Selectivity and kinetic modeling of penicillin G acylase variants for the synthesis of cephalexin under a broad range of substrate concentrations

Abstract

The kinetics of cephalexin synthesis and hydrolysis of the activated acyl‐donor precursor phenylglycine methyl ester (PGME) were characterized under a broad range of substrate concentrations. A previously developed model by Youshko‐Svedas involving the formation of the acyl‐enzyme complex followed by binding of the nucleophilic β‐lactam donor does not fully estimate the maximum reaction yields for cephalexin synthesis at different concentrations using initial‐rate data. 7‐aminodesacetoxycephalosporanic acid (7‐ADCA) was discovered to be a potent inhibitor of cephalexin hydrolysis, which may account for the deviation from model predictions. Three kinetic models were compared for cephalexin synthesis, with the model incorporating competitive inhibition due to 7‐ADCA yielding the best fit. Additionally, the βF24A variant and Assemblase® did not exhibit significantly different kinetics for the synthesis of cephalexin compared to the wild‐type, for the concentration range evaluated and for both initial‐rate experiments and time‐course synthesis experiments. Lastly, a continuous stirred‐tank reactor for cephalexin synthesis was simulated using the model incorporating competitive inhibition by 7‐ADCA, with clear tradeoffs observed between productivity, fractional yield, and PGME conversion.

Identification and Validation of Magnolol Biosynthesis Genes in Magnolia officinalis

“…Mohamad et al reported that magnolol was synthesized chemically through aryl coupling and the methoxymethyl ether of 4-allyl-2-lithiophenol using the zinc chloride method, as well as from 5,5 ′ -dibromo-2,2 ′ -dimethoxybiphenyl by allylation with allyltributylstannane, followed by ether cleavage [45]. While a potential biosynthetic route for magnolol has been suggested previously, with relevant genes initially explored via bioinformatics [21,34], the complete biosynthesis pathway had not been experimentally validated until now [46]. Our current results show that laccase can catalyze the synthesis of magnolol from chavicol, thereby confirming the last step of this new biosynthesis pathway within the M. officinalis plant.…”

“…It is therefore of paramount importance to maintain the equilibrium between the substrate concentration and the magnolol yield of MoLAC14 enzyme. Excessive substrate concentrations may inhibit the enzyme, reducing conversion rates, while lower concentrations could impede catalysis due to the enzyme's high-affinity constant [33][34][35]. Intriguingly, our exploration within synthetic biology led to the detection of a byproduct.…”

Pushing the boundaries of phosphorylase cascade reaction for cellobiose production II: Model‐based multiobjective optimization

“…A more robust approach uses design of experiments which can be performed in a purely data-driven fashion based on statistical models or under support from kinetic models of the synthetic reactions (Siedentop et al, 2021;Taylor, Pomberger, et al, 2023). Mechanism-based kinetic models achieve the most comprehensive and in-depth description of the reactions analyzed (Almquist et al, 2014;Gernaey et al, 2010;Harris et al, 2022;Sigg et al, 2021Sigg et al, , 2023. These models are powerful engineering tools of optimization in their own right.…”