ABSTRACT
Conformational analysis of biosynthetic and biodegradable poly(3-hydroxyvalerate) (P3HV), an analog of poly(3-hydroxybutyrate) (P3HB), was performed. The only structural difference between the two polymers consists in the side chain, which is either methyl (P3HB) or ethyl (P3HV). Molecular orbital calculations and NMR experiments were conducted on a monomeric model compound to determine the bond conformations of the main and side chains of P3HV. The refined rotational isomeric state (RIS) scheme was applied to derive configurational properties of P3HV. The characteristic ratio of P3HV (2.1-3.0) is smaller than that of P3HB (5.4-5.6), indicating greater conformational flexibility of the P3HV chain. The increased flexibility due to the internal rotation of the ethyl side group of P3HV results in a lower equilibrium melting point (130 °C) compared with P3HB (203 °C). The RIS calculations on P3HV also suggested its potential for rubber-like properties. Periodic density functional theory calculations were used to optimize the crystal structures of P3HB and P3HV and obtain their elastic moduli. Three-dimensional Young's modulus distributions of both crystals are closer to isotropic than anisotropic. In conclusion, the material design and usage of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) are discussed.
ABSTRACT
Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.
Subject(s)
Genetic Engineering/methods , Plants, Genetically Modified/metabolism , Pseudomonas/metabolism , Brachypodium/metabolism , Brachypodium/microbiology , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Plant Roots/metabolism , Plant Roots/microbiology , Plants, Genetically Modified/genetics , Plasmids/genetics , Plasmids/metabolism , Recombinases/genetics , Recombination, Genetic , RhizosphereABSTRACT
The cellular activities of gymnosperms monoterpene synthases are largely compromised due to their requirement for manganese, which is deficient in microbial cells. Through site-saturation mutagenesis of the residue adjacent to metal-binding glutamate, we found that pinene synthase is highly mutable at this position yet drastically alter their metal binding preference, thereby quickly improving the cellular performance in heterologous hosts.
