Evolution-guided optimization of biosynthetic pathways Srivatsan Ramana,b,1,2, Jameson K. Rogersa,c,1, Noah D. Taylora,b,1, and George M. Churcha,b aWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; bDepartment of Genetics, Harvard Medical School, Boston, MA 02115; and cSchool of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02143 Edited by David Baker, University of Washington, Seattle, WA, and approved October 30, 2014 (received for review May 21, 2014) Engineering biosynthetic pathways for chemical production readout (7). The resulting expression of a fluorescent reporter requires extensive optimization of the host cellular metabolic gene or antibiotic resistance gene allows facile identification of machinery. Because it is challenging to specify a priori an optimal mutant cells with increased production of the target chemical. design, metabolic engineers often need to construct and evaluate Sensor reporters have been used to screen for increased mi- a large number of variants of the pathway. We report a general crobial production of several chemicals, including the isoprenoid strategy that combines targeted genome-wide mutagenesis to precursor mevalonate (8), L-lysine (9, 10), 1-butanol (11), and generate pathway variants with evolution to enrich for rare high triacetic acid lactone (12). These studies evaluated a set of variants that altered the expression or coding sequences of one or two producers. We convert the intracellular presence of the target − chemical into a fitness advantage for the cell by using a sensor do- key enzyme genes encoded on a plasmid (8, 10 12). Similarly, a lysine-responsive sensor reporter was used to uncover new main responsive to the chemical to control a reporter gene necessary endogenous enzyme mutants in Corynebacterium glutamicum for survival under selective conditions. Because artificial selection implicated in higher L-lysine production (9). tends to amplify unproductive cheaters, we devised a negative We sought to expand the scope of sensor-directed metabolic selection scheme to eliminate cheaters while preserving library engineering to the directed evolution of whole endogenous diversity. This scheme allows us to perform multiple rounds of evo- pathways. Using FBA as a guide, we simultaneously targeted up 9 lution (addressing ∼10 cells per round) with minimal carryover of to 18 Escherichia coli genomic loci to induce mutations in reg- cheaters after each round. Based on candidate genes identified by ulatory or coding sequence of genes implicated in biosynthesis flux balance analysis, we used targeted genome-wide mutagenesis to of a target molecule. We established a robust selection, using a vary the expression of pathway genes involved in the production of sensor protein responsive to the target chemical to regulate the naringenin and glucaric acid. Through up to four rounds of evolution, expression of an antibiotic resistance gene. Nearly a billion path- we increased production of naringenin and glucaric acid by 36- and way variants could be evaluated simultaneously, enriching for the 22-fold, respectively. Naringenin production (61 mg/L) from glucose best producers when selection pressure was applied. was more than double the previous highest titer reported. Whole- A major challenge faced by this selection approach (and a genome sequencing of evolved strains revealed additional untar- difficulty for most genetic selections) is the incidence of cheater geted mutations that likely benefit production, suggesting new cells that survive without producing the target molecule. These routes for optimization. cheaters evolve to survive selection by mutating the sensor or se- lection machinery, rather than through higher target molecule syn- “ ” evolution | metabolic engineering | synthetic biology | sensors | thesis. Lacking a metabolic burden, these evolutionary escapees biosynthetic pathways Significance icrobial production of chemicals presents an alternative Microbes can be made to produce industrially valuable chem- Mto ubiquitous chemical synthesis methods. Biosynthetic SCIENCES production is attractive because it can use a broad assortment of icals in high quantities by engineering their central metabolic organic feedstocks, proceed under benign physiological condi- pathways. This process may require evaluating billions of cells, APPLIED BIOLOGICAL tions, and avoid environmentally deleterious byproducts. Biosyn- each containing a unique pathway design, to identify the rare thetic alternatives are being pursued for a wide range of chemicals, cells with high production phenotypes. We mutated targeted from bulk commodity building blocks to specialty chemicals. locations across the genome to modify several genes