Metabolic engineering of microorganisms for production of aromatic compounds

Abstract

Metabolic engineering has been enabling development of high performance microbial strains for the efficient production of natural and non-natural compounds from renewable non-food biomass. Even though microbial production of various chemicals has successfully been conducted and commercialized, there are still numerous chemicals and materials that await their efficient bio-based production. Aromatic chemicals, which are typically derived from benzene, toluene and xylene in petroleum industry, have been used in large amounts in various industries. Over the last three decades, many metabolically engineered microorganisms have been developed for the bio-based production of aromatic chemicals, many of which are derived from aromatic amino acid pathways. This review highlights the latest metabolic engineering strategies and tools applied to the biosynthesis of aromatic chemicals, many derived from shikimate and aromatic amino acids, including L-phenylalanine, L-tyrosine and L-tryptophan. It is expected that more and more engineered microorganisms capable of efficiently producing aromatic chemicals will be developed toward their industrial-scale production from renewable biomass.

Keywords: Aromatic compounds; Metabolic engineering; Phenylalanine; Shikimate pathway; Synthetic biology; Tryptophan; Tyrosine.

Fig. 1

De novo biosynthesis of various aromatic compounds derived from the SHK and aromatic amino acid biosynthesis pathway. Abbreviations for metabolites: 4-HPPA: 4-hydroxyphenylpyruvate; AA: anthranilic acid; ABC: ATP-binding cassette transporter; l-ARA: l-arabinose; CHA: chorismate; CLB: cellobiose; DAHP: 3-deoxy-d-arabinoheptulosonate 7-phosphate; DHAP: dihydroxyacetone phosphate; DHQ: 3-dehydroquianate; DHS: 3-dehydroshikimate; E4P: erythrose 4-phosphate; EPSP: 5-enolpyruvyl-shikimate 3-phosphate; FBP: fructose 1,6-biphosphate; G3P: glyceraldehyde 3-phosphate; G6P: glucose 6-phosphate; GLC: glucose; GLY: glycerol; PCA: protocatechuic acid; PEP: phosphoenolpyruvate; l-PHE: l-phenylalanine; PP: pentose phosphate; PPA: phenylpyruvate; PTS: phosphotransferase system; PYR: pyruvate; S3P: shikimate-3-phosphate; SHK: shikimate; SMP: proton symporter; TCA: tricarboxylic acid; l-TRP: l-tryptophan; l-TYR: l-tyrosine; X5P: xylose 5-phosphate; XYL: xylose. Abbreviations for enzymes: 4ADCL: 4-amino-4-deoxychorismate lyase; 4-CL: 4-coumarate:CoA ligase; AAAH: aromatic amino acid hydroxylase; AAS: aromatic acetaldehyde synthase; AAT: aromatic amino acid transaminase; ACAR: aromatic carboxylic acid reductase; ADCS: aminodeoxychorismate synthase; ADH: alcohol dehydrogenase; AntABC: anthranilate 1,2-dioxygenase; AROE: shikimate dehydrogenase; AS: hydroquinone glucosyl transferase; BDC: 2,3-DHBA decarboxylase; C3H: p-coumarate 3-hydroxylase; C4H: cinnamic acid decarboxylase; CAR: carboxylic acid reductase; CCR: cinnamoyl-CoA reductase; CDO: catechol 1,2-dioxygenase; CHI: chalcone isomerase; CHS: chalcone synthase; Comt: caffeate O-methyltransferase; COMT: catechol-O-methyltransferase; CPR: cytochrome P450 reductase; CS: chorismate synthase; DAT: d-amino acid transferase; DBR: double bond reductase; DDC: l-DOPA decarboxylase; DHAPS: 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase; DHQD: 3-dehydroquianate dehydratase; DHQS: 3-dehydroquianate synthase; DHS-DH: 3-dehydroshikimate dehydratase; Dmd: d-mandelate dehydrogenase; ECH: feruloyl-CoA hydratase/lyase; EntA: 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase; EntB: isochorismatase; EntC: isochorismate synthase; EntD: phosphopantetheinyl transferase; EPSPS: 5-enolpyruvyl-shikimate 3-phosphate synthase; ER: enoate reductase; F3H: flavanone 3-hydroxylase; F3′H: flavonoid 3-hydroxylase; F3′5′H: F3′5′H-encoded 3′,5′-hydroxylase; FCS: feruloyl-CoA synthetase; FDC: ferulate decarboxylase; FLDH: d-phenyllactate dehydrogenase; FLS: flavanol synthase; FMO: flavin-containing monooxygenase; FNS: flavone synthase; GT2: gallic acid glucosyltransferase; GTF: glucosyltransferase; HA-DH: hydroxyacyl-dehydrogenase; HmaS: l-4-hydroxymandelate synthase; Hmo: l-4-hydroxymandelate oxidase; HPAH: 4-hydroxyphenylacetate 3-hydroxylase; HpgAT: l-4-(hydroxyl)-phenylglycine aminotransferase; HpgT: l-4-hydroxyphenylglycine aminotransferase; ICS: isochorismate synthase; IDH: indole acetic acid dehydrogenase; IpdC: indole-3-pyruvic acid decarboxylase; IPL: isochorismate pyruvate lyase; d-LDH: d-lactate dehydrogenase; l-LDH: l-lactate dehydrogenase; MNX1: 4-hydroxybenzoate 1-hydroxylase; PABAS: p-aminobenzoate synthase; Pad: phenolic acid decarboxylase; PAD: phenylacrylate decarboxylase; PAL: phenylalanine ammonia lyase; PCD: protocatechuic acid 4,5-dioxygenase; PHBD: 4-hydroxybenzoate decarboxylase; PHBH: p-hydroxybenzoate hydroxylase; QAD: quinate dehydrogenase; RAS: rosmarinic acid synthase; SDC: salicylate decarboxylase; SK: shikimate kinase; SMO: salicylate monooxygenase; STS: stilbene synthase; T-5H: tryptamine 5-hydroxylase; TAL: tyrosine ammonia-lyase; TDC: tryptophan decarboxylase; Tnase: tryptophanase; TPH: tryptophan 5-hydroxylase; TPL: tyrosine phenol-lyase; TrpE: anthranilate synthase; TYDC: tyrosine decarboxylase; TYO: tyramine oxidase; UbiC: chorismate lyase; VioA: l-tryptophan oxidase; VioB: iminophenyl-pyruvate dimer synthase; VioC: violacein synthase; VioD: protoviolaceinate synthase; VioE: violacein biosynthesis protein. Continuous arrows show single enzymatic reactions, and dashed arrows show multiple enzymatic reactions

