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Background <p><i>Bacillus methanolicus</i> MGA3 is a thermotolerant methylotroph that utilizes methanol, a renewable C₁ substrate, as its sole carbon and energy source. The strain naturally overproduces and secretes L-glutamate, making it a promising platform for engineering pathways toward L-glutamate-derived amino acids such as L-proline, which has applications in nutrition, stress protection, and industry.</p> Results <p>Heterologous expression of an osmotic stress–responsive L-proline biosynthetic operon from the mesophile <i>Bacillus licheniformis</i> in the thermotolerant <i>B. methanolicus</i> strain MGA3 did not increase L-proline levels but instead led to accumulation of L-citrulline. This was likely due to heat sensitivity of pyrroline-5-carboxylate reductase (ProH), the last enzyme of the osmoregulatory L-proline biosynthetic route, and metabolic crosstalk between L-proline and L-arginine pathways operating in Bacilli. To overcome these limitations, a synthetic operon containing the native anabolic <i>proBA</i> and <i>proI</i> L-proline biosynthetic genes from <i>B. methanolicus</i> MGA3 was engineered to remove transcriptional T-box regulation and biochemical feedback inhibition of ProB enzyme activity. Expression of this engineered operon enhanced L-proline synthesis and triggered its secretion during methanol-based growth of <i>B. methanolicus</i> MGA3 at 50° C. In fed-batch fermentation with methanol as carbon and energy source, extracellular L-proline levels reached 262 ± 20&#xa0;mg L⁻<sup>1</sup> after 40&#xa0;h. During the fermentation process, a stepwise increase in medium osmolarity was observed, likely due to large-scale L-glutamate excretion, which impaired cellular growth.</p> Conclusions <p>This study links osmolarity dynamics to methanol-based fermentation in <i>B. methanolicus</i> MGA3 and demonstrates its potential as a cell factory for L-proline and L-citrulline production. These findings support further strain optimization for producing value-added amino acids and highlight the relevance of methylotrophic thermophiles in sustainable biotechnology.</p>

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Harnessing the thermotolerant methylotroph Bacillus methanolicus for methanol-based synthetic L-proline production

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月23日

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  • Christine Frank,
  • David Virant,
  • Gregor Kosec,
  • Tamara Hoffmann,
  • Oskar Zelder,
  • Max F. Felle,
  • Erhard Bremer

摘要

Background

Bacillus methanolicus MGA3 is a thermotolerant methylotroph that utilizes methanol, a renewable C₁ substrate, as its sole carbon and energy source. The strain naturally overproduces and secretes L-glutamate, making it a promising platform for engineering pathways toward L-glutamate-derived amino acids such as L-proline, which has applications in nutrition, stress protection, and industry.

Results

Heterologous expression of an osmotic stress–responsive L-proline biosynthetic operon from the mesophile Bacillus licheniformis in the thermotolerant B. methanolicus strain MGA3 did not increase L-proline levels but instead led to accumulation of L-citrulline. This was likely due to heat sensitivity of pyrroline-5-carboxylate reductase (ProH), the last enzyme of the osmoregulatory L-proline biosynthetic route, and metabolic crosstalk between L-proline and L-arginine pathways operating in Bacilli. To overcome these limitations, a synthetic operon containing the native anabolic proBA and proI L-proline biosynthetic genes from B. methanolicus MGA3 was engineered to remove transcriptional T-box regulation and biochemical feedback inhibition of ProB enzyme activity. Expression of this engineered operon enhanced L-proline synthesis and triggered its secretion during methanol-based growth of B. methanolicus MGA3 at 50° C. In fed-batch fermentation with methanol as carbon and energy source, extracellular L-proline levels reached 262 ± 20 mg L⁻1 after 40 h. During the fermentation process, a stepwise increase in medium osmolarity was observed, likely due to large-scale L-glutamate excretion, which impaired cellular growth.

Conclusions

This study links osmolarity dynamics to methanol-based fermentation in B. methanolicus MGA3 and demonstrates its potential as a cell factory for L-proline and L-citrulline production. These findings support further strain optimization for producing value-added amino acids and highlight the relevance of methylotrophic thermophiles in sustainable biotechnology.