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Engineering Escherichia Coli Towards De Novo Production of Gatekeeper (2S)-Flavanones: Naringenin, Pinocembrin, Eriodictyol and Homoeriodictyol Mark S

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Engineering Escherichia Coli Towards De Novo Production of Gatekeeper (2S)-Flavanones: Naringenin, Pinocembrin, Eriodictyol and Homoeriodictyol Mark S Synthetic Biology, 2020, 5(1): ysaa012 doi: 10.1093/synbio/ysaa012 Advance Access Publication Date: 6 August 2020 Research Article Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol Mark S. Dunstan1,†, Christopher J. Robinson 1,†, Adrian J. Jervis1,†, Cunyu Yan1, Pablo Carbonell 1,2, Katherine A. Hollywood1, Andrew Currin 1, Neil Swainston1, Rosalind Le Feuvre1, Jason Micklefield1, Jean-Loup Faulon1,3, Rainer Breitling1, Nicholas Turner1, Eriko Takano1,*, and Nigel S. Scrutton1,* 1Manchester aaSynthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology and Department of Chemistry, The University of Manchester, Manchester M1 7DN, UK, 2Current affiliation: Instituto Universitario de Automa´tica e Informa´tica Industrial, Universitat Polite`cnica de Vale`ncia, 46022 Valencia, Spain and 3MICALIS, INRA-AgroParisTech, Domaine de Vilvert, 78352 Jouy en Josas Cedex, France *Corresponding authors: E-mails: [email protected] and [email protected] †These authors contributed equally to this work. Abstract Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health bene- fits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoerio- dictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimiza- tion of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocem- brin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoerio- dictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets. Key words: pathway engineering; flavonoids; synthetic biology; metabolic engineering; flavanones 1. Introduction stresses, including ultraviolet radiation, microbial infections, and both physical and radical damage. Humans consume flavo- In nature, flavonoids are produced by a broad range of plant noids through plant-derived foods and these play crucial roles species to help combat various organic and non-organic in helping prevent the onset of numerous diseases (1). Submitted: 2 June 2020; Received (in revised form): 15 July 2020; Accepted: 22 July 2020 VC The Author(s) 2020. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 2|Synthetic Biology, 2020, Vol. 5, No. 1 Naringenin, pinocembrin, eriodictyol and homoeriodictyol are metabolism has proven more challenging. Studies describing important naturally occurring flavonoids found in many edible naringenin and pinocembrin production have reported titers of fruits and plants, and they have far reaching applications in nu- 100 mg/l (21) and 97 mg/l (22), respectively, requiring matB trition and human health. The beneficial effects of flavonoids (malonyl-CoA synthetase) and matC (malonate carrier protein) have been well studied, and recent reviews have highlighted genes, or genes encoding the subunits of acetyl-CoA carboxyl- over 30 known applications for flavonoids in treating human ase (accA, accB, accC, and accD) to boost malonyl-CoA availability. disease, with properties ranging from anti-inflammatory, anti- Here, we demonstrate E. coli production chassis and opti- hyperlipidemic, anti-cancer, anti-malarial and anti-stroke dam- mized processes towards the overproduction of four key gate- age, to weight loss and radioprotection (DNA repair) (2–4). In ad- keeper flavonoids. Utilizing a semi-automated Design-Build- dition, flavonoids are attracting research interest as potential Test-Learn (DBTL) synthetic biology pipeline, we present micro- biomaterials in tissue engineering applications (5). Current in- bial production strains towards the de novo production of narin- dustry production of flavonoids comes from extraction from genin, pinocembrin, eriodictyol and homoeriodictyol, removing plant-based sources or de novo chemical synthesis; however, the need for precursor feeding. We report competitive produc- both routes have their disadvantages. The limited availability of tion titers for naringenin and pinocembrin, directly from glyc- flavonoids in plant tissues, prolonged and unpredictable crop erol with titers of 484 mg/l and 214 mg/l, respectively. We harvesting and multiple solvent-based purification steps affect demonstrate