Fig. 2

The metabolic engineering approaches to microbial production of SHK pathway derivatives. A microbial co-culture system developed for the biosynthesis of cis,cis-muconic acid from a mixture of glucose and xylose (shown in the upper panel). Metabolic design for the production of a series of chorismate-derived aromatic amines (shown in the bottom panel). Abbreviations for metabolites: 4-ACA: 4-aminocinnamic acid; 4-APAA: 4-aminophenyl acetic acid; 4-APE: 4-aminophenylethanol; 4-APEA: 4-aminophenylamine; 4-APhe: 4-aminophenylalanine; 4-APheAL: 4-aminophenyl acetaldehyde; 4-APheP: 4-aminophenyl pyruvate; AcCoA: acetyl-CoA; CA: catechol; CHA: chorismate; CMA: cis,cis-muconic acid; DAHP: 3-deoxy-d-arabino-heptulosonate 7-phosphate; DHQ: 3-dehydroquinate; DHS: 3-dehydroshikimate; E4P: erythrose 4-phosphate; F6P: fructose 6-phosphate; G6P: glucose 6-phosphate; GLC: glucose; GLY: glycerol; PCA: protocatechuic acid; PEP: phosphoenolpyruvate; l-PHE: l-phenylalanine; PYR: pyruvate; SHK: shikimate; TCA: tricarboxylic acid; l-TRP: l-tryptophan: l-TYR: l-tyrosine; X5P: xylose 5-phosphate; XYL: xylose. Abbreviations for enzymes: AAAD: aromatic amino acid decarboxylase; ADH: aldehyde dehydrogenase; ALDH: alcohol dehydrogenase; AroE: shikimate dehydrogenase; AroF: feedback sensitive DHAP synthase; AroG^fbr: feedback resistance isozyme of DHAP synthase; AroH: chorismate mutase; AT: aminotransferase; AroY: protocatechuate decarboxylase; AroZ: 3-dehydroshikimate dehydratase; CatA: catechol 1,2-dioxygenase; PapA: 4-amino-4-deoxychorismate synthase; PapB: 4-amino-4-deoxychorismate mutase; PapC: 4-amino-4-deoxyprephenate dehydrogenase; PDC: phenylpyruvate decarboxylase; PpsA: phosphoenolpyruvate synthase; ShiA: shikimate transporter, PAL: phenylalanine ammonia lyase; TktA: transketolase; YdiB: Quinate/shikimate dehydrogenase. The inactivated metabolic pathways are indicated by “X”. Dotted lines indicate feedback inhibition. Native metabolic pathways are indicated by black arrows, and non-native pathways are indicated by blue arrows. Multiple metabolic reactions are indicated by sequential arrows. Blue boxes represent feedback inhibition resistant mutants of endogenous enzymes. Pink line or pink boxes represent overexpressed enzymes

