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Optimization of dilution rate and mixed carbon feed for continuous production of recombinant plant sucrose:sucrose 1-fructosyltransferase in Komagataella phaffii

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Abstract

The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h−1. The methanol–sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h−1 was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.

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Acknowledgements

The authors would like to acknowledge the help received from the staff members in the Bioprocess Laboratory (Department of Biochemical Engineering and Biotechnology) for reactor operations.

Funding

This work was funded through a research grant received from the Department of Biotechnology (Ministry of Science and Technology, Govt. of India), Grant number: BT/PR13813/BBE/117/59/2015). ASJ acknowledges the JRF fellowship received from the Department of Biotechnology.

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  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz-Khas, New-Delhi, 110016, India

    Avijeet S. Jaswal, Ravikrishnan Elangovan & Saroj Mishra

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Avijeet S. Jaswal: conceptualization, designing, methodology, conducting experiments, data analyses, writing—first draft, final draft. Ravikrishnan Elangovan: conceptualization, bioinformatics, data analyses, final draft preparation. Saroj Mishra: designing, data analyses, project fund acquirement and management, final draft preparation. All the authors read the final version and have approved the manuscript.

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Correspondence to Saroj Mishra.

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Jaswal, A.S., Elangovan, R. & Mishra, S. Optimization of dilution rate and mixed carbon feed for continuous production of recombinant plant sucrose:sucrose 1-fructosyltransferase in Komagataella phaffii. Bioprocess Biosyst Eng (2024). https://doi.org/10.1007/s00449-024-03045-w

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