The gene cluster responsible for grincamycin (GCN, 1) biosynthesis in Streptomyces lusitanus SCSIO LR32 was identified; heterologous expression of the GCN cluster in S. coelicolor M512 yielded P-1894B (1b) as a predominant product. The ΔgcnQ mutant accumulates intermediate 1a and two shunt products 2a and 3a bearing L-rhodinose for L-cinerulose A substitutions. In vitro data demonstrated that GcnQ is capable of iteratively tailoring the two L-rhodinose moieties into L-aculose moieties, supporting divergent roles of GcnQ in different hosts.

During production of therapeutic monoclonal antibodies (mAbs) in mammalian cell culture, it is important to ensure that viral impurities and potential viral contaminants will be removed during downstream purification. Anion exchange chromatography provides a high degree of virus removal from mAb feedstocks, but the mechanism by which this is achieved has not been characterized. In this work, we have investigated the binding of three viruses to Q sepharose fast flow (QSFF) resin to determine the degree to which electrostatic interactions are responsible for viral clearance by this process. We first used a chromatofocusing technique to determine the isoelectric points of the viruses and established that they are negatively charged under standard QSFF conditions. We then determined that virus removal by this chromatography resin is strongly disrupted by the presence of high salt concentrations or by the absence of the positively charged Q ligand, indicating that binding of the virus to the resin is primarily due to electrostatic forces, and that any non-electrostatic interactions which may be present are not sufficient to provide virus removal. Finally, we determined the binding profile of a virus in a QSFF column after a viral clearance process. These data indicate that virus particles generally behave similarly to proteins, but they also illustrate the high degree of performance necessary to achieve several logs of virus reduction. Overall, this mechanistic understanding of an important viral clearance process provides the foundation for the development of science-based process validation strategies to ensure viral safety of biotechnology products.

Both 22:4n-6 and 22:5n-3 are synthesized from n-6 and n-3 fatty acid precursors in the endoplasmic reticulum. The synthesis of both 22:5n-6 and 22:6n-3 requires that 22:4n-6 and 22:5n-3 are metabolized, respectively, to 24:5n-6 and 24:6n-3 in the endoplasmic reticulum. These two 24-carbon acids must then move to peroxisomes for partial degradation followed by the movement of 22:5n-6 and 22:6n-3 back to the endoplasmic reticulum for use as substrates in membrane lipid biosynthesis. Clearly an understanding of the control of intracellular fatty acid movement as well as of the reactions carried out by microsomes, peroxisomes, and mitochondria are all required in order to understand not only what regulates the biosynthesis of 22:5n-6 and 22:6n-3 but also why most tissue lipids selectively accumulate 22:6n-3.