production of eriodictyol from caffeic acid (88 mg/ the yield and cost of production (6). In contrast, chemical syn- l). And finally, we show the first example of homoeriodictyol thesis requires the use of toxic solvents and extreme chemical production (17 mg/l) from central metabolism using glycerol as reaction conditions, which is unsustainable and yields a non- a carbon source, overcoming the need for trans-phenylacrylic green product, which can limit downstream applications (7). acid feedstocks, by coupling to a 3-step ferulic acid production Biosynthesis via microbial fermentation offers an attractive al- pathway. This study provides a platform for further down- ternative, with great strides having been made over the last 10 stream modification by enzymes to support the production of years in engineering biological systems to produce a number of natural and non-natural flavanones, flavones, flavonols and industrial targets. Engineering microbial hosts for the isoflavones. production of fine and specialty chemicals, including flavo- noids, offers an attractive and green route to these compounds 2. Materials and methods and has become an ongoing focus of the synthetic biology community (8, 9). 2.1 Bacterial strains and media Four major flavonoids, naringenin, pinocembrin, eriodictyol Escherichia coli DH5a (New England Biolabs) was used for routine and homoerydictiol, act as gatekeeper molecules due to their cloning and pathway propagation. Strains were maintained on pivotal positions at important branch points in plant flavonoid Lysogeny broth (LB) or LB agar containing antibiotics for plasmid biosynthesis pathways. Naringenin is commercially produced selection. Production experiments were conducted in a variety by extraction from grapefruit peel (Citrus  paradisi L.) a waste of media and included: Terrific broth (TB, Formedium TRB0102); product of the juicing process (10), pinocembrin is obtained TBP (TB phosphate-buffered, Formedium TBP0102); TBsb (TB from Populus and Euphorbia plants (11), and both eriodictyol and supplemented with 0.5 M sorbitol and 5 mM betaine), EZ (EZ- homoeriodictyol are flavanones primarily extracted from the rich defined medium kit, Teknova M2105), MOPS (prepared as mountain balm plant (Eriodictyon californicum) or produced by EZ but excluding 10 ACGU and 5 Supplement EZ), Super multi-step chemical synthesis (12). Homoeriodictyol has many Optimal Broth (SOB, Formedium SOB0202) and M9 (23). All me- health-promoting properties including anti-oxidation, anti-in- flammatory, anti-bacterial and anti-cancer effects (2, 3, 13). In dia were supplemented with either 0.4% w/v glucose or glycerol, m addition, homoeriodictyol has applications as a taste enhancer/ and antibiotics as appropriate: ampicillin (100 g/ml), kanamy- m m modifier for the food industry, and has the useful property of cin (50 g/ml) and/or chloramphenicol (20 g/ml). masking the bitter taste of foods without imparting any strong flavor of its own (14). In plants, flavanones are naturally pro- 2.2 Plasmid assembly duced from L-tyrosine (naringenin, eriodictyol, homoeriodic- The pathways were built using an automated ligase cycling re- tyol) or L-phenylalanine (pinocembrin), through the action of action assembly method as described previously (8, 9, 24). tyrosine ammonia-lyase (TAL) or phenylalanine ammonia-lyase Automated, worklist-driven liquid handling was implemented (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS) for bridging oligo pooling and ligase cycling reaction setup. and chalcone isomerase (CHI) (Figure 1A). Completed reactions were transformed into high-efficiency NEB Bioproduction routes to flavonoids production have also 5-alpha cells, and correct plasmid assemblies identified by been reported using trans-phenylacrylic acid precursors, such as high-throughput, next-generation sequencing using Nanopore coumaric acid, cinnamic acid, caffeic acid and ferulic acid, technology (18). Some assemblies (Supplementary Table S3) through metabolically engineered strains of Escherichia coli. were also verified using Sanger sequencing (GATC Biotech). Naringenin titers of 474 mg/l have been reported from coumaric Through this assembly pipeline, we typically obtained >70% of acid (2.6 mM) (15). Feeding cinnamic acid has resulted in pino- sequence-perfect plasmid targets at the first attempt. Routine cembrin
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