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Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

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High-Level 5-Methyltetrahydrofolate Bioproduction in Bacillus subtilis by Combining Modular Engineering and

Han Yang^1,2,3,4, Jinning Yang^1,2,3,4, Cheng Liu^1,2,3,4

¹Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.

Journal of Agricultural and Food Chemistry

|May 6, 2022

Summary

Engineered *Bacillus subtilis* now efficiently produces 5-Methyltetrahydrofolate (5-MTHF), a vital nutraceutical. This breakthrough in microbial synthesis offers a sustainable method for producing this important folate form.

Keywords:

5-methyltetrahydrofolate Bacillus subtilis CRISPRi technology modular pathway engineering transcriptome

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Light-Controlled Fermentations for Microbial Chemical and Protein Production

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Published on: March 22, 2022

Area of Science:

Biotechnology
Metabolic Engineering
Microbial Fermentation

Background:

5-Methyltetrahydrofolate (5-MTHF) is the primary folate in human plasma and a key nutraceutical.
Current microbial synthesis of 5-MTHF is inefficient, hindering sustainable production.

Purpose of the Study:

To engineer *Bacillus subtilis* for efficient microbial production of 5-MTHF.
To optimize precursor supply and identify key genes for enhanced 5-MTHF biosynthesis.

Main Methods:

Modular engineering to enhance guanosine-5-triphosphate (GTP) and p-aminobenzoic acid (pABA) supply.
Transcriptome analysis to identify key genes for 5-MTHF production.
Gene expression modulation using strong promoters and CRISPR interference (CRISPRi).

Main Results:

Optimized precursor supply modules in *B. subtilis*.
Identified and validated key genes, including *comGC*, for 5-MTHF biosynthesis.
Achieved a record microbial production of 3.41 ± 0.10 mg/L 5-MTHF with 0.21 mg/L/h productivity.

Conclusions:

Engineered *B. subtilis* provides a robust platform for sustainable 5-MTHF production.
This work establishes a foundation for further improvements in microbial 5-MTHF fermentation.
The produced 5-MTHF can be utilized as a food and nutraceutical ingredient.