Engineering Escherichia coli to overproduce aromatic amino acids and derived compounds

Abstract

The production of aromatic amino acids using fermentation processes with recombinant microorganisms can be an advantageous approach to reach their global demands. In addition, a large array of compounds with alimentary and pharmaceutical applications can potentially be synthesized from intermediates of this metabolic pathway. However, contrary to other amino acids and primary metabolites, the artificial channelling of building blocks from central metabolism towards the aromatic amino acid pathway is complicated to achieve in an efficient manner. The length and complex regulation of this pathway have progressively called for the employment of more integral approaches, promoting the merge of complementary tools and techniques in order to surpass metabolic and regulatory bottlenecks. As a result, relevant insights on the subject have been obtained during the last years, especially with genetically modified strains of Escherichia coli. By combining metabolic engineering strategies with developments in synthetic biology, systems biology and bioprocess engineering, notable advances were achieved regarding the generation, characterization and optimization of E. coli strains for the overproduction of aromatic amino acids, some of their precursors and related compounds. In this paper we review and compare recent successful reports dealing with the modification of metabolic traits to attain these objectives.

Figure 1

Schematic representation of the AAA pathway in Escherichia coli including its transcriptional and allosteric regulatory control circuits. Central carbon metabolism intermediates and genes shown: PPP (pentose phosphate pathway); TCA (tricarboxylic acid cycle); E4P (erythrose-4-P); PGNL (6-phospho D-glucono-1,5-lactone); PEP (phosphoenolpyruvate); PYR (pyruvate); ACoA (acetyl-CoA); CIT (citrate); OAA (oxaloacetate); zwf (glucose 6-phosphate-1-dehydrogenase); tktA (transketolase I); pykA, pykF (pyruvate kinase II and pyruvate kinase I, respectively); lpdA, aceE, and aceF (coding for PYR dehydrogenase subunits); gltA (citrate synthase); pckA (PEP carboxykinase); ppc (PEP carboxylase); ppsA (PEP synthetase). Shikimate pathway intermediates and genes shown: DAHP (3-deoxy-D-arabino-heptulosonate-7-phosphate); DHQ (3-dehydroquinate); DHS (3-dehydroshikimate); SHK (shikimate); S3P (SHK-3-phosphate); EPSP (5-enolpyruvyl-shikimate 3-phosphate); CHA (chorismate); aroF, aroG, aroH (DAHP synthase AroF, AroG and AroH, respectively); aroB (DHQ synthase); aroD (DHQ dehydratase); aroE, ydiB (SHK dehydrogenase and SHK dehydrogenase / quinate dehydrogenase, respectively); aroA (3-phosphoshikimate-1-carboxyvinyltransferase); aroC (CHA synthase). Terminal AAA biosynthetic pathways intermediates and genes shown: ANT (anthranilate); PRANT (N-(5-phosphoribosyl)-anthranilate); CDP (1-(o-carboxyphenylamino)-1'-deoxyribulose 5'-phosphate); IGP ((1S,2R)-1-C-(indol-3-yl)glycerol 3-phosphate); trpE, trpD (ANT synthase component I and II, respectively); trpC (indole-3-glycerol phosphate synthase / phosphoribosylanthranilate isomerase); trpA (indoleglycerol phosphate aldolase); trpB (tryptophan synthase); PRE (prephenate); PPN (phenylpyruvate); HPP (4-hydroxyphenylpyruvate); tyrA, pheA (TyrA and PheA subunits of the CHA mutase, respectively); ilvE (subunit of the branched-chain amino acid aminotransferase); aspC (subunit of aspartate aminotransferase); tyrB (tyrosine aminotransferase). Continuous arrows show single enzymatic reactions, black dashed arrows show several enzymatic reactions, long-dashed blue arrows indicate allosteric regulation and dotted blue arrows indicate transcriptional repression. Adapted from EcoCyc database [1].

