Optimization of tyrosol-producing pathway with tyrosine decarboxylase and tyramine oxidase in high-tyrosine-producing Escherichia coli.

Tyrosol (4-hydroxyphenylethanol) is a phenolic compound used in the pharmaceutical and chemical industries. However, current supply methods, such as extraction from natural resources and chemical synthesis, have disadvantages from the viewpoint of cost and environmental protection. Here, we developed a tyrosol-producing Escherichia coli cell factory from a high-tyrosine-producing strain by expressing selected tyrosine decarboxylase-, tyramine oxidase (TYO)-, and medium-chain dehydrogenase/reductase (YahK)-encoding genes. The genes were controlled by the strong T7 promoter and integrated into the chromosome because of the advantages over plasmid-based systems. The strain produced a melanin-like pigment as a by-product, which is suggested to be formed from 4-hydroxyphenylacetaldehyde (a TYO product/YahK substrate). By using a culture medium containing a high concentration of glycerol, which was reported to enhance NADH supply required for YahK activity, the final titer of tyrosol reached 2.42 g/L in test tube-scale cultivation with a concomitant decrease in the amount of pigment. These results indicate that chromosomally integrated and T7 promoter-controlled gene expression system in E. coli is useful for high production of heterologous enzymes and might be applied for industrial production of useful compounds including tyrosine and tyrosol.

Engineered Escherichia coli platforms for tyrosine-derivative production from phenylalanine using phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system.

BACKGROUND: Aromatic compounds derived from tyrosine are important and diverse chemicals that have industrial and commercial applications. Although these aromatic compounds can be obtained by extraction from natural producers, their growth is slow, and their content is low. To overcome these problems, many of them have been chemically synthesized from petroleum-based feedstocks. However, because of the environmental burden and depleting availability of feedstock, microbial cell factories are attracting much attention as sustainable and environmentally friendly processes. RESULTS: To facilitate development of microbial cell factories for producing tyrosine derivatives, we developed simple and convenient tyrosine-producing Escherichia coli platforms with a bacterial phenylalanine hydroxylase, which converted phenylalanine to tyrosine with tetrahydromonapterin as a cofactor, using a synthetic biology approach. By introducing a tetrahydrobiopterin-regeneration system, the tyrosine titer of the plasmid-based engineered strain was 4.63 g/L in a medium supplemented with 5.00 g/L phenylalanine with a test tube. The strains were successfully used to produce industrially attractive compounds, such as tyrosol with a yield of 1.58 g/L by installing a tyrosol-producing module consisting of genes encoding tyrosine decarboxylase and tyramine oxidase on a plasmid. Gene integration into E. coli chromosomes has an advantage over the use of plasmids because it increases genetic stability without antibiotic feeding to the culture media and enables more flexible pathway engineering by accepting more plasmids with artificial pathway genes. Therefore, we constructed a plasmid-free tyrosine-producing platform by integrating five modules, comprising genes encoding the phenylalanine hydroxylase and tetrahydrobiopterin-regeneration system, into the chromosome. The platform strain could produce 1.04 g/L of 3,4-dihydroxyphenylalanine, a drug medicine, by installing a gene encoding tyrosine hydroxylase and the tetrahydrobiopterin-regeneration system on a plasmid. Moreover, by installing the tyrosol-producing module, tyrosol was produced with a yield of 1.28 g/L. CONCLUSIONS: We developed novel E. coli platforms for producing tyrosine from phenylalanine at multi-gram-per-liter levels in test-tube cultivation. The platforms allowed development and evaluation of microbial cell factories installing various designed tyrosine-derivative biosynthetic pathways at multi-grams-per-liter levels in test tubes.

Ergothioneine production by Corynebacterium glutamicum harboring heterologous biosynthesis pathways.

In this study, Corynebacterium glutamicum was engineered to produce ergothioneine, an amino acid derivative with high antioxidant activity. The ergothioneine biosynthesis genes, egtABCDE, from Mycolicibacterium smegmatis were introduced into wild-type and l-cysteine-producing strains of C. glutamicum to evaluate their ergothioneine production. In the l-cysteine-producing strain, ergothioneine production reached approximately 40 mg L-1 after 2 weeks, and the amount was higher than that in the wild-type strain. As C. glutamicum possesses an ortholog of M. smegmatis egtA, which encodes an enzyme responsible for γ-glutamyl-l-cysteine synthesis, the effect of introducing egtBCDE genes on ergothioneine production in the l-cysteine-producing strain was evaluated, revealing that a further increase to more than 70 mg L-1 was achieved. As EgtBs from Methylobacterium bacteria are reported to use l-cysteine as a sulfur donor in ergothioneine biosynthesis, egtB from Methylobacterium was expressed with M. smegmatis egtDE in the l-cysteine-producing strain. As a result, ergothioneine production was further improved to approximately 100 mg L-1. These results indicate that utilization of the l-cysteine-producing strain and introduction of heterologous biosynthesis pathways from M. smegmatis and Methylobacterium bacteria are effective for improved ergothioneine production by C. glutamicum.

Biosynthetic Gene Cluster of Linaridin Peptides Contains Epimerase Gene.

Salinipeptins belong to the type-A linaridin class of ribosomally synthesized and post-translationally modified peptides (RiPPs) comprising 22 amino acid residues with multiple D-amino acids. Although chirality of other type-A linaridins, such as grisemycin and cypemycin, has not been reported, the biosynthetic gene clusters of type-A linaridins have identical gene organization. Here, we report heterologous expression of grisemycin biosynthetic gene cluster (grm) and show that grisemycin contains multiple D-amino acids, similar to salinipeptins. The heterologous expression experiments also confirm the involvement of a novel peptide epimerase in grisemycin biosynthesis. Gene-deletion experiments indicate that grmL, a single gene with unknown function, is indispensable for grisemycin production. We also show that the presence of D-amino acids is likely a common feature of linaridin natural products by analyzing two other type-A linaridin clusters.

High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains.

We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.

Recent advances in functional analysis of polyunsaturated fatty acid synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid are essential fatty acids for humans. PUFAs are biosynthesized by either desaturases/elongases from oleic acid or PUFA synthases from acetyl units. PUFA synthases are composed of three or four subunits, and each creates a specific PUFA even though the multiple catalytic domains in each subunit are very similar. We recently dissected these PUFA synthases by in vivo and in vitro experiments and elucidated how the enzymes control PUFA profiles. Moreover, for the first time, we converted a practical microalgal docosahexaenoic acid synthase into an eicosapentaenoic acid synthase based on the results.

Off-Loading Mechanism of Products in Polyunsaturated Fatty Acid Synthases.

Marine microorganisms de novo biosynthesize polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid by polyunsaturated fatty acid (PUFA) synthases composed of three or four polypeptides in a manner similar to fatty acid synthases (FASs). FASs usually possess thioesterase (TE) domains to release free fatty acids from acyl carrier protein (ACP)-tethered intermediates. Here, we investigated the off-loading mechanism with microalgal and bacterial PUFA synthases through in vivo and in vitro experiments. The in vitro experiments with acyltransferase (AT)-like domains and acyl-ACP substrates clearly demonstrated that the AT-like domains catalyzed the hydrolysis of acyl-ACPs to yield free fatty acids.

Crystal structures clarify cofactor binding of plant tyrosine decarboxylase.

Plant tyrosine decarboxylase (TyrDC) is a group II pyridoxal 5'-phosphate (PLP)-dependent decarboxylase that mainly catalyzes the decarboxylation of tyrosine to tyramine. This is biologically important for diverting essential primary metabolites into secondary metabolic pathways. Intensive studies have characterized the effective of PLP-binding and the substrate specificity of mammalian 3,4-dihydroxyphenyl-l-alanine (Dopa) decarboxylases, a member of group II PLP-dependent decarboxylase. However, the characteristics of PLP binding and substrate specificity of plant TyrDCs remain unknown. In this study, we focus on the PLP binding manner, and determined the crystal structures of the apo and PLP binding form of type II TyrDC from Papaver somniferum (PsTyrDCII and PsTyrDCII-PLP). The structures showed that, unlike mammalian Dopa decarboxylase, the binding of PLP does not induce distinct conformational changes of PsTyrDCII regarding the overall structure, but the PLP binding pocket displays conformational changes at Phe124, His203 and Thr262. Combining structural comparation and the obtained biochemical findings, it is demonstrated that PsTyrDCII does not binds PLP tightly. Such characteristics of PLP binding may be required by its catalytic reaction and substrate binding. The activity of TyrDC probably regulated by the concentration of PLP in cells.

Subtle Control of Carbon Chain Length in Polyunsaturated Fatty Acid Synthases.

Some marine bacteria synthesize docosahexaenoic acid (DHA; C22) and eicosapentaenoic acid (EPA; C20) by enzyme complexes composed of four subunits (A-D). We recently revealed that ß-ketoacyl synthase (KSC)/chain length factor (CLF)-like domains in the "C" subunit of DHA synthase catalyzed the last elongation step (C20 to C22) even though their amino acid sequences are very similar to those of EPA synthase. To investigate the amino acid residues controlling the product chain length, conserved residues in the KSC/CLF-like domains in DHA synthase were replaced with corresponding EPA synthase residues. Among 12 mutants, two CLF-like domain-mutated genes completely lost DHA productivity and produced trace amounts of EPA when coexpressed with dha-ABD in Escherichia coli, whereas when coexpressed with epa-ABD, they produced the same amounts of EPA as epa-ABCD. These results suggest that the product profiles were subtly controlled by several amino acid residues.

Amino Acid Residues Recognizing Isomeric Glutamate Substrates in UDP- N-acetylmuramic acid-l-alanine-glutamate Synthetases.

We recently revealed that a previously unknown pathway for peptidoglycan biosynthesis operates in some microorganisms, including Xanthomonas oryzae. It involves two enzymes, MurD2 and MurL, which catalyze the ligation of l-glutamate (l-Glu) to UDP- N-acetylmuramic acid-l-alanine and the epimerization of the terminal l-Glu of the product, respectively. MurD2 of X. oryzae possesses a 26% identity with MurD of Escherichia coli (MurDec), which ligates d-Glu to UDP- N-acetylmuramic acid-l-alanine. To understand how X. oryzae MurD2 recognizes the isomer substrate, we estimated its structure based on that of MurDec during docking simulations. Several amino acid residues, which may be responsible for l-Glu recognition, were replaced with their corresponding amino acid residues in MurDec. Consequently, we obtained a mutated MurD2 enzyme that contained two amino acid substitutions and accepted only d-Glu as the substrate. We next tried to convert the substrate specificity of MurDec using the same strategy, but the mutant enzyme still accepted only d-Glu. Then, MurD of Streptococcus mutans (MurDsm), which possesses the key amino acid residue for l-Glu recognition identified in MurD2, was used for random screenings of mutant enzymes accepting l-Glu. We obtained a mutated MurDsm that had one amino acid substitution and slightly accepted l-Glu. A mutated MurDec possessing the corresponding one amino acid substitution also accepted l-Glu. Thus, we revealed that a few amino acid residues in MurD/MurD2 might control the acceptability of substrates with different stereochemistries.

Control Mechanism for Carbon-Chain Length in Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are essential fatty acids. PUFA synthases are composed of three to four subunits and each create a specific PUFA without undesirable byproducts. However, detailed biosynthetic mechanisms for controlling final product profiles have been obscure. Here, the bacterial DHA and EPA synthases were carefully dissected by in vivo and in vitro experiments. In vitro analysis with two KS domains (KSA and KSC ) and acyl-acyl carrier protein (ACP) substrates showed that KSA accepted short- to medium-chain substrates while KSC accepted medium- to long-chain substrates. Unexpectedly, condensation from C18 to C20 , the last elongation step in EPA biosynthesis, was catalyzed by KSA domains in both EPA and DHA synthases. Conversely, condensation from C20 to C22 , the last elongation step for DHA biosynthesis, was catalyzed by the KSC domain in DHA synthase. KSC domains therefore determine the chain lengths.

Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli.

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (L-cysteine (Cys), L-histidine, and L-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Control Mechanism for cis Double-Bond Formation by Polyunsaturated Fatty-Acid Synthases.

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final-product profiles are still obscure. In this study, the FabA-type dehydratase domain (DHFabA ) in the C-subunit and the polyketide synthase-type dehydratase domain (DHPKS ) in the B-subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C4 - to C8 -acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DHPKS and DHFabA , depending on the carbon-chain length, to introduce either saturation or cis double bonds to growing acyl chains.

Ergothioneine production with Aspergillus oryzae.

To establish a reliable and practical ergothioneine (ERG) supply, we employed fermentative ERG production using Aspergillus oryzae, a fungus used for food production. We heterologously overexpressed the egt-1 and -2 genes of Neurospora crassa in A. oryzae and succeeded in producing ERG (231.0 mg/kg of media, which was 20 times higher than the wild type). Abbreviations: ERG: ergothioneine; HER: hercynine; Cys-HER: hercynylcysteine-sulfoxide; SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; l-His: l-histidine; l-Cys: l-cysteine; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry.

Heterologous and High Production of Ergothioneine in Escherichia coli.

Ergothioneine (ERG) is a histidine-derived thiol compound suggested to function as an antioxidant and cytoprotectant in humans. Therefore, experimental trials have been conducted applying ERG from mushrooms in dietary supplements and as a cosmetic additive. However, this method of producing ERG is expensive; therefore, alternative methods for ERG supply are required. Five Mycobacterium smegmatis genes, egtABCDE, have been confirmed to be responsible for ERG biosynthesis. This enabled us to develop practical fermentative ERG production by microorganisms. In this study, we carried out heterologous and high-level production of ERG in Escherichia coli using the egt genes from M. smegmatis. By high production of each of the Egt enzymes and elimination of bottlenecks in the substrate supply, we succeeded in constructing a production system that yielded 24 mg/L (104 µM) secreted ERG.

N-Phenylacetylation and Nonribosomal Peptide Synthetases with Substrate Promiscuity for Biosynthesis of Heptapeptide Variants, JBIR-78 and JBIR-95.

JBIR-78 (1) and JBIR-95 (2), both of which are heptapeptide derivatives isolated from Kibdelosporangium sp. AK-AA56, have the same amino acid sequences except for the second amino acid: phenylacetic acid (Paa)-l-Val-d-Asp (1)/d-cysteic acid (2)-l-Ala-(3S)-3-hydroxy-d-Leu-Gly-d-Ala-l-Phe. Heterologous expression of the biosynthetic gene cluster including genes encoding nonribosomal peptide synthetases (NRPS) and in vitro assays with recombinant Orf3, an l-cysteic acid synthase homologue, suggested the single A domain in module 2 activates both l-Asp and l-cysteic acid to yield 1 and 2, respectively, although the substrate specificities of the A domains of NRPSs are usually strict. Biosynthetic mechanism of introduction of N-terminal Paa was also investigated. Recombinant Orf1 and Orf2 similar to subunits of pyruvate dehydrogenase complex catalyzed the conversion of phenylpyruvate into phenylacetyl-CoA together with dihydrolipoyl dehydrogenase whose encoding gene is located outside of the gene cluster. Moreover, we showed that phenylacetyl-CoA was directly condensed with l-Val, which was tethered to a peptidyl carrier protein, at the first condensation domain in the NRPS.

A Glycopeptidyl-Glutamate Epimerase for Bacterial Peptidoglycan Biosynthesis.

d-Glutamate (Glu) supplied by Glu racemases or d-amino acid transaminase is utilized for peptidoglycan biosynthesis in microorganisms. Comparative genomics has shown that some microorganisms, including Xanthomonas oryzae, perhaps have no orthologues of these genes. We performed shotgun cloning experiments with a d-Glu auxotrophic Escherichia coli mutant as the host and X. oryzae as the DNA donor. We obtained complementary genes, XOO_1319 and XOO_1320, which are annotated as a hypothetical protein and MurD (UDP-MurNAc-l-Ala-d-Glu synthetase), respectively. By detailed in vitro analysis, we revealed that XOO_1320 is an enzyme to ligate l-Glu to UDP-MurNAc-l-Ala, providing the first example of MurD utilizing l-Glu, and that XOO_1319 is a novel enzyme catalyzing epimerization of the terminal l-Glu of the product in the presence of ATP and Mg2+. We investigated the occurrence of XOO_1319 orthologues and found that it exists in some categories of microorganisms, including pathogenic ones.

Enhanced production of polyunsaturated fatty acids by enzyme engineering of tandem acyl carrier proteins.

In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity.

In vitro synthesis of polyhydroxyalkanoates using thermostable acetyl-CoA synthetase, CoA transferase, and PHA synthase from thermotorelant bacteria.

Thermostable enzymes are required for the rapid and sustainable production of polyhydroxyalkanoate (PHA) in vitro. The in vitro synthesis of PHA using the engineered thermostable synthase PhaC1SG(STQK) has been reported; however, the non-thermostable enzymes acetyl-CoA synthetase (ACS) and CoA transferase (CT) from mesophilic strains were used as monomer-supplying enzymes in this system. In the present study, acs and ct were cloned from the thermophilic bacteria Pelotomaculum thermopropionicum JCM10971 and Thermus thermophilus JCM10941 to construct an in vitro PHA synthesis system using only thermostable enzymes. ACS from P. thermopropionicum (ACSPt) and CT from T. thermophilus (CTTt) were confirmed to have high thermostability, and their optimal temperatures were around 60°C and 75°C, respectively. The in vitro PHA synthesis was successfully performed by ACSPt, CTTt, PhaC1SG(STQK), and poly(3-hydroxybutyrate) [P(3HB)] was synthesized at 45°C. Furthermore, the yields of P(3HB) and P(lactate-co-3HB) at 37°C were 1.4-fold higher than those of the in vitro synthesis system with non-thermostable ACS and CT from mesophilic strains. Overall, the thermostable ACS and CT were demonstrated to be useful for the efficient in vitro PHA synthesis at relatively high temperatures.

Exploring Peptide Ligase Orthologs in Actinobacteria-Discovery of Pseudopeptide Natural Products, Ketomemicins.

We recently identified a novel peptide ligase (PGM1), an ATP-grasp-ligase, that catalyzes amide bond formation between (S)-2-(3,5-dihydroxy-4-methoxyphenyl)-2-guanidinoacetic acid and ribosomally supplied oligopeptides in pheganomycin biosynthesis. This was the first example of an ATP-grasp-ligase utilizing peptides as nucleophiles. To explore the potential of this type of enzyme, we performed a BLAST search and identified many orthologs. The orthologs of Streptomyces mobaraensis, Salinispora tropica, and Micromonospora sp. were found in similar gene clusters consisting of six genes. To probe the functions of these genes, we heterologously expressed each of the clusters in Streptomyces lividans and detected novel and structurally similar pseudotripeptides in the broth of all transformants. Moreover, a recombinant PGM1 ortholog of Micromonospora sp. was demonstrated to be a novel dipeptide ligase catalyzing amide bond formation between amidino-arginine and dipeptides to yield tripeptides; this is the first report of a peptide ligase utilizing dipeptides as nucleophiles.