CRISPR/Cas9-Based amyE Regulatory Engineering for Enhanced α-Amylase Production to Support Sustainable Starch-Rich Industrial Crop Bioprocessing

Document Type : Original Article

Authors

1 Department of Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

2 Department of Biochemistry, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran

10.22126/atic.2026.12202.1222

Abstract

The sustainable processing of starch-rich industrial crops, such as potatoes, maize, and wheat, is central to the production of value-added products, including bioethanol and high-fructose corn syrup, from these feedstocks. Efficient conversion of crop-derived starch into fermentable sugars relies on robust α-amylase enzymes and their optimal concentrations. Here, we present a proof-of-concept CRISPR/Cas9-based strategy aimed at engineering the regulatory region of the amyE gene in Bacillus subtilis, a GRAS-certified bacterium widely used for industrial enzyme production. Our modular synthetic construct features combined elements, including a high-strength promoter, multiple ribosomal binding sites, and optimized secretion signal, enabling optimization of α-amylase expression and secretion. The modular plasmid was successfully assembled and validated in Escherichia coli, which is used for its rapid cloning capabilities and compatibility with plasmid construction, before transformation into the final B. subtilis production host. This integrated engineering approach in a single construct is expected to yield higher extracellular α-amylase titers than native systems, supporting more efficient industrial starch bioprocessing of starch-rich agricultural feedstocks and advancing green technologies for the industrial crop sector. Future work will focus on quantifying α-amylase activity in engineered strains and validating the approach on agricultural substrates to assess industrial scalability.

Graphical Abstract

CRISPR/Cas9-Based amyE Regulatory Engineering for Enhanced α-Amylase Production to Support Sustainable Starch-Rich Industrial Crop Bioprocessing

Highlights

  • Developed a CRISPR/Cas9-based strategy to engineer the regulatory region of the amyE gene in Bacillus subtilis for enhanced α-amylase production.
  • Designed and assembled a modular synthetic construct combining a high-strength promoter (NBP351015), multiple optimized ribosomal binding sites, and an efficient secretion signal peptide (SPyojL).
  • Successfully validated the construct in Escherichia coli before its intended transformation into Bacillus subtilis for industrial enzyme production.
  • The approach aims to significantly increase extracellular α-amylase titers, supporting more efficient and sustainable starch bioprocessing of crops like potato, maize, and wheat.
  • The CRISPR/Cas9 system enables precise, marker-free chromosomal integration, ensuring stable and consistent enzyme expression suitable for industrial scaling.

Keywords

Main Subjects

References

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Agrotechniques in Industrial Crops

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Available Online from 02 October 2025

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