Novel scheme for biosynthesis of aryl metabolites from L-phenylalanine in the fungus Bjerkandera adusta

Aryl metabolite biosynthesis was studied in the white rot fungus Bjerkandera adusta cultivated in a liquid medium supplemented with L-phenylalanine. Aromatic compounds were analyzed by gas chromatography-mass spectrometry following addition of labelled precursors ((14)C- and (13)C-labelled L-phenylalanine), which did not interfere with fungal metabolism. The major aromatic compounds identified were benzyl alcohol, benzaldehyde (bitter almond aroma), and benzoic acid. Hydroxy- and methoxybenzylic compounds (alcohols, aldehydes, and acids) were also found in fungal cultures. Intracellular enzymatic activities (phenylalanine ammonia lyase, aryl-alcohol oxidase, aryl-alcohol dehydrogenase, aryl-aldehyde dehydrogenase, lignin peroxidase) and extracellular enzymatic activities (aryl-alcohol oxidase, lignin peroxidase), as well as aromatic compounds, were detected in B. adusta cultures. Metabolite formation required de novo protein biosynthesis. Our results show that L-phenylalanine was deaminated to trans-cinnamic acid by a phenylalanine ammonia lyase and trans-cinnamic acid was in turn converted to aromatic acids (phenylpyruvic, phenylacetic, mandelic, and benzoylformic acids); benzaldehyde was a metabolic intermediate. These acids were transformed into benzaldehyde, benzyl alcohol, and benzoic acid. Our findings support the hypothesis that all of these compounds are intermediates in the biosynthetic pathway from L-phenylalanine to aryl metabolites. Additionally, trans-cinnamic acid can also be transformed via beta-oxidation to benzoic acid. This was confirmed by the presence of acetophenone as a beta-oxidation degradation intermediate. To our knowledge, this is the first time that a beta-oxidation sequence leading to benzoic acid synthesis has been found in a white rot fungus. A novel metabolic scheme for biosynthesis of aryl metabolites from L-phenylalanine is proposed.     

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.     

植物戊糖磷酸途径及其两个关键酶的研究进展

戊糖磷酸途径是植物体中糖代谢的重要途径,主要生理功能是产生供还原性生物合成需要的NADPH,可供核酸代谢的磷酸戊糖以及一些中间产物可参与氨基酸合成和脂肪酸合成等.葡萄糖-6-磷酸脱氢酶和6-磷酸葡萄糖酸脱氢酶是戊糖磷酸途径的两个关键酶,广泛的分布于高等植物的胞质和质体中.本文综述了植物戊糖磷酸途径及其两个关键酶的分子生物学的研究进展,讨论了该途径在植物生长发育和环境胁迫应答中的作用.     

Yeast factories for the production of aromatic compounds: from building blocks to plant secondary metabolites

The aromatic amino acid biosynthesis pathway is a source to a plethora of commercially relevant chemicals with very diverse industrial applications. Tremendous efforts in microbial engineering have led to the production of compounds ranging from small aromatic molecular building blocks all the way to intricate plant secondary metabolites. Particularly, the yeast Saccharomyces cerevisiae has been a great model organism given its superior capability to heterologously express long metabolic pathways, especially the ones containing cytochrome P450 enzymes. This review contains a collection of state-of-the-art metabolic engineering work devoted towards unraveling the mechanisms for enhancing the flux of carbon into the aromatic pathway. Some of the molecules discussed include the polymer precursor muconic acid, as well as important nutraceuticals (flavonoids and stilbenoids), and opium-derived drugs (benzylisoquinoline alkaloids).     

Phenylalanine catabolism in Archaeoglobus fulgidus VC-16

Evidence is presented for a pathway of phenylalanine catabolism in the hyperthermophilic archaeon Archaeoglobus fulgidus involving the following enzymes—phenylalanine:2-oxoglutarate aminotransferase, phenyllactate dehydrogenase, radical iron–sulphur 3-phenyllactyl-CoA dehydratase, phenylpropionyl-CoA dehydrogenase, aryl pyruvate ferredoxin oxidoreductase, ADP-forming acetyl-CoA synthetase and family III CoA-transferase. Hitherto amino acid degradation pathways involving radical iron–sulphur dehydratases have been characterised only in mesophilic clostridia and related bacteria. The difference here is that the pathway is not fermentative but coupled to sulphate reduction. Initial experiments also show the utilisation of tryptophan as a growth substrate and the decarboxylation of caffeate by cell extracts, suggesting the potential to catabolise different classes of aromatic compounds.     

