Chimeric Fatty Acyl-Acyl Carrier Protein Thioesterases Provide Mechanistic Insight into Enzyme Specificity and Expression.

Medium-chain fatty acids are commodity chemicals. Increasing and modifying the activity of thioesterases (TEs) on medium-chain fatty acyl-acyl carrier protein (acyl-ACP) esters may enable a high-yield microbial production of these molecules. The plant Cuphea palustris harbors two distinct TEs: C. palustris FatB1 (CpFatB1) (C8 specificity, lower activity) and CpFatB2 (C14 specificity, higher activity) with 78% sequence identity. We combined structural features from these two enzymes to create several chimeric TEs, some of which showed nonnatural fatty acid production as measured by an enzymatic assay and gas chromatography-mass spectrometry (GC-MS). Notably, chimera 4 exhibited an increased C8 fatty acid production in correlation with improved microbial expression. This chimera led us to identify CpFatB2-specific amino acids between positions 219 and 272 that lead to higher protein levels. Chimera 7 produced a broad range of fatty acids and appeared to combine a fatty acid binding pocket with long-chain specificity and an ACP interaction site that may activate fatty acid extrusion. Using homology modeling and in silico docking with ACP, we identified a "positive patch" within amino acids 162 to 218, which may direct the ACP interaction and regulate access to short-chain fatty acids. On the basis of this modeling, we transplanted putative ACP interaction sequences from CpFatB1 into CpFatB2 and created a chimeric thioesterase that produced medium-chain as well as long-chain fatty acids. Thus, the engineering of chimeric enzymes and characterizing their microbial activity and chain-length specificity suggested mechanistic insights into TE functions and also generated thioesterases with potentially useful properties. These observations may inform a rational engineering of TEs to allow alkyl chain length control.IMPORTANCE Medium-chain fatty acids are important commodity chemicals. These molecules are used as plastic precursors and in shampoos and other detergents and could be used as biofuel precursors if production economics were favorable. Hydrocarbon-based liquid fuels must be optimized to have a desired boiling point, low freezing point, low viscosity, and other physical characteristics. Similarly, the solubility and harshness of detergents and the flexibility of plastic polymers can be modulated. The length and distribution of the carbon chains in the hydrophobic tails determine these properties. The biological synthesis of cell membranes and fatty acids produces chains of primarily 16 to 18 carbons, which give rise to current biofuels. The ultimate goal of the work presented here is to engineer metabolic pathways to produce designer molecules with the correct number of carbons in a chain, so that such molecules could be used directly as specialty commodity chemicals or as fuels after minimal processing.

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells.

Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

Production of fatty acids in Ralstonia eutropha H16 by engineering ß-oxidation and carbon storage.

Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB's physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into ß-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous ß-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha.

Eliminating tyrosine sequence variants in CHO cell lines producing recombinant monoclonal antibodies.

Amino acid sequence variants are defined as unintended amino acid sequence changes that contribute to product variation with potential impact to product safety, immunogenicity, and efficacy. Therefore, it is important to understand the propensity for sequence variant (SV) formation during the production of recombinant proteins for therapeutic use. During the development of clinical therapeutic products, several monoclonal antibodies (mAbs) produced from Chinese Hamster Ovary (CHO) cells exhibited SVs at low levels (≤3%) in multiple locations throughout the mAbs. In these examples, the cell culture process depleted tyrosine, and the tyrosine residues in the recombinant mAbs were replaced with phenylalanine or histidine. In this work, it is demonstrated that tyrosine supplementation eliminated the tyrosine SVs, while early tyrosine starvation significantly increased the SV level in all mAbs tested. Additionally, it was determined that phenylalanine is the amino acid preferentially misincorporated in the absence of tyrosine over histidine, with no other amino acid misincorporated in the absence of tyrosine, phenylalanine, and histidine. The data support that the tyrosine SVs are due to mistranslation and not DNA mutation, most likely due to tRNA(Tyr) mischarging due to the structural similarities between tyrosine and phenylalanine.

CaV1.3-selective L-type calcium channel antagonists as potential new therapeutics for Parkinson's disease.

L-type calcium channels expressed in the brain are heterogeneous. The predominant class of L-type calcium channels has a Ca(V)1.2 pore-forming subunit. L-type calcium channels with a Ca(V)1.3 pore-forming subunit are much less abundant, but have been implicated in the generation of mitochondrial oxidant stress underlying pathogenesis in Parkinson's disease. Thus, selectively antagonizing Ca(V)1.3 L-type calcium channels could provide a means of diminishing cell loss in Parkinson's disease without producing side effects accompanying general antagonism of L-type calcium channels. However, there are no known selective antagonists of Ca(V)1.3 L-type calcium channel. Here we report high-throughput screening of commercial and 'in-house' chemical libraries and modification of promising hits. Pyrimidine-2,4,6-triones were identified as a potential scaffold; structure-activity relationship-based modification of this scaffold led to 1-(3-chlorophenethyl)-3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione (8), a potent and highly selective Ca(V)1.3 L-type calcium channel antagonist. The biological relevance was confirmed by whole-cell patch-clamp electrophysiology. These studies describe the first highly selective Ca(V)1.3 L-type calcium channel antagonist and point to a novel therapeutic strategy for Parkinson's disease.