ABSTRACT
While bacterial natural products are a valuable source of therapeutics, the molecules produced by most biosynthetic gene clusters remain unknown. Tambjamine YP1, produced by Pseudoalteromonas tunicata, is partially derived from fatty acids siphoned from primary metabolism. A structurally similar tambjamine produced by Streptomyces, BE-18591, had not been linked to a gene cluster. Using enzymes putatively implicated in the construction of these two tambjamines, we used sequence similarity networks and gene knockout experiments to identify the biosynthetic gene cluster responsible for the production of tambjamine BE-18591 in Streptomyces albus. Despite the structural similarities between YP1 and BE-18591, the biosynthesis of the alkylamine tails of these molecules differs significantly, with the S. albus gene cluster putatively encoding a dedicated system for the construction of the fatty acid precursor to BE-18591. These different pathways in Pseudoalteromonas and Streptomyces suggest that evolutionary convergence is operative, with similar selective pressures leading to the emergence of structurally similar tambjamine natural products using different biosynthetic logic.
Subject(s)
Biological Products , Streptomyces , Biological Products/metabolism , Streptomyces/genetics , Streptomyces/metabolism , Multigene FamilyABSTRACT
Digital microfluidics employs water-repellant surfaces to exquisitely manipulate droplets of water for chemical analysis. However, the actuation and manipulation of organic droplets is still relatively unexplored as it is significantly more difficult to synthesize organic-repellent surfaces compared to water-repellent surfaces. Here, we present the fabrication of slippery liquid-infused porous surfaces (SLIPS) based on a porous polymer monolithic approach. The synthesized SLIPS were able to repel organic liquids such as hexane and methanol with a contact angle of 42.1 ± 0.4° and 69.0 ± 1.8°, respectively, as well as water with a contact angle of 115.8 ± 0.8°. More importantly for digital microfluidic applications, the sliding angle of liquids tested was between 4° and 6°. As a result, droplets containing magnetically susceptible material could be facilely manipulated on the SLIPS surface. A systematic actuation study was carried out to explore how actuation parameters including speed, paramagnetic particle (PMP) concentrations, and droplet volume impacted the outcomes (droplet actuation, disengagement, and PMP extraction). Two different applications were used to demonstrate the utility of actuating organic droplets on SLIPS surfaces including on-chip liquid-liquid extractions of natural products (NPs) from marine bacteria and droplet-based polymer synthesis with different polymerization conditions. Both applications employ an aqueous droplet and organic droplet interface at which either phase transfer or a chemical reaction is carried out. Two NPs (prodigiosin from Pseudoalteromonas rubra and violacein from Pseudoalteromonas luteoviolacea) were extracted, from aqueous droplets containing the bacteria, into butanol droplets and characterized with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Nylon 6,6 was synthesized on-chip via magnetic actuation of organic droplets containing adipoyl chloride and hexamethylamine. Relative intensities of the characteristic polymer masses suggest that droplet-based microfluidic synthesis on slips can be used to probe reaction conditions. The compatibility of SLIPS with both aqueous and organic solutions opens up a wider number of droplet-based sample preparation protocols and chemical transformations.