identified Natural cells are seldom optimized to produce a desired as key players. We used sensory proteins responsive to molecule. To achieve economically viable production, extensive a number of target chemicals to couple the concentration of modifications to host cell metabolism are often required to im- the target chemical in each cell to individual cell fitness. This prove metabolite titer, production rate, and yield. The opti- coupling of chemical production to fitness allows us to harness mizations of biosynthetic pathways for 1,3-propanediol (1), evolution to progressively enrich superior pathway designs. flavonoids (2, 3), L-tyrosine (4), and 1,4-butanediol (5) illus- Through iterations of genetic diversification and selection, we trate this complexity. Fortunately, computational models of increased the production of naringenin and glucaric acid 36- cellular metabolism, such as flux balance analysis (FBA), aid in and 22-fold, respectively. predicting metabolic changes likely to improve the production of a target molecule. Powerful methods including oligonucle- Author contributions: S.R., J.K.R., N.D.T., and G.M.C. designed research; S.R., J.K.R., and otide-directed genome engineering (6) (multiplex automated N.D.T. performed research; S.R., J.K.R., N.D.T., and G.M.C. analyzed data; and S.R., J.K.R., N.D.T., and G.M.C. wrote the paper. genome engineering, MAGE) and Cas9-mediated editing can specifically mutate genomic targets predicted by FBA. The Conflict of interest statement: The authors have a pending patent application. combinatorial space of these genomic mutations quickly out- This article is a PNAS Direct Submission. strips the throughput of current analytical methods for evaluat- Freely available online through the PNAS open access option. 3 ing chemical production in individual clones (<10 samples per Data deposition: The sequences reported in this paper have been deposited in the machine per day). GenBank database, www.ncbi.nlm.nih.gov/bioproject/267705. Biosensors that report on the concentration of a chemical 1S.R., J.K.R., and N.D.T. contributed equally to this work. within each individual cell can alleviate this screening bottleneck 2To whom correspondence should be addressed. Email: [email protected]. (10). Such sensor reporters transduce the binding of a target small This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. molecule by a sensory protein or RNA into a gene expression 1073/pnas.1409523111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1409523111 PNAS | December 16, 2014 | vol. 111 | no. 50 | 17803–17808 Downloaded by guest on September 29, 2021 outcompete the top producers during a selection. Multiple se- compound operational range (µM) sensor escape cv A -5 lection cycles compound escape, obscuring productive cells and muconate benM 6.0x10 11% theophylline theRR 3.9x10-3 22% making further pathway evolution infeasible. We therefore de- ado-B12 btuB 7.3x10-5 13% octane alkS 3.1x10-6 25% vised a selection scheme that, by toggling between negative and IPTG lacI 9.8x10-4 5% positive selection, allows us to remove escapees from the pop- xylose xylR 5.2x10-5 23% glucarate cdaR 5.5x10-8 9% ulation when they arise. This strategy maintained high selection erythromycin mphR 6.4x10-5 5% fidelity, permitting multiple rounds of evolution to progressively josamycin mphR ATc tetR 5.8x10-5 6% enrich for higher-producing cells. tetracycline tetR For sensor reporter metabolic engineering to be generalizable, phloretin ttgR 5.8x10-5 10% genistein ttgR sensor domains specific to many different target molecules must pinocembrin ttgR be available. Fortunately, natural sensors exist for a wide array naringenin ttgR 10-5 10-3 10-1 101 103 105 of industrially relevant chemicals, including aliphatic hydro- operational r -4 escape (cfu / cells plated) rate 10 -2 carbons, short-chain alcohols, sugars, amino acids, polymer B 128 C 10 64 -3 building blocks, and vitamins. Many more sensor domains are 10-5 10 likely to be present among the thousands of additional bacterial 32 10-4 -6 16 regulators known from sequence (13−15) that remain to be 10 ange ratio -5 escape rate 8 10 characterized. We adapted 10 regulators to our selection sys- -7 (cfu / cells plated) 10 4 10-6 tem, creating synthetic dependence on their cognate inducer 2 molecules, and demonstrated the utility of two of these for 10-8 1 10-7 14 27 55 273 genome-wide metabolic engineering. tion colicin treatment strain parent (µg/mL) unmodified RBS RBSmod RBS mod8 RBS mod9 mod6 7 dual sensor library of Results RBSdual mod selector 10 weak degradation MAGE strains Sensor selectors are a specific example of the sensor reporter mediumstrong degrada degradation paradigm that use a gene whose product