Conor McClune

All Publications

• Multiplexed perturbation of yew reveals cryptic proteins that enable a total biosynthesis of baccatin III and Taxol precursors. bioRxiv : the preprint server for biology McClune, C. J., Liu, J. C., Wick, C., De La Peña, R., Lange, B. M., Fordyce, P. M., Sattely, E. S. 2024

  Abstract

  Plants make complex and potent therapeutic molecules, but difficulties in sourcing from natural producers or chemical synthesis can challenge their use in the clinic. A prominent example is the anti-cancer therapeutic paclitaxel (Taxol®). Identification of the full paclitaxel biosynthetic pathway would enable heterologous drug production, but it has eluded discovery despite a half century of intensive research. Within the search space of Taxus' large, enzyme-rich genome, we suspected the complex paclitaxel pathway would be difficult to resolve using conventional gene co-expression analysis and small sample sets. To improve the resolution of gene set identification, we developed a multiplexed perturbation strategy to transcriptionally profile cell states spanning tissues, cell types, developmental stages, and elicitation conditions. This approach revealed a set of paclitaxel biosynthetic genes that segregate into expression modules that suggest consecutive biosynthetic sub-pathways. These modules resolved seven new genes that, when combined with previously known enzymes, are sufficient for the de novo biosynthesis and isolation of baccatin III, an industrial precursor for Taxol, in Nicotiana benthamiana leaves at levels comparable to the natural abundance in Taxus needles. Included are taxane 1β-hydroxylase (T1βH), taxane 9α-hydroxylase (T9αH), taxane 7β-O-acyltransferase (T7AT), taxane 7β-O-deacetylase (T7dA), taxane 9α-O-deacetylase (T9dA), and taxane 9-oxidase (T9ox). Importantly, the T9αH we discovered is distinct and independently evolved from those recently reported, which failed to yield baccatin III with downstream enzymes. Unexpectedly, we also found a nuclear transport factor 2 (NTF2)-like protein (FoTO1) crucial for high yields of taxanes; this gene promotes the formation of the desired product during the first taxane oxidation step, resolving a longstanding bottleneck in paclitaxel pathway reconstitution. Together with a new β-phenylalanine-CoA-ligase, the eight genes discovered in this study enables the complete reconstitution of 3'-N-debenzoyl-2'-deoxy-paclitaxel with a 20-enzyme pathway in Nicotiana plants. More broadly, we establish a generalizable approach for pathway discovery that scales the power of co-expression studies to match the complexity of specialized metabolism, enabling discovery of gene sets responsible for high-value biological functions.

  View details for DOI 10.1101/2024.11.06.622305

  View details for PubMedID 39574719

  View details for PubMedCentralID PMC11580873

• Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets ANALYTICAL CHEMISTRY Khariton, M., McClune, C. J., Brower, K. K., Klemm, S., Sattely, E. S., Fordyce, P. M., Wang, B. 2023; 95 (2): 935-945

  View details for DOI 10.1021/acs.analchem.2c03475935Anal

  View details for Web of Science ID 000923878900001

• Alleviating Cell Lysate-Induced Inhibition to Enable RT-PCR from Single Cells in Picoliter-Volume Double Emulsion Droplets. Analytical chemistry Khariton, M., McClune, C. J., Brower, K. K., Klemm, S., Sattely, E. S., Fordyce, P. M., Wang, B. 2023

  Abstract

  Microfluidic droplet assays enable single-cell polymerase chain reaction (PCR) and sequencing analyses at unprecedented scales, with most methods encapsulating cells within nanoliter-sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) allows sorting droplets with commercially available fluorescence-activated cell sorter (FACS) machines, making it possible to isolate single cells based on phenotypes of interest for downstream analyses. However, sorting DE droplets with standard cytometers requires small droplets that can pass FACS nozzles. This poses challenges for molecular biology, as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, which can be efficiently alleviated through heat lysis. We further show that the magnitude of inhibition depends on the cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most mouse and human cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL DE droplets with fluorescence quantifiable via FACS. These results open up new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet-based single-cell analysis technologies.

  View details for DOI 10.1021/acs.analchem.2c03475

  View details for PubMedID 36598332

• Constraints on the expansion of paralogous protein families. Current biology : CB McClune, C. J., Laub, M. T. 2020; 30 (10): R460–R464

  Abstract

  Duplication and divergence is a major mechanism by which new proteins and functions emerge in biology. Consequently, most organisms, in all domains of life, have genomes that encode large paralogous families of proteins. For recently duplicated pathways to acquire different, independent functions, the two paralogs must acquire mutations that effectively insulate them from one another. For instance, paralogous signaling proteins must acquire mutations that endow them with different interaction specificities such that they can participate in different signaling pathways without disruptive cross-talk. Although duplicated genes undoubtedly shape each other's evolution as they diverge and attain new functions, it is less clear how other paralogs impact or constrain gene duplication. Does the establishment of a new pathway by duplication and divergence require the system-wide optimization of all paralogs? The answer has profound implications for molecular evolution and our ability to engineer biological systems. Here, we discuss models, experiments, and approaches for tackling this question, and for understanding how new proteins and pathways are born.

  View details for DOI 10.1016/j.cub.2020.02.075

  View details for PubMedID 32428482

• Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space. Nature McClune, C. J., Alvarez-Buylla, A., Voigt, C. A., Laub, M. T. 2019; 574 (7780): 702-706

  Abstract

  Gene duplication is a common and powerful mechanism by which cells create new signalling pathways1,2, but recently duplicated proteins typically must become insulated from each other and from other paralogues to prevent unwanted crosstalk3. A similar challenge arises when new sensors or synthetic signalling pathways are engineered within cells or transferred between genomes. How easily new pathways can be introduced into cells depends on the density and distribution of paralogous pathways in the sequence space that is defined by their specificity-determining residues4,5. Here we directly investigate how crowded this sequence space is, by generating novel two-component signalling proteins in Escherichia coli using cell sorting coupled to deep sequencing to analyse large libraries designed on the basis of coevolutionary patterns. We produce 58 insulated pathways comprising functional kinase-substrate pairs that have different specificities than their parent proteins, and demonstrate that several of these new pairs are orthogonal to all 27 paralogous pathways in E. coli. Additionally, from the kinase-substrate pairs generated, we identify sets consisting of six pairs that are mutually orthogonal to each other, which considerably increases the two-component signalling capacity of E. coli. These results indicate that sequence space is not densely occupied. The relative sparsity of paralogues in sequence space suggests that new insulated pathways can arise easily during evolution, or be designed de novo. We demonstrate the latter by engineering a signalling pathway in E. coli that responds to a plant cytokinin, without crosstalk to extant pathways. Our work also demonstrates how coevolution-guided mutagenesis and the mapping of sequence space can be used to design large sets of orthogonal protein-protein interactions.

  View details for DOI 10.1038/s41586-019-1639-8

  View details for PubMedID 31645757

  View details for PubMedCentralID PMC6858568

• Permanent genetic memory with >1-byte capacity. Nature methods Yang, L., Nielsen, A. A., Fernandez-Rodriguez, J., McClune, C. J., Laub, M. T., Lu, T. K., Voigt, C. A. 2014; 11 (12): 1261-6

  Abstract

  Genetic memory enables the recording of information in the DNA of living cells. Memory can record a transient environmental signal or cell state that is then recalled at a later time. Permanent memory is implemented using irreversible recombinases that invert the orientation of a unit of DNA, corresponding to the [0,1] state of a bit. To expand the memory capacity, we have applied bioinformatics to identify 34 phage integrases (and their cognate attB and attP recognition sites), from which we build 11 memory switches that are perfectly orthogonal to each other and the FimE and HbiF bacterial invertases. Using these switches, a memory array is constructed in Escherichia coli that can record 1.375 bytes of information. It is demonstrated that the recombinases can be layered and used to permanently record the transient state of a transcriptional logic gate.

  View details for DOI 10.1038/nmeth.3147

  View details for PubMedID 25344638

  View details for PubMedCentralID PMC4245323