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Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium

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Abstract

High-yielding chemical and chemo-enzymatic methods of D-pantothenic acid (DPA) synthesis are limited by using poisonous chemicals and DL-pantolactone racemic mixture formation. Alternatively, the safe microbial fermentative route of DPA production was found promising but suffered from low productivity and precursor supplementation. In this study, Bacillus megaterium was metabolically engineered to produce DPA without precursor supplementation. In order to provide a higher supply of precursor D-pantoic acid, key genes involved in its synthesis are overexpressed, resulting strain was produced 0.53 ± 0.08 g/L DPA was attained in shake flasks. Cofactor CH2-THF was found to be vital for DPA biosynthesis and was regenerated through the serine-glycine degradation pathway. Enhanced supply of another precursor, β-alanine was achieved by codon optimization and dosing of the limiting L-asparate-1-decarboxylase (ADC). Co-expression of Pantoate-β-alanine ligase, ADC, phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate ammonia-lyase enhanced DPA concentration to 2.56 ± 0.05 g/L at shake flasks level. Fed-batch fermentation in a bioreactor with and without the supplementation of β-alanine increased DPA concentration to 19.52 ± 0.26 and 4.78 ± 0.53 g/L, respectively. This present study successfully demonstrated a rational approach combining precursor supply engineering with cofactor regeneration for the enhancement of DPA titer in recombinant B. megaterium.

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Acknowledgements

Funding from the Department of Biotechnology, Govt. of India (Grant No: BT/PR15946/NER/95/485/2016) is gratefully acknowledged. Authors acknowledge the Ministry of Human Resource and Development, New Delhi, India, for fellowship.

Funding

This study was financially supported by Department of Biotechnology, Govt. of India (Grant No: BT/PR15946/NER/95/485/2016). Authors acknowledge Ministry of Human Resource and Development, New Delhi, India for fellowship.

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Authors and Affiliations

  1. BioPAT Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, Assam, 781039, India

    Subbi Rami Reddy Tadi, Ganesh Nehru, Satya Sai Pavan Allampalli & Senthilkumar Sivaprakasam

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  1. Subbi Rami Reddy Tadi
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  2. Ganesh Nehru
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  3. Satya Sai Pavan Allampalli
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  4. Senthilkumar Sivaprakasam
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SRRT: conceptualization, methodology, investigation, writing—original draft of the manuscript, review and editing. GN: methodology and investigation support, review and editing, SSPA: methodology and investigation support, review and editing, SS: project administration, Supervision, writing—review and editing.

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Correspondence to Senthilkumar Sivaprakasam.

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Tadi, S.R.R., Nehru, G., Allampalli, S.S.P. et al. Engineering precursor and co-factor supply to enhance D-pantothenic acid production in Bacillus megaterium. Bioprocess Biosyst Eng 45, 843–854 (2022). https://doi.org/10.1007/s00449-022-02701-3

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