In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments

Area of Science:

• Biotechnology
• Biochemical Engineering
• Microbial Physiology

Background:

• The penicillin biosynthesis pathway in *Penicillium chrysogenum* is complex, involving multiple enzymes and transporters.
• Understanding the *in vivo* kinetics is crucial for optimizing penicillin production.
• Previous studies often lacked dynamic insights into pathway regulation and metabolite transport.

Purpose of the Study:

• To elucidate the *in vivo* kinetic properties of enzymes and transporters in the penicillin biosynthesis pathway.
• To construct and validate a dynamic mathematical model of penicillin biosynthesis.
• To analyze enzyme behavior, fluxes, and flux control within the pathway under specific conditions.

Main Methods:

• Combined experimentation (step response with phenyl acetic acid) and mathematical modeling.
• Utilized a glucose-limited chemostat in a high-yielding *Penicillium chrysogenum* strain.
• Simultaneously estimated model parameters and enzyme levels using multi-timescale experimental data (seconds, minutes, hours).

Main Results:

• All penicillin pathway enzymes were expressed constitutively, producing 6-aminopenicillanic acid (6APA) in the absence of phenyl acetic acid (PAA).
• Penicillin G (PenG) was produced within seconds after PAA addition, indicating rapid pathway activation.
• Derived hypotheses for secretion mechanisms of pathway metabolites based on extra- and intracellular measurements.
• Quantified *in vivo* enzyme behavior, fluxes, and flux control, leading to a reassessment of Acyl-CoA:Isopenicillin N Acyltransferase (AT) function.

Conclusions:

• The dynamic model accurately represents penicillin biosynthesis *in vivo*.
• Enzyme levels and kinetic properties were determined under physiological conditions.
• The study provides a refined understanding of penicillin pathway regulation and offers insights for strain engineering and process optimization.