Evaluating pathway enumeration algorithms in metabolic engineering case studies

The design of cell factories for the production of compounds involves the search for suitable heterologous pathways. Different strategies have been proposed to infer such pathways, but most are optimization approaches with specific objective functions, not suited to enumerate multiple pathways. In this work, we analyze two pathway enumeration algorithms based on graph representations: the Solution Structure Generation and the Find Path algorithms. Both are capable of enumerating exhaustively multiple pathways using network topology. We study their capabilities and limitations when designing novel heterologous pathways, by applying these methods on two case studies of synthetic metabolic engineering related to the production of butanol and vanillin

Systems-wide prediction of enzyme promiscuity reveals a new underground alternative route for pyridoxal 5'-phosphate production in E. coli

Recent insights suggest that non-specific and/or promiscuous enzymes are common and active across life. Understanding the role of such enzymes is an important open question in biology. Here we develop a genome-wide method, PROPER, that uses a permissive PSI-BLAST approach to predict promiscuous activities of metabolic genes. Enzyme promiscuity is typically studied experimentally using multicopy suppression, in which over-expression of a promiscuous 'replacer' gene rescues lethality caused by inactivation of a 'target' gene. We use PROPER to predict multicopy suppression in Escherichia coli, achieving highly significant overlap with published cases (hypergeometric p = 4.4e-13). We then validate three novel predicted target-replacer gene pairs in new multicopy suppression experiments. We next go beyond PROPER and develop a network-based approach, GEM-PROPER, that integrates PROPER with genome-scale metabolic modeling to predict promiscuous replacements via alternative metabolic pathways. GEM-PROPER predicts a new indirect replacer (thiG) for an essential enzyme (pdxB) in production of pyridoxal 5'-phosphate (the active form of Vitamin B6), which we validate experimentally via multicopy suppression. We perform a structural analysis of thiG to determine its potential promiscuous active site, which we validate experimentally by inactivating the pertaining residues and showing a loss of replacer activity. Thus, this study is a successful example where a computational investigation leads to a network-based identification of an indirect promiscuous replacement of a key metabolic enzyme, which would have been extremely difficult to identify directly.Funding agencies: (MO) Whitaker Foundation (Whitaker International Scholars Program) (http://www.whitaker.org/grants/fellows-scholars) (MO) Dan David Fellowship (http://www.dandavidprize.org/scholarship-applications) (ER) European Union FP7 INFECT project (http://www.fp7infect.eu/) ERA-Net Plant project (http://www.erapg.org/publicpage.m?key=everyone&trail=/everyone) (ER) I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation (grant No 41/11) (www.i-core.org.il/ISF) (UG) McDonnell foundation (https://www.jsmf.org/) (UG) German-Israeli Project Cooperation (DIP) (http://www.dfg.de/en/research_funding/programmes/international_cooperation/german_israeli_cooperation/) (MD) Spanish FPU grant (http://cepima.upc.edu/positions/FPU_2013) (MD) FEBS short term fellowship (http://www.febs.org/our-activities/fellowships/febs-short-term-fellowships/guidelines-for-febs-short-term-fellowships) (NBT) Grant No. 1775/12 of the I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundatio