Disregarded Free Chains Affect Bacterial Adhesion on Cross-Linked Polydimethylsiloxane Surfaces.

The surface properties exhibited by chemically cross-linked polydimethylsiloxanes (CPDMS) such as morphology, stiffness, and wettability have garnered great interest in the study of bacteria-material interactions. Nevertheless, the hidden factor of uncross-linked free PDMS chains that dissociate in CPDMS has often been overlooked when studying the biofilm formation on these polymeric elastomer surfaces. Here, we undertake a comparative characterization of the effects of free chains in CPDMS on bacterial adhesion to both flat and textured Sharklet CPDMS surfaces. Surprisingly, compared to unextracted surfaces, removing free chains from flat and textured CPDMS through solvent extraction results in a tremendous increase in bacterial colony-forming units for both Gram-negative and Gram-positive bacteria up to 2-3 orders in the initial adhesion stage of 2 h. These findings demonstrate that the solvent extraction of free chains from CPDMS is essential in studying the interactions between bacteria and silicone elastomer materials when focusing on a single variable.

Multiplex gene knockout raises Ala-Gln production by Escherichia coli expressing amino acid ester acyltransferase.

L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA, pepB, pepD, pepN, dpp, and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)-ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, BpADNPA (BPA-ΔpepADN) was built, and the production of Ala-Gln was 129% of the BPA's accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. KEY POINTS: • Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis • The balanced gene knockout combination is pepA, pepD, and pepN • The accumulation of Ala-Gln with BpADNPA was 129% of the control.

Clean Production of l-Alanyl-l-glutamine by an Efficient Yeast Biocatalyst Expressing α-Amino Acid Ester Acyltransferase without N-Glycosylation.

l-Alanyl-l-glutamine (Ala-Gln) is a widely used value-added dipeptide whose production relies heavily upon an efficient biocatalyst. The currently available yeast biocatalysts that express α-amino acid ester acyltransferase (SsAet) possess relatively low activity, which may be attributed to glycosylation. Here, to promote SsAet activity in yeast, we identified the N-glycosylation site as the Asn residue at position 442 and subsequently eliminated the negative effect of N-glycosylation on SsAet by removing artificial and native signal peptides to obtain K3A1, a novel yeast biocatalyst with significantly improved activity. Additionally, the optimal reaction conditions of strain K3A1 were determined (25 °C, pH 8.5, AlaOMe/Gln = 1:2), resulting in a maximum molar yield and productivity of approximately 80% and 1.74 g·(L·min)-1, respectively. Therefore, we developed a promising system to cleanly produce Ala-Gln in a safe, efficient, and sustainable manner, which may contribute to the future industrial production of Ala-Gln.