.nlm.nih.gov/patent/US-2018208935-A1.

[27] Liu J, Li J, Gao N, Zhang X, Zhao G, Song X. Identification and characterization of a protein Bro1 essential for sophorolipids synthesis in Starmerella bombicola. J Ind Microbiol Biotechnol. 2020;47:437–48.

Funding

the Key Research and Development Program of China(2024YFA0917901-05)

the Key Research and Development Program of China (2023YFC3402400)

Innovative Research Group Project of the National Natural Science Foundation of China(No. 32271533)

RIGHTS & PERMISSIONS

Jiangnan University