Figure 2

Biosynthetic pathways for the production of diverse aromatic metabolites by combination of heterologous expression modules with the overproduction of intermediates from SHK- and terminal AAA pathways in Escherichia coli. Salvianic acid from HPP: (a) hpaBC (codes for an endogenous hydroxylase) of E. coli and ldh (lactate dehydrogenase) of Lactobacillus pentosus[94]. 2S-pinocembrin from L-PHE and malonyl-CoA: (b) aroF and pheA ^fbr of E. coli; (c) PAL (phenylalanine ammonia lyase) of Rhodotorula glutinis and 4CL (4-coumarate-CoA ligase) of Petroselium crispum; (d) CHS (chalcone synthase) of Petunia x hybrida and CHI (chalcone isomerase) of Medicago sativa; (e) matB and matC (coding for malonate synthetase and malonate carrier protein) of Rhizobium trifolii[98]. δ-tocotrienol (f) via MGGBQ (2-methyl-6-geranylgeranyl-benzoquinol) (g) from HPP and δ-tocopherol via GGPP (geranylgeranylpyrophosphate): ggh (geranylgeranylpyrophosphate reductase) of Synechocystis sp., crtE (geranylgeranylpyrophosphate synthase) of Pantoea ananatis, hpt (homogentisate phytyltransferase) of Synechocystis sp., hpd (p-hydroxyphenylpyruvate dioxygenase) of Pseudomonas putida, vte1 (tocopherol-cyclase) of Arabidopsis thaliana[95], idi (isopentenyl-diphosphate isomerase) and dxs (1-deoxyxylulose-5-phosphate synthase) of E. coli[96]. Caffeic and ferulic acids from L-TYR: (h) TAL (tyrosine ammonia lyase) and Sam5 (4-coumarate hydroxylase) of Saccharothrix espanaensis and COM (caffeic acid methyltransferase) of Arabidopsis thaliana[103]; (i) TAL of R. glutinis and (j) Coum3H (4-coumarate hydroxylase) of S. espanaensis[104]. Resveratrol from L-TYR and malonyl-CoA: (k) TAL of R. glutinis and 4CL of P. crispum; (l) STS (stilbene synthase) of Vitis vinifera; (m) matB and matC of R. trifolii[99]. Deoxyviolacein and violacein from L-TRP: (n) vioABCD genes of Chromobacterium violaceum and (o) vioE of Janthinobacterium lividum[82]. Continuous arrows show unique enzymatic reactions; dashed arrows show several enzymatic reactions. GAP: glyceraldehyde-3-phosphate. c, indicates chromosomal integration. p, indicates plasmid expression module. ^fbr, feedback resistant gene. ^op, codon-optimized gene. ↱, promoter.

Figure 3

Biosynthetic pathways for the production of diverse aromatic metabolites by combination of heterologous expression modules with the overproduction of intermediates from SHK- and terminal L-TYR pathways in Escherichia coli. PDC (2-pyrone-4,6-dicarboxylic acid) from DHS and CHA: (a) aroF ^fbr and aroB of E. coli; (b) ubiC (chorismate pyruvate-lyase) and pobA (4-hydroxybenzoate hydroxylase) of E. coli and Pseudomonas putida, respectively; (c) LigAB (protocatechuate 4,5-dioxygenase) and LigC (CHMS dehydrogenase) of Sphingobium sp. SYK-6 and qutC (dehydroshikimate dehydratase) of Emericella (Aspergillus) nidulans[100]. (S)-Reticuline from L-TYR: (d) tyrA ^fbr, aroG ^fbr, tktA and ppsA of E. coli; (e) NCS (norcoclaurine synthetase) of Coptis japonica, TYR (tyrosinase) of Streptomyces castaneoglobisporus, DODC (DOPA decarboxylase) of Pseudomonas putida and MAO (monoamine oxidase) of Micrococcus luteus; (f) 6OMT (norcoclaurine 6-O-methyltransferase), 4′OMT (3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase) and CNMT (coclaurine-N-methyltransferase) of C. japonica[97]. Hydroxytyrosol from L-TYR via 3,4-DHPAA (3,4-dihydroxyphenylacetaldehyde): (g) PCD (pterin-4 alpha-carbinolamine dehydratase) and DHPR (dihydropteridine reductase) of human and TH (tyrosine hydroxylase) of mouse; (h) DDC (L-DOPA decarboxylase) of pig and TYO (tyramine oxidase) of M. luteus[106]. Avenanthramides AvnD [N-(4′-hydroxycinnamoyl)-anthranilic acid] and AvnF [N-(3′,4′-dihydroxycinnamoyl)-anthranilic acid] from L-TYR and ANT: AvnDF module, tal (tyrosine ammonia-lyase) of Rhodotorula glutinis, 4CL1 (4-coumarate-CoA ligase) of Nicotiana tabacum, COUA3H (SAM5) (p-coumarate 3-hydroxylase) of Saccharothrix espanesis, HCBT (hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase) of Dianthus caryophyllus and hpaBC (code for an endogenous hydroxylase) of E. coli. SHK and TYR modules contain endogenous genes of E. coli[56]. PCA (protocatechuate); CHMS (4-carboxy-2-hydroxymuconate-6-semialdehyde); CAFA (caffeate); CAF-CoA (caffeoyl-CoA); COUA (p-coumarate); COU-CoA (p-coumaroyl-CoA); adhP (alcohol dehydrogenase of E. coli). p, indicates plasmid expression module. ^fbr, feedback resistant gene. ^op, codon-optimized gene. ↱, promoter. NER, Non-enzymatic reaction.