Fig. 3

The metabolic engineering approaches to microbial production of aromatic amino acid derivatives. A systematic study for the de novo microbial production of aromatic-containing polyester derived from phenylalanine pathway (shown in the upper panel). De novo biosynthesis of the aromatic plant natural products, fisetin and quercetin, derived from phenylalanine and tyrosine pathways (shown in the bottom panel). Abbreviations for metabolites: 3HB-CoA: 3-hydroxybutyryl-CoA; 4-HPPA: 4-hydroxyphenylpyruvic acid; Ac-CoA: acetyl-CoA; CHA: chorismate; DAHP: 3-deoxy-d-arabino-heptulosonate 7-phosphate; E4P: erythrose 4-phosphate; FUM: fumarate; G6P: glucose 6-phosphate; Na-3HB: sodium 3-hydroxybutyrate; p-CA: p-coumaric acid; p-CA-CoA: p-coumaroyl-CoA; PEP: phosphoenolpyruvate; l-PHE: l-phenylalanine; PhLA: d-phenyllactate; PhLA-CoA: d-phenyllactatyl-CoA; PPA: phenylpyruvate; PYR: pyruvate; SUC: succinate; l-TYR: l-tyrosine. Abbreviations for enzymes: 4CL: 4-coumaric acid CoA ligase; AdhE: aldehyde-alcohol dehydrogenase; Aro3: 3-deoxy-7-phosphoheptulonate synthase; Aro4: feedback resistance isozyme of phosphote-3-dehydro-3-deoxyheptonate aldolase; Aro7: feedback resistance isozyme of chorismate mutase; Aro10: transaminated amino acid decarboxylase; AroF: feedback sensitive DHAP synthase; AroG^fbr: feedback resistance isozyme of DHAP synthase; AspC: aspartate aminotransferase; C4H: cinnamic acid decarboxylase; CHI: chalcone isomerase; CHR: chalcone reductase; CHS: chalcone synthase; CPR: cytochrome P450 reductase; F3H: flavanone 3-hydroxylase; FldA: cinnamoyl-CoA:phenyllactate CoA-transferase; FldH: d-phenyllactate dehydrogenase; FLS: flavanol synthase; FMO: flavonoid 3′-monooxygenase; FrdB: fumarate reductase iron–sulfur subunit; HadA: isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase; LdhA: d-lactate dehydrogenase; PAL: phenylalanine ammonia lyase; PflB: formate acetyltransferase 1; Pdc5: pyruvate decarboxylase isozyme; PhaA: acetyl-CoA acetyltransferase; PhaB: acetoacetyl-CoA reductase; PhaC: poly(3-hydroxyalkanoate) polymerase subunit; PhaC1437: variant of PhaC; PheA^fbr: feedback resistance isozyme of bifunctional chorismate mutase/prephenete dehydratase; PoxB: pyruvate dehydrogenase; TAL: tyrosine ammonia lyase; TyrA: prephenate dehydrogenase; TyrB: tyrosine aminotransferase; TyrR: transcriptional regulatory protein 2. The inactivated metabolic pathways are indicated by “X”. Native metabolic pathways are indicated by black arrows, and non-native pathways are indicated by blue arrows. Sequential arrows indicate multiple metabolic reactions. Orange eclipse displays other native metabolism. Blue boxes represent feedback inhibition resistant mutants of endogenous enzymes. Pink line or pink boxes represent overexpressed enzymes. Green boxes represent aromatic polymers