A novel expression system for lytic polysaccharide monooxygenases.

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Lytic polysaccharide monooxygenases (LPMOs) are crucial enzymes for breaking down lignocellulosic biomass.
• Current methods for recombinant LPMO production in bacteria often suffer from low yields due to issues like catabolite repression and lack of controlled expression.
• Efficient production of correctly processed LPMOs is essential for their application in lignocellulose saccharification.

Purpose of the Study:

• To develop and evaluate a novel inducible expression system for high-yield recombinant LPMO production in bacteria.
• To optimize conditions for controlled LPMO translocation and processing in the periplasmic space.
• To assess the scalability of the developed expression system for industrial applications.

Main Methods:

• Construction and evaluation of a cassette vector utilizing the XylS/Pm inducible system.
• Testing of different LPMO signal sequences, including that of LPMO10A from Serratia marcescens.
• Optimization of induction conditions (inducer dosage and temperature) for mature LPMO production.
• Analysis of recombinant LPMO integrity using NMR spectroscopy.
• Evaluation of the system in shaking flasks and high cell-density cultivations.

Main Results:

• The XylS/Pm system enabled controlled and reliable production of mature, translocated LPMOs.
• The signal sequence of LPMO10A from Serratia marcescens resulted in the highest production and translocation levels.
• Optimized conditions (0.1 mM m-toluic acid, 16°C) yielded 7-22 mg/L of LPMOs in shaking flasks.
• NMR analysis confirmed the correct processing and folding of recombinant LPMOs.
• High cell-density cultivations demonstrated stable production yields and scalability (42-1298 mg/L).

Conclusions:

• The developed XylS/Pm based expression cassette is a robust system for producing correctly processed LPMOs.
• This system overcomes limitations of previous methods, offering improved yields and controlled expression.
• The demonstrated scalability indicates significant potential for industrial applications in biomass conversion.