Efficient biosynthesis of α-aminoadipic acid via lysine catabolism in Escherichia coli

α-Aminoadipic acid (AAA) is a nonproteinogenic amino acid with potential applications in pharmaceutical, chemical and animal feed industries. Currently, AAA is produced by chemical synthesis, which suffers from high cost and low production efficiency. In this study, we engineered Escherichia coli for high-level AAA production by coupling lysine biosynthesis and degradation pathways. First, the lysine-α-ketoglutarate reductase and saccharopine dehydrogenase from Saccharomyces cerevisiae and α-aminoadipate-δ-semialdehyde dehydrogenase from Rhodococcus erythropolis were selected by in vitro enzyme assays for pathway assembly. Subsequently, lysine supply was enhanced by blocking its degradation pathway, overexpressing key pathway enzymes and improving nicotinamide adenine dineucleotide phosphate (NADPH) regeneration. Finally, a glutamate transporter from Corynebacterium glutamicum was introduced to elevate AAA efflux. The final strain produced 2.94 and 5.64 g/L AAA in shake flasks and bioreactors, respectively. This work provides an efficient and sustainable way for AAA production.     

微生物降解4-羟基苯甲酸的研究进展

4-羟基苯甲酸(4HBA)是在自然界中广泛存在的芳香族化合物,也是很多天然产物和人工合成化合物的中间代谢产物。4HBA的代谢途径有原儿茶酸开环途径、脱碳酸途径和厌氧微生物的苯甲酰-CoA还原途径,以及尚未完全阐明的龙胆酸开环途径。从4HBA转化为龙胆酸的过程包含NIH重排反应步骤,本综述重点介绍NIH重排反应的研究进展并初步介绍了涉及4HBA降解过程中的酶。在本综述中,结合我们的研究工作介绍了一个嗜热Bacillus sp.B1菌株降解4HBA等芳香族化合物的代谢途径,最后对4HBA降解过程中的NIH重排反应研究进行了展望。     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Cloning and heterologous expression of two aryl-aldehyde dehydrogenases from the white-rot basidiomycete Phanerochaete chrysosporium

We identified two aryl-aldehyde dehydrogenase proteins (PcALDH1 and PcALDH2) from the white-rot basidiomycete Phanerochaete chrysosporium. Both PcALDHs were translationally up-regulated in response to exogenous addition of vanillin, one of the key aromatic compounds in the pathway of lignin degradation by basidiomycetes. To clarify the catalytic functions of PcALDHs, we isolated full-length cDNAs encoding these proteins and heterologously expressed the recombinant enzymes using a pET/Escherichia coli system. The open reading frames of both PcALDH1 and PcALDH2 consisted of 1503 nucleotides. The deduced amino acid sequences of both proteins showed high homologies with aryl-aldehyde dehydrogenases from other organisms and contained ten conserved domains of ALDHs. Moreover, a novel glycine-rich motif “GxGxxxG” was located at the NAD⁺-binding site. The recombinant PcALDHs catalyzed dehydrogenation reactions of several aryl-aldehyde compounds, including vanillin, to their corresponding aromatic acids. These results strongly suggested that PcALDHs metabolize aryl-aldehyde compounds generated during fungal degradation of lignin and various aromatic xenobiotics.     

Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture

For the complete biodegradation of a mixture of benzene, toluene, and p-xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis-glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis-glycol compounds by the action of toluate-cis-glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p-xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. (c) 1994 John Wiley & Sons, Inc.     

大肠杆菌高效合成L-苯甘氨酸的研究进展

自然界存在着多种氨基酸,除用于蛋白质合成的20种外,大量用于合成具有生物活性的物质,广泛应用于食品、医药等多个领域。其中,非天然芳香族氨基酸L-苯甘氨酸作为一种重要的组成单元广泛的应用于盘尼西林、维吉霉素S、原始霉素I等β-内酰胺类抗生素的生物合成当中。目前L-苯甘氨酸主要通过化学法合成,但该方法合成收率低、污染大,且不易得到单一手性的化合物。由于生物合成L-苯甘氨酸具有反应条件温和、产物立体选择性好的优势,因此受到了广泛的关注。通过对L-苯甘氨酸两条生物合成途径的解析,合成所需相关酶的筛选及辅因子平衡再生等,逐步形成了以苯乙酮酸、扁桃酸和L-苯丙氨酸为底物的合成线路。主要对L-苯甘氨酸的生物合成途径及生物合成策略展开综述,为研究者提供优化方向,以期为高效工业化生物合成L-苯甘氨酸提供理论参考。     

Regulation of enzyme synthesis in the aromatic amino acid pathway of Bacillus subtilus

The control of the synthesis of certain key enzymes of aromatic amino acid biosynthesis was studied. Tyrosine represses the first enzyme of the 3-deoxy-d-arabino heptulosonic acid 7-phosphate pathway, DAHP synthetase, as well as shikimate kinase and chorismate mutase about fivefold in cultures grown under conditions limiting the synthesis of the aromatic amino acids. A mixture of tyrosine and phenylalanine represses twofold further. Tryptophan does not appear to be involved in the control of these enzymes. The specific activity of at least one early enzyme, dehydroquinase, remains essentially constant under a variety of nutritional supplementations. Two enzymes in the terminal branches are repressed by the amino acids they help to synthesize: prephenate dehydrogenase can be repressed fourfold by tyrosine, and anthranilate synthetase can be repressed over 200-fold by tryptophan. There is no evidence that phenylalanine represses prephenate dehydratase. Regulatory mutants have been isolated in which various enzymes of the pathway are no longer repressible. One class is derepressed for several of the prechorismate enzymes, as well as chorismate mutase and prephenate dehydrogenase. In another mutant, several enzymes of tryptophan biosynthesis are no longer repressible. Thus, the rate of synthesis of enzymes at every stage of the pathway is under control of various aromatic amino acids. Tyrosine and phenylalanine control the synthesis of enzymes involved in the synthesis of the three aromatic amino acids. Each terminal branch is under the control of its end product.     

Bacterial aerobic degradation of benzene,toluene, ethylbenzene and xylene

Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the “upper” pathway are then mineralized by eitherortho-ormeta-ring cleavage (“lower” pathway). BTEX are relatively water-soluble and there-fore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.     

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC₅₀ 15 μM) and D3 for MhpB (IC₅₀ 110 μM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1 mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the β-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Genomic insights into the metabolic potential of the polycyclic aromatic hydrocarbon degrading sulfate‐reducing Deltaproteobacterium N47

Anaerobic degradation of polycyclic aromatic hydrocarbons (PAHs) is an important process during natural attenuation of aromatic hydrocarbon spills. However, knowledge about metabolic potential and physiology of organisms involved in anaerobic degradation of PAHs is scarce. Therefore, we introduce the first genome of the sulfate‐reducing Deltaproteobacterium N47 able to catabolize naphthalene, 2‐methylnaphthalene, or 2‐naphthoic acid as sole carbon source. Based on proteomics, we analysed metabolic pathways during growth on PAHs to gain physiological insights on anaerobic PAH degradation. The genomic assembly and taxonomic binning resulted in 17 contigs covering most of the sulfate reducer N47 genome according to general cluster of orthologous groups (COGs) analyses. According to the genes present, the Deltaproteobacterium N47 can potentially grow with the following sugars including d ‐mannose, d ‐fructose, d ‐galactose, α‐d ‐glucose‐1P, starch, glycogen, peptidoglycan and possesses the prerequisites for butanoic acid fermentation. Despite the inability for culture N47 to utilize NO₃^‐ as terminal electron acceptor, genes for nitrate ammonification are present. Furthermore, it is the first sequenced genome containing a complete TCA cycle along with the carbon monoxide dehydrogenase pathway. The genome contained a significant percentage of repetitive sequences and transposase‐related protein domains enhancing the ability of genome evolution. Likewise, the sulfate reducer N47 genome contained many unique putative genes with unknown function, which are candidates for yet‐unknown metabolic pathways.     

Bifunctional isocitrate-homoisocitrate dehydrogenase: a missing link in the evolution of beta-decarboxylating dehydrogenase

Beta-decarboxylating dehydrogenases comprise 3-isopropylmalate dehydrogenase, isocitrate dehydrogenase, and homoisocitrate dehydrogenase. They share a high degree of amino acid sequence identity and occupy equivalent positions in the amino acid biosynthetic pathways for leucine, glutamate, and lysine, respectively. Therefore, not only the enzymes but also the whole pathways should have evolved from a common ancestral pathway. In Pyrococcus horikoshii, only one pathway of the three has been identified in the genomic sequence, and PH1722 is the sole beta-decarboxylating dehydrogenase gene. The organism does not require leucine, glutamate, or lysine for growth; the single pathway might play multiple (i.e., ancestral) roles in amino acid biosynthesis. The PH1722 gene was cloned and expressed in Escherichia coli and the substrate specificity of the recombinant enzyme was investigated. It exhibited activities on isocitrate and homoisocitrate at near equal efficiency, but not on 3-isopropylmalate. PH1722 is thus a novel, bifunctional beta-decarboxylating dehydrogenase, which likely plays a dual role in glutamate and lysine biosynthesis in vivo.