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<p><i>Pseudomonas putida</i> is an attractive synthetic biology platform organism for chemical synthesis from low-grade feedstocks due to its high tolerance to chemical solvents and lignin-derived small molecules that are often inhibitory to other biotechnologically relevant microorganisms. However, there are few molecular tools available for engineering <i>P. putida</i> and other gram-negative bacteria to secrete non-native enzymes for extracellular feedstock depolymerisation. In this study, <i>P. putida</i> was transformed to secrete cellulase enzymes and evaluated for growth on polymeric or oligomeric cellulose substrates. Active exo- and endocellulase enzymes were secreted into the culture supernatant, and a preferred set of twin-arginine translocase secretion signal peptides were identified. Extracellular cellulase activity was sufficient to support growth of <i>P. putida</i> using cellotriose or cellotetraose as the sole source of carbon and energy. This work supports progress in engineering <i>P. putida</i> to catabolise extracellular polymers including cellulosic polymers, demonstrating functional secretion of large multi-domain enzymes into the culture medium.</p>

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Cellulase secretion by engineered Pseudomonas putida enables growth on cellulose oligomers

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月25日

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  • Madeline R. Smith,
  • Kaylee Moffitt,
  • William Holdsworth,
  • Carlos H. Luna-Flores,
  • Mansi Goyal,
  • Alex Beliaev,
  • Robert E. Speight,
  • James B. Behrendorff

摘要

Pseudomonas putida is an attractive synthetic biology platform organism for chemical synthesis from low-grade feedstocks due to its high tolerance to chemical solvents and lignin-derived small molecules that are often inhibitory to other biotechnologically relevant microorganisms. However, there are few molecular tools available for engineering P. putida and other gram-negative bacteria to secrete non-native enzymes for extracellular feedstock depolymerisation. In this study, P. putida was transformed to secrete cellulase enzymes and evaluated for growth on polymeric or oligomeric cellulose substrates. Active exo- and endocellulase enzymes were secreted into the culture supernatant, and a preferred set of twin-arginine translocase secretion signal peptides were identified. Extracellular cellulase activity was sufficient to support growth of P. putida using cellotriose or cellotetraose as the sole source of carbon and energy. This work supports progress in engineering P. putida to catabolise extracellular polymers including cellulosic polymers, demonstrating functional secretion of large multi-domain enzymes into the culture medium.