Precision Fermentation for Sustainable Bioproduction of Bisabolol using Engineered Escherichia coli

Bisabolol – A Versatile Bioactive Compound With Skincare Benefits

Allozymes employed an enzyme-directed approach to engineer a precision fermentation for the sustainable biosynthesis of (−)-α-bisabolol, a monocyclic sesquiterpene alcohol with notable anti-inflammatory and antioxidant properties. Naturally derived from Chamomile and Candeia, bisabolol is traditionally extracted from essential oils for cosmetic applications. However, this natural extraction process is challenged by resource depletion and limited supply. While chemical synthesis is an alternative, it leads to the formation of diastereomers. Precision fermentation offers an enantiopure and a sustainable solution to produce bisabolol, addressing these challenges.

Modular Design of the Engineered Strain for Bisabolol Biosynthesis

We engineered a biosynthetic pathway in E. coli to convert glucose into bisabolol by assembling and expressing nearly 15 enzymes. The pathway was designed and tested in a modular approach, comprising three key modules: Module 1 included three enzymes converting IPP and DMAPP to bisabolol; Module 2a contained seven native MEP pathway enzymes to enhance precursor availability; Module 2b featured six MVA pathway enzymes for further precursor augmentation; and Module 3 incorporated native glycolysis enzymes to generate active precursors for the MEP and MVA pathways.

Improvement in Production Through Enzyme Engineering

During experimental validation, we identified several metabolic bottlenecks that limited bisabolol production. To overcome these challenges, we discovered novel enzymes and engineered two rate-limiting enzymes, a reductase and a kinase, to enhance pathway efficiency. Using Allozymes proprietary ultrahigh-throughput microfluidics-based platform, we screened and optimized enzyme variants, enabling the rapid identification of improved mutants with higher activity. Additionally, we employed promoter engineering to fine-tune enzyme expression levels, ensuring balanced metabolic flux and minimizing accumulation of intermediates. By integrating strategies such as enzyme discovery, directed evolution, and pathway optimization, we achieved a 28-fold increase in bisabolol production at the flask scale, compared to the production from the version 1 proof-of-concept strain.

Significant Enhancements in Bisabolol Production Through Engineered Strain and Process Optimization

The study optimized both upstream and downstream bioprocesses to enhance bisabolol production. Using a design of experiments (DoE) approach, key fermentation parameters were systematically refined, leading to a 252-fold overall increase in titer, along with a 2.5-fold boost in productivity and a 7-fold increase in yield at the bioreactor scale. Additionally, a distillation-based downstream process was developed and optimized to ensure high-purity bisabolol with efficient recovery, integrating solvent extraction and filtration to support large-scale production.

Pilot Scale Validation and In-House Process Development Scaled Up to 150L

Finally, we validated the engineered system in-house at the 150L pilot scale, demonstrating its industrial scalability and commercial feasibility for precision fermentation-based bisabolol production.

Conclusion

This project successfully scaled bisabolol production from microgram levels to kilograms through an integrated approach combining biosynthetic pathway design, enzyme engineering, and bioprocess optimization. The 252-fold increase in titer, along with improvements in productivity and yield, demonstrates the effectiveness of these strategies. Allozymes' enzyme-targeted approach played a key role in accelerating development, optimizing fermentation, and ensuring a seamless transition to industrial-scale production. This methodology underscores the potential of precision fermentation for sustainable and efficient biomanufacturing.