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In this study, the influence of some factors on the heterogeneous nucleation of hen egg-white lysozyme (E.C. 3.2.1.17) on a series of chemically modified surfaces has been investigated. Microbatch crystallization experiments were conducted on the microscope glass slides that were treated with poly-L-glutamic acid (PLG), poly(2-hydroxyethyl methacrylate) (P2HEMA), poly(methyl methacrylate) (PMMA), poly(4-vinyl pyridine) (P4VP), and (3-aminopropyl)triethoxysilane (APTES). An optical microscope with a heating/cooling stage was employed to measure the induction time of heterogeneous nucleation. The surface topography and roughness were characterized by atomic force microscopy. Contact angles for crystallization solution on the investigated surfaces were measured by a contact angle meter. From the theoretical analysis, the energetic barrier to heterogeneous nucleation was found to increase at higher contact angles and to decrease at higher roughness. Experimentally, a qualitative increase of the induction time of the heterogeneous nucleation on P2HEMA, APTES, and PMMA surfaces with the contact angle was observed. Such surfaces as P2HEMA, PLG, and APTES, which were of higher roughness, were shown to promote the heterogeneous nucleation. In addition, the surface with specific topography is expected to increase the possibility of the formation of a critical nucleus. Finally, the P4VP surface appeared to suppress the heterogeneous nucleation as a result of the electrostatic interaction between the lysozyme and P4VP molecules.

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BackgroundTo achieve high-level production of non-native isoprenoid products, it requires the metabolic flux to be diverted from the production of sterols to the heterologous metabolic reactions. However, there are limited tools for restricting metabolic flux towards ergosterol synthesis. In the present study, we explored dynamic control of ERG9 expression using different ergosterol-responsive promoters to improve the production of non-native isoprenoids.ResultsSeveral ergosterol-responsive promoters were identified using quantitative real-time PCR (qRT-PCR) analysis in an engineered strain with relatively high mevalonate pathway activity. We found mRNA levels for ERG11, ERG2 and ERG3 expression were significantly lower in the engineered strain over the reference strain BY4742, indicating these genes are transcriptionally down-regulated when ergosterol is in excess. Further replacement of the native ERG9 promoter with these ergosterol-responsive promoters revealed that all engineered strains improved amorpha-4,11-diene by 2 ~ 5-fold over the reference strain with ERG9 under its native promoter. The best engineered strain with ERG9 under the control of PERG1 produced amorpha-4,11-diene to a titer around 350 mg/L after 96 h cultivation in shake-flasks.ConclusionsWe envision dynamic control at the branching step using feedback regulation at transcriptional level could serve as a generalized approach for redirecting the metabolic flux towards product-of-interest.