Elías R. Olivera

The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l-Phenylalanine, l-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

The phenylacetyl‐CoA catabolon: a complex catabolic unit with broad biotechnological applications

The term catabolon was introduced to define a complex functional unit integrated by different catabolic pathways, which are, or could be, co‐ordinately regulated, and that catalyses the transformation of structurally related compounds into a common catabolite. The phenylacetyl‐CoA catabolon encompasses all the routes involved in the transformation of styrene, 2‐phenylethylamine, trans‐styrylacetic acid, phenylacetaldehyde, phenylacetic acid, phenylacetyl amides, phenylacetyl esters and n‐phenylalkanoic acids containing an even number of carbon atoms, into phenylacetyl‐CoA. This common intermediate is subsequently catabolized through a route of convergence, the phenylacetyl‐CoA catabolon core, into general metabolites. The genetic organization of this central route, the biochemical significance of the whole functional unit and its broad biotechnological applications are discussed.

Two different pathways are involved in the β-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications

In Pseudomonas putida U, the degradation of n‐alkanoic and n‐phenylalkanoic acids is carried out by two sets of β‐oxidation enzymes (βI and βII). Whereas the first one (called βI) is constitutive and catalyses the degradation of n‐alkanoic and n‐phenylalkanoic acids very efficiently, the other one (βII), which is only expressed when some of the genes encoding βI enzymes are mutated, catabolizes n‐phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the βI genes handicaps the growth of P. putida U in media containing n‐alkanoic or n‐phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n‐alkanoates as a result of the induction of βII, but they were still unable to catabolize n‐phenylalkanoates completely, as the βI‐FadBA enzymes are essential for the β‐oxidation of certain n‐phenylalkanoyl‐CoA derivatives when they reach a critical size. Owing to the existence of the βII system, mutants lacking βIfadB/A are able to synthesize new poly 3‐OH‐n‐alkanoates (PHAs) and poly 3‐OH‐n‐phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.

Production of 3-hydroxy-n-phenylalkanoic acids by a genetically engineered strain of Pseudomonas putida

Overexpression of the gene encoding the poly-3-hydroxy-n-phenylalkanoate (PHPhA) depolymerase (phaZ) in Pseudomonas putida U avoids the accumulation of these polymers as storage granules. In this recombinant strain, the 3-OH-acyl-CoA derivatives released from the different aliphatic or aromatic poly-3-hydroxyalkanoates (PHAs) are catabolized through the β-oxidation pathway and transformed into general metabolites (acetyl-CoA, succinyl-CoA, phenylacetyl-CoA) or into non-metabolizable end-products (cinnamoyl-CoA). Taking into account the biochemical, pharmaceutical and industrial interest of some PHA catabolites (i.e., 3-OH-PhAs), we designed a genetically engineered strain of P. putida U (P. putida U ΔfadBA-phaZ) that efficiently bioconverts (more than 80%) different n-phenylalkanoic acids into their 3-hydroxyderivatives and excretes these compounds into the culture broth.

Molecular cloning and expression in different microbes of the DNA encoding Pseudomonas putida U phenylacetyl-CoA ligase. Use of this gene to improