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Background <p>The economic viability of enzymatic lignocellulosic biomass conversion is hindered by high production costs and energy-intensive recovery of thermostable enzymes. Traditional optimization approaches treat strain engineering, fermentation, and downstream processing as separate domains, leading to suboptimal system performance. This study aimed to develop an integrated platform that synergistically combines synthetic biology, machine learning-guided bioprocess optimization, and continuous downstream processing for sustainable cellulase production.</p> Results <p>We engineered a <i>Bacillus subtilis</i> chassis with a Quorum-Lysis-Modulation (QLM) system for auto-induction of hyperthermostable cellulase CtCel9A and CRISPRi-mediated cell wall weakening. The QLM strain achieved a CtCel9A titer of 9.32 ± 0.29&#xa0;IU/mL, a 3.6-fold increase over conventional methods. Fermentation was optimized using Gaussian Process Regression in high-throughput microbioreactors, yielding excellent predictive performance (R<sup>2</sup> = 0.94). A novel continuous Pulsed Electric Field-Assisted Synergistic Lysis (PEF-ASL) system was developed and optimized via Response Surface Methodology (Box-Behnken Design), achieving 96.8% enzyme recovery with specific energy consumption of 0.11 kWh/g, a 68% reduction compared to bead milling. The PEF system was operated at 100&#xa0;Hz pulse frequency with 30 ± 2&#xa0;µs pulse width using monopolar exponential decay waveform, and the specific energy input was calculated as 0.098 kWh/g, consistent with the measured value. Multi-objective optimization using NSGA-III balanced yield, energy consumption, water intensity, and production cost. After re-optimization with a full cost function including waste treatment, quality control, contingency, and facility overhead, Pareto front analysis identified a knee-point solution representing the optimal compromise among competing objectives. System-wide optimization reduced production costs by 52–58% and carbon footprint by 57% compared to conventional enzyme production, with techno-economic analysis for a 500&#xa0;kg/year facility indicating a manufacturing cost of $132/kg, net present value of $4.7 million, and payback period of 3.8&#xa0;years. The sfGFP fusion at the C-terminus reduced specific activity by 8.3% compared to unfused enzyme but enabled real-time fluorescence monitoring critical for auto-induction timing.</p> Conclusions <p>The SynBio-DSP platform establishes a new paradigm for sustainable biocatalyst manufacturing by co-optimizing cellular design and process configuration. The integrated approach demonstrates that simultaneous improvements in productivity, cost, and environmental impact are achievable for scalable, economically viable biofuel enzyme production.</p>

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SynBio-DSP: an integrated synthetic biology platform with fed-batch fermentation and continuous downstream processing for sustainable production of thermostable cellulases

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月14日

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  • Shah Faisal Mohammad,
  • Fawad Ali,
  • Mamirkulova Shynara

摘要

Background

The economic viability of enzymatic lignocellulosic biomass conversion is hindered by high production costs and energy-intensive recovery of thermostable enzymes. Traditional optimization approaches treat strain engineering, fermentation, and downstream processing as separate domains, leading to suboptimal system performance. This study aimed to develop an integrated platform that synergistically combines synthetic biology, machine learning-guided bioprocess optimization, and continuous downstream processing for sustainable cellulase production.

Results

We engineered a Bacillus subtilis chassis with a Quorum-Lysis-Modulation (QLM) system for auto-induction of hyperthermostable cellulase CtCel9A and CRISPRi-mediated cell wall weakening. The QLM strain achieved a CtCel9A titer of 9.32 ± 0.29 IU/mL, a 3.6-fold increase over conventional methods. Fermentation was optimized using Gaussian Process Regression in high-throughput microbioreactors, yielding excellent predictive performance (R2 = 0.94). A novel continuous Pulsed Electric Field-Assisted Synergistic Lysis (PEF-ASL) system was developed and optimized via Response Surface Methodology (Box-Behnken Design), achieving 96.8% enzyme recovery with specific energy consumption of 0.11 kWh/g, a 68% reduction compared to bead milling. The PEF system was operated at 100 Hz pulse frequency with 30 ± 2 µs pulse width using monopolar exponential decay waveform, and the specific energy input was calculated as 0.098 kWh/g, consistent with the measured value. Multi-objective optimization using NSGA-III balanced yield, energy consumption, water intensity, and production cost. After re-optimization with a full cost function including waste treatment, quality control, contingency, and facility overhead, Pareto front analysis identified a knee-point solution representing the optimal compromise among competing objectives. System-wide optimization reduced production costs by 52–58% and carbon footprint by 57% compared to conventional enzyme production, with techno-economic analysis for a 500 kg/year facility indicating a manufacturing cost of $132/kg, net present value of $4.7 million, and payback period of 3.8 years. The sfGFP fusion at the C-terminus reduced specific activity by 8.3% compared to unfused enzyme but enabled real-time fluorescence monitoring critical for auto-induction timing.

Conclusions

The SynBio-DSP platform establishes a new paradigm for sustainable biocatalyst manufacturing by co-optimizing cellular design and process configuration. The integrated approach demonstrates that simultaneous improvements in productivity, cost, and environmental impact are achievable for scalable, economically viable biofuel enzyme production.