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Publications and source records attributed to Vickers, C. E..
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Enzyme spatial organisation and compartmentalisation are naturally evolved mechanisms for facilitating multi-step biocatalysis. We explored the synthetic in vivo co-encapsulation of two different cargo proteins in yeast using a self-assembling virus-like particle. Co-encapsulation was verified using single particle techniques for both end-to-end fusion of the cargo proteins with the encapsulation anchor at one end, and coexpression of each cargo protein with their individual anchors. The co-encapsulation of a bifunctional geranyl diphosphate/farnesyl diphosphate synthase and a bifunctional linalool/nerolidol synthase delivered nerolidol titres up to 30 times that of an unorganised free enzyme control, a remarkable improvement from a single engineering step. Interestingly, striking differences in the ratio of products (linalool and nerolidol) were observed with each spatial organisation approach. This work presents the largest reported titre fold increases from in vivo enzyme compartmentalisation and suggests that enzyme spatial organisation could be used to modulate the product profile of promiscuous enzymes.
The end-to-end fusion of enzymes that catalyse successive steps in a reaction pathway is a metabolic engineering strategy that has been successfully applied in a variety of pathways and is particularly common in terpene bioproduction. Despite its popularity, limited work has been done to interrogate the mechanism of metabolic enhancement from enzyme fusion. We observed a remarkable >110-fold improvement in nerolidol production upon translational fusion of nerolidol synthase (a sesquiterpene synthase) to farnesyl diphosphate synthase. This delivered a titre increase from 29.6 mg/L up to 4.2 g/L nerolidol in a single engineering step. Whole-cell proteomic analysis revealed that nerolidol synthase levels in the fusion strains were greatly elevated compared to the non-fusion control. Similarly, the fusion of nerolidol synthase to non-catalytic domains also produced comparable increases in titre, which coincided with improved enzyme expression. When farnesyl diphosphate synthase was fused to other terpene synthases, we observed more modest improvements in terpene titre (1.9- and 3.8-fold), which corresponds to increases of a similar magnitude in terpene synthase expression. Therefore, increased in vivo enzyme levels - resulting from improved expression and/or stability - is likely to be a major driver of catalytic enhancement from enzyme fusion.
Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons, and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside virus-like particles can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here we present a set of acceptor vectors that provide a quick and efficient way to build, test and optimise cargo loading inside P22 virus-like particles. We prototyped the system using yellow fluorescent protein then applied it to mevalonate kinases, a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different mevalonate kinases required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimise cargo enzyme behaviour in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behaviour. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/479872v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@172138corg.highwire.dtl.DTLVardef@616de5org.highwire.dtl.DTLVardef@75ea19org.highwire.dtl.DTLVardef@15c19cd_HPS_FORMAT_FIGEXP M_FIG C_FIG
Metabolic pathways are commonly organised by sequestration into discrete cellular compartments. Compartments prevent unfavourable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behaviour and for metabolic engineering. Here, we expand the intracellular compartmentalisation toolbox for budding yeast (Saccharomyces cerevisiae) with engineered Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell; and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilised GFP fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. In order to access metabolites of native and engineered yeast metabolism, VLP-based nanocompartments were directed to assemble in the cytosol by removal of the VP1 nuclear localisation signal. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in D-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ~20% more D-glucaric acid compared to controls expressing free MIOX - despite accumulating dramatically less expressed protein - and also grew to higher cell densities. These effects were linked to enzyme stabilisation and mitigation of cellular toxicity by the engineered compartment. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.
Strigolactones are plant hormones and rhizosphere signalling molecules with key roles in plant development, mycorrhizal fungal symbioses, and plant parasitism. Currently, sensitive, specific, and high-throughput methods of detecting strigolactones are limited. Here, we developed genetically encoded fluorescent strigolactone biosensors based on the strigolactone receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica via domain insertion of circularly permuted GFP. The DAD2 biosensor exhibited loss of cpGFP fluorescence in vitro upon treatment with the strigolactones 5-deoxystrigol and orobanchol, or the strigolactone analogue GR24. The biosensor likewise responded to strigolactones in an in vivo protoplast system, and retained strigolactone hydrolysis activity. The ShHTL7 biosensor exhibited loss of cpGFP fluorescence upon GR24 treatment in vitro, and responded to a specific inhibitor of ShHTL7 but not DAD2, indicating that the biosensors retained the specificity of their parent receptors. These biosensors have applications in high-throughput screening, and may also have utility for studying strigolactone biology.
Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nano-scale beer production. Samples included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.