4-hydroxyphenylacetate 3-hydroxylase Research Articles

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives.

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented.

Efficient production of hydroxytyrosol by directed evolution of HpaB in Escherichia coli

Hydroxytyrosol (HT) is an olive-derived phenolic phytochemical that has gained increasing commercial interest due to its natural antioxidant properties. It is widely used in the field of food supplement and medicine. It is reported that 4-hydroxyphenylacetate 3-hydroxylase (EcHpaB) and flavin reductase (EcHpaC) from E. coli BL21(DE3) can successfully express and catalyze the production of HT from tyrosol. In this study, the tyrosol production strain YMG5∗R as chassis cells, and a random mutant library of EcHpaB was established using error-prone PCR to improve the ability of EcHpaB to convert tyrosol to HT. Finally, a highly efficient HT synthetic mutant strainYMG5∗R-HpaBTLEHC with high transformation efficiency was screened by directed evolution. The YMG5∗R-HpaBTLEHC strain efficiently converted 50 mM tyrosol, with a yield of hydroxytyrosol reaching 48.2 mM (7.43 g/L) and a space-time yield reached 0.62 g/L·h. Overall, our study demonstrates the successful development of a highly efficient synthetic enzyme mutant for the production of HT, which has the potential to significantly improve the commercial viability of this natural antioxidant.

Caffeic acid production from glucose using metabolically engineered Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) is a precursor for high-valued compounds with anticancer, antiviral activities, and anti-inflammatory making it an important substance in the food additive, cosmetics, and pharmaceutical industries. Here, we developed an engineered Escherichia coli strain capable of directly producing high levels of caffeic acid from glucose. Tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) and p-coumaric acid 3-hydroxylase from Saccharothrix espanaensis (SeC3H) were expressed. Next, feedback-resistant chorismate mutase/prephenate dehydrogenase, was introduced to promote l-tyrosine synthesis. This engineered strain CA3 produced 1.58 g/L of caffeic acid from glucose without tyrosine supplemented to the medium. Furthermore, to reduce p-coumaric acid accumulation, 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa (PaHpaBC) was introduced. Finally, an engineered strain CA8 directly produced 6.17 g/L of caffeic acid from glucose using a jar fermenter. The E. coli developed in this study would be helpful as a chassis strain to produce value-added caffeic acid-derivatives.

Engineering a Prokaryotic Non-P450 Hydroxylase for 3'-Hydroxylation of Flavonoids.

Plant-derived cytochrome P450-dependent flavonoid 3'-hydroxylases are the rate-limiting enzymes in flavonoid biosynthesis. In this study, the large component (HpaB) of a prokaryotic 4-hydroxyphenylacetate (4-HPA) 3-hydroxylase was engineered for efficient 3'-hydroxylation of naringenin. First, we selected four HpaBs through database search and phylogenetic analysis and compared their catalytic activities toward 4-HPA and naringenin. HpaB from Rhodococcus opacus B-4 (RoHpaB) showed better preference toward naringenin. To elucidate the underlying mechanism, we analyzed the structural differences of HpaBs through homologous modeling, molecular docking, and molecular dynamics simulation, and the substrate preference of RoHpaB was mainly attributed to the shorter chain length of loop 212-222 and the larger substrate binding pocket. RoHpaB was further engineered by alanine scanning and structural replacement, and the RoHpaBY215A variant was obtained, whose catalytic efficiency (kcat/Km) toward naringenin is 25.3 times higher than that of RoHpaB. In addition, RoHpaBY215A also showed significantly improved activity toward flavonoids apigenin and kaempferol. This work opens the possibility of using engineered HpaB as a versatile hydroxylase to produce functionalized flavonoids.

High-density fermentation of Escherichia coli to express 4-hydroxyphenylacetate 3-hydroxylase and efficient biosynthesis of caffeic acid

The 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H), originated from Escherichia coli, converts p-coumaric acid to caffeic acid. In order to improve the efficiency of caffeic acid biosynthesis, we engineered E. coli for overexpression of 4HPA3H. The high-density fermentation of the engineered E. coli was conducted in a 5 L bioreactor. Subsequently, the conditions for whole-cell biocatalysis were optimized. The dry cell weight of the 4HPA3H-expressed strain reached 34.80 g/L. After incubated in the bioreactor for 6 h, 18.74 g/L (0.85 g/(L·OD600)) of caffeic acid was obtained, with a conversion rate of 78.81% achieved. To the best of our knowledge, the titer of caffeic acid is the highest reported to date. The high-density fermentation of E. coli for overexpression of 4HPA3H and the efficient biosynthesis of caffeic acid may facilitate future large-scale production of caffeic acid.

Biosynthesis of eriodictyol from tyrosine by Corynebacterium glutamicum

BackgroundEriodictyol is a bioactive flavonoid compound that shows potential applications in medicine development and food processing. Microbial synthesis of eriodictyol has been attracting increasing attention due to several benefits. In this study, we employed a GRAS strain Corynebacterium glutamicum as the host to produce eriodictyol directly from tyrosine.ResultsWe firstly optimized the biosynthetic module of naringenin, the upstream intermediate for eriodictyol production, through screening of different gene orthologues. Next, to improve the level of the precursor malonyl-CoA necessary for naringenin production, we introduced matB and matC from Rhizobium trifolii into C. glutamicum to convert extracellular malonate to intracellular malonyl-CoA. This combinatorial engineering resulted in around 35-fold increase in naringenin production from tyrosine compared to the initial recombinant C. glutamicum. Subsequently, the hpaBC genes from E. coli encoding 4-hydroxyphenylacetate 3-hydroxylase were expressed in C. glutamicum to synthesize eriodictyol from naringenin. Further optimization of the biotransformation process parameters led to the production of 14.10 mg/L eriodictyol.ConclusionsThe biosynthesis of the ortho-hydroxylated flavonoid eriodictyol in C. glutamicum was achieved for the first time via functional expression of E. coli hpaBC, providing a baseline strain for biosynthesis of other complex flavonoids. Our study demonstrates the potential application of C. glutamicum as a host microbe for the biosynthesis of value-added natural compounds from tyrosine.

Development of an artificial biosynthetic pathway for biosynthesis of (S)-reticuline based on HpaBC in engineered Escherichia coli.

Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionallythese compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)-Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)-reticuline biosynthetic pathway based on 4-hydroxyphenylacetate 3-hydroxylase (HpaBC)in Escherichia coli. Then, we further improved the (S)-reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4-dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.

De novo biosynthesis of tyrosol acetate and hydroxytyrosol acetate from glucose in engineered Escherichia coli

Tyrosol and hydroxytyrosol derived from virgin olive oil and olives extract, have wide applications both as functional food components and as nutraceuticals. However, they have low bioavailability due to their low absorption and high metabolism in human liver and small intestine. Acetylation of tyrosol and hydroxytyrosol can effectively improve their bioavailability and thus increase their potential use in the food and cosmeceutical industries. There is no report on the bioproductin of tyrosol acetate and hydroxytyrosol acetate so far. Thus, it is of great significance to develop microbial cell factories for achieving tyrosol acetate or hydroxytyrosol acetate biosynthesis. In this study, a de novo biosynthetic pathway for the production of tyrosol acetate and hydroxytyrosol acetate was constructed in Escherichia coli. First, an engineered E. coli that allows production of tyrosol from simple carbon sources was established. Four aldehyde reductases were compared, and it was found that yeaE is the best aldehyde reductase for tyrosol accumulation. Subsequently, the pathway was extended for tyrosol acetate production by further overexpression of alcohol acetyltransferase ATF1 for the conversion of tyrosol to tyrosol acetate. Finally, the pathway was further extended for hydroxytyrosol acetate production by overexpression of 4-hydroxyphenylacetate 3-hydroxylase HpaBC.

Optimization of the Biosynthesis of B-Ring Ortho-Hydroxy Lated Flavonoids Using the 4-Hydroxyphenylacetate 3-Hydroxylase Complex (HpaBC) of Escherichia coli.

Flavonoids are important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho-hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L−1, an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.

A single amino acid substitution in aromatic hydroxylase (HpaB) of Escherichia coli alters substrate specificity of the structural isomers of hydroxyphenylacetate

BackgroundA broad range of aromatic compounds can be degraded by enteric bacteria, and hydroxyphenylacetic acid (HPA) degrading bacteria are the most widespread. Majority of Escherichia coli strains can use both the structural isomers of HPA, 3HPA and 4HPA, as the sole carbon source, which are catabolized by the same pathway whose associated enzymes are encoded by hpa gene cluster. Previously, we observed that E. coli B REL606 grew only on 4HPA, while E. coli B BL21(DE3) grew on 3HPA as well as 4HPA.ResultsIn this study, we report that a single amino acid in 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of E. coli determines the substrate specificity of HPA isomers. Alignment of protein sequences encoded in hpa gene clusters of BL21(DE3) and REL606 showed that there was a difference of only one amino acid (position 379 in HpaB) between the two, viz., Arg in BL21(DE3) and Cys in REL606. REL606 cells expressing HpaB having Arg379 could grow on 3HPA, whereas those expressing HpaB with Gly379 or Ser379 could not. Structural analysis suggested that the amino acid residue at position 379 of HpaB is located not in the active site, but in the vicinity of the 4HPA binding site, and that it plays an important role in mediating the entrance and stable binding of substrates to the active site.ConclusionsThe arginine residue at position 379 of HpaB is critical for 3HPA recognition. Information regarding the effect of amino acid residues on the substrate specificity of structural isomers can facilitate in designing hydoxylases with high catalytic efficiency and versatility.

Tuning of pKa values activates substrates in flavin-dependent aromatic hydroxylases

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

Structural Insights into Catalytic Versatility of the Flavin-dependent Hydroxylase (HpaB) from Escherichia coli

4-Hydroxyphenylacetate 3-hydroxylase (EcHpaB) from Escherichia coli is capable of efficient ortho-hydroxylation of a wide range of phenolic compounds and demonstrates great potential for broad chemoenzymatic applications. To understand the structural and mechanistic basis of its catalytic versatility, we elucidated the crystal structure of EcHpaB by X-ray crystallography, which revealed a unique loop structure covering the active site. We further performed mutagenesis studies of this loop to probe its role in substrate specificity and catalytic activity. Our results not only showed the loop has great plasticity and strong tolerance towards extensive mutagenesis, but also suggested a flexible loop that enables the entrance and stable binding of substrates into the active site is the key factor to the enzyme catalytic versatility. These findings lay the groundwork for editing the loop sequence and structure for generation of EcHpaB mutants with improved performance for broader laboratory and industrial use.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg·L−1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.

Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY

BackgroundAs an environmental pollutant, 4-hydroxyphenylacetate (4-HPA) was a product of softwood lignin decomposition and was found in industrial effluents from olive oil production. Sulfobacillus acidophilus TPY was a moderately thermoacidophilic bacterium capable of degrading aromatic compounds including 4-HPA. The enzymes involved in the degradation of 4-HPA and the role of this strain in the bioremediation of marine pollutants need to be illustrated.Results3,4-dihydroxyphenylacetate dioxygenase (DHPAO) encoded by mhpB2 and two components of 4-hydroxydroxyphenylacetate (4-HPA) 3-hydroxylase encoded by hpaB and hpaC from S. acidophilus TPY, a moderately thermoacidophilic bacterium, involved in the degradation of 4-HPA possessed quite low amino acid sequence identity (22–53%) with other ever reported corresponding enzymes, which suggest their novelty. These two enzymes were expressed in E. coli and purified to homogeneity. DHPAO activity in E. coli was revealed by spraying with catechol or 3,4-dihydroxyphenylacetate (3,4-DHPA) on the colonies to make them turn brilliant yellow color. DHPAO possessed total activity of 7.81 U and 185.95 U/mg specific activity at the first minute when 3,4-DHPA was served as substrate. DHPAO was a thermophilic enzyme with optimum temperature of 50 °C and optimum substrate of 3,4-DHPA. The small component (HpaC) was a flavoprotein, and both HpaB and HpaC of 4-HPA 3-hydroxylase were NADH-dependent and essential in the conversion of 4-HPA to 3,4-DHPA. 4-HPA 3-hydroxylase possessed 3.59 U total activity and 27.37 U/mg specific activity at the first minute when enzymatic coupled assay with DHPAO was applied in the enzymatic determination.ConclusionsThe ability of this extreme environmental marine strain to degrade catechol and substituted catechols suggest its applications in the bioremediation of catechol and substituted catechols polluted marine environments.

Caffeic acid production enhancement by engineering a phenylalanine over‐producing Escherichia coli strain

Caffeic acid is a plant-specific phenylpropanoic acid with multiple health-improving effects reported, and its therapeutic derivatives have also been studied throughout the last decade. To meet its market need and achieve high-level production, microbial production of caffeic acid approaches have been developed in metabolically engineered Escherichia coli. In our previous work, we have established the first artificial pathway that realized de novo production of caffeic acid using E. coli endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HP3H). In this work, we exploited the catalytic potential of 4HPA3H in the whole-cell bioconversion study and produced 3.82 g/L (461.12 mg/L/OD) caffeic acid from p-coumaric acid, a direct precursor. We further engineered a phenylalanine over-producer into a tyrosine over-producer and then introduced the artificial pathway. After adjusting the expression strategy and optimizing the inoculants timing, de novo production of caffeic acid reached 766.68 mg/L. Both results from the direct precursor and simple carbon sources represent the highest titers of caffeic acid from microbial production so far.

The C-terminal Domain of 4-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii Is an Autoinhibitory Domain

p-Hydroxyphenylacetate (HPA) 3-hydroxylase from Acinetobacter baumannii consists of a reductase component (C(1)) and an oxygenase component (C(2)). C(1) catalyzes the reduction of FMN by NADH to provide FMNH(-) as a substrate for C(2). The rate of reduction of flavin is enhanced ∼20-fold by binding HPA. The N-terminal domain of C(1) is homologous to other flavin reductases, whereas the C-terminal domain (residues 192-315) is similar to MarR, a repressor protein involved in bacterial antibiotic resistance. In this study, three forms of truncated C(1) variants and single site mutation variants of residues Arg-21, Phe-216, Arg-217, Ile-246, and Arg-247 were constructed to investigate the role of the C-terminal domain in regulating C(1). In the absence of HPA, the C(1) variant in which residues 179-315 were removed (t178C(1)) was reduced by NADH and released FMNH(-) at the same rates as wild-type enzyme carries out these functions in the presence of HPA. In contrast, variants with residues 231-315 removed behaved similarly to the wild-type enzyme. Thus, residues 179-230 are involved in repressing the production of FMNH(-) in the absence of HPA. These results are consistent with the C-terminal domain in the wild-type enzyme being an autoinhibitory domain that upon binding the effector HPA undergoes conformational changes to allow faster flavin reduction and release. Most of the single site variants investigated had catalytic properties similar to those of the wild-type enzyme except for the F216A variant, which had a rate of reduction that was not stimulated by HPA. F216A could be involved with HPA binding or in the required conformational change for stimulation of flavin reduction by HPA.

Biosynthesis of caffeic acid in Escherichia coli using its endogenous hydroxylase complex

BackgroundCaffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources.ResultsWe first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation.ConclusionWe have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from caffeic acid. In addition, we have identified that TAL is the rate-limiting enzyme in this pathway. Thus, exploration for more active TALs via bio-prospecting and protein engineering approaches is necessary for further improvement of caffeic acid production.

Metabolic engineering of Escherichia coli for improving l-3,4-dihydroxyphenylalanine (l-DOPA) synthesis from glucose

L-3,4-dihydroxyphenylalanine (L-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of L-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of L-tyrosine (L-Tyr), an L-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on L-Tyr production of PTS inactivation (PTS(-) gluc(+) phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of L-Tyr production (q( L-Tyr)), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q( L-Tyr) in the PTS(+) and the PTS(-) gluc(+) strains, respectively. An 8.6-fold increase in L-Tyr yield from glucose was observed in the PTS(-) gluc(+) tyrR (-) strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for L-Tyr production caused the synthesis of L-DOPA. One of such strains, having the PTS(-) gluc(+) tyrR (-) phenotype, displayed the best production parameters in minimal medium, with a specific rate of L-DOPA production of 13.6 mg/g/h, L-DOPA yield from glucose of 51.7 mg/g and a final L-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of L-DOPA in 50 h.

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1-2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5-20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.