Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (V HH s) José I. Jiménez a,b , Sofía Fraile a , Olga Zafra a,1 , Víctor de Lorenzo a,n Q1 a Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain b Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom article info Article history: Received 20 November 2014 Received in revised form 16 February 2015 Accepted 6 April 2015 Keywords: Networks Systems Biology V HH Nanobodies Pseudomonas putida Nicotinic acid NicX abstract Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (V HH s, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised V HH s against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. V HH s were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting V HH s allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study. & 2015 International Metabolic Engineering Society. Published by Elsevier Inc. 1. Introduction The customary approach for studying and eventually redesign metabolic pathways involves entering perturbations in specific nodes of otherwise balanced networks (Long and Antoniewicz, 2014). In practical terms, this is brought about by either mutating/ deleting a selected site of the pathway (knock-out), by entering a new activity (knock-in) or by making the onset of a given activity dependent on an external signal (as is typical of inducible expres- sion systems). Although conditional production of a new protein (or even a whole route) in an existing organism, e.g. a bacterium is easy to engineer, deliberate removal or inhibition of an ongoing enzymatic activity poses a more intricate technical challenge (Holtz and Keasling, 2010). Active proteins may remain in the bacterial cells for much longer than their mRNA has ceased to be produced or its translation has been inhibited with a riboswitch (Desai and Gallivan, 2004), small RNAs (Na et al., 2013) or siRNA in the bacterial cytoplasm (Man et al.), thereby letting the network to resettle and thus conceal the effects of the perturbation. Further- more, the presence of many RNAses in different bacteria is often a bottleneck to engineer artificial RNA-based conditional expression systems (Conrad and Sonnewald, 2003). The issue is therefore how to inhibit specific biological functions while they are well in action, but keeping the integrity of the proteins involved. To this end, what could be the active agents for such a selective post- translational inhibition of enzymatic activity without destroying the proteins properly? On this background we entertained the possibility of using antibodies (AB) specific for a given enzymatic target and which could be expressed intracellularly for that purpose. Various types of recombinant ABs have been used in yeasts (Visintin et al., 1999), animal cells (Lo et al., 2008) and plant cells (Jobling et al., 2003) for controlling diverse intracellular activities. However, the same strategy has not found any signifi- cant application in prokaryotes due to the difficulty of folding the business part of the ABs (e.g. recombinant scF v domains, which hold disulfide bonds) in the reducing milieu of the bacterial cytoplasm. Luckily, the last few years has witnessed different strategies for intracellularly producing active AB fragments in Escherichia coli and in other Gram-negative bacteria by either manipulating genetically the redox potential of the cytoplasm (Jurado et al., 2006b) or by fusing the AB fragment to a thioredoxin moiety (Zafra et al., 2011), respectively. These observations, how- ever, have not been translated into a general stratagem for making post-translational activity knockouts. The approaches presented below capitalize our previous research on intracellular expression of recombinant ABs (Jurado et al., 2006a) for enabling the functional suppression of selected metabolic pathways – but in a fashion that keeps intact the protein structure of the targeted enzyme. The strategy involves the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ymben Metabolic Engineering http://dx.doi.org/10.1016/j.ymben.2015.04.002 1096-7176/& 2015 International Metabolic Engineering Society. Published by Elsevier Inc. n Corresponding author. Fax: þ34 91 585 45 06. E-mail address: vdlorenzo@cnb.csic.es (V. de Lorenzo). 1 Current address: Centro de Biología Molecular Severo Ochoa (CSIC), Uni- versidad Autónoma, Madrid 28049, Spain. Please cite this article as: Jiménez, J.I., et al., Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel- derived nanobodies (V HH s). Metab. Eng. (2015), http://dx.doi.org/10.1016/j.ymben.2015.04.002i Metabolic Engineering ∎ (∎∎∎∎) ∎∎∎–∎∎∎