Engineered biofilms composed of a single recombinant bacterial species have emerged as highly effective biocatalysts across diverse applications. This study focuses on the enzymatic conversion of 5-haloindole to 5-halotryptophan—a key pharmaceutical intermediate—using recombinant Escherichia coli expressing tryptophan synthase encoded by plasmid pSTB7. To enhance reaction efficiency, two E. coli K-12 strains, MG1655 and MC4100, along with their isogenic ompR234 mutants (PHL644 and PHL628), were evaluated. The ompR234 mutation significantly increased biofilm formation in both strain backgrounds by overproducing curli adhesins. All biotransformations required the presence of pSTB7, as no conversion occurred in cells lacking the plasmid. Engineered biofilms of PHL628 pSTB7 and PHL644 pSTB7 consistently outperformed their planktonic counterparts in halotryptophan yield. Flow cytometry confirmed high cell viability (>90%) after 24 hours in both biofilm and planktonic states, indicating that enhanced performance was not due to superior survival under reaction conditions. Monitoring substrate depletion, product synthesis, and conversion selectivity revealed intrinsic metabolic differences between MG1655 and MC4100, as well as between planktonic and biofilm lifestyles. Notably, MC4100 exhibited rapid haloindole uptake, while biofilm formation altered transport dynamics and enzyme accessibility. These findings highlight the importance of comprehensive physiological characterization when selecting host strains for biotransformation processes. The results demonstrate that engineered biofilms offer superior catalytic performance not due to increased viability but because of optimized metabolic flux and improved enzyme stability within the structured matrix. This work underscores the necessity of integrating strain-specific physiology into biocatalyst design, particularly for industrial-scale biosynthesis of complex pharmaceutical intermediates.
Initial reaction rates normalized per dry cell mass showed that fluoroindole was converted faster than chloro- or bromoindole, consistent with steric hindrance effects on the TrpBA enzyme. While biofilm reactions generally matched planktonic rates, exceptions were observed: PHL628 pSTB7 biofilms exhibited three to four times higher initial activity than its planktonic form. This suggests that structural immobilization enhances catalytic efficiency beyond mere protection from harsh conditions. Despite similar initial rates, biofilms sustained longer reaction durations, likely due to reduced enzyme degradation and efficient substrate channeling through the extracellular matrix. In contrast, planktonic systems showed declining conversion efficiency over time, possibly due to product inhibition or cellular stress. The data also indicate that halotryptophan export mechanisms differ between growth forms, with biofilms potentially facilitating more efficient product release.Hyaluronic acid Epigenetic Reader Domain Furthermore, the presence of pSTB7 slightly suppressed biofilm biomass in PHL628, likely due to intracellular indole depletion via active tryptophan synthesis.Polyoxin D Description However, this did not compromise overall productivity.PMID:34823118 Overall, the study establishes that strain selection must consider not only biofilm-forming capacity but also metabolic regulation, enzyme expression dynamics, and transport characteristics. Future efforts should focus on engineering regulatory networks to decouple biofilm formation from metabolic burden while maximizing catalytic output. These insights are crucial for advancing synthetic biology platforms toward scalable, sustainable biomanufacturing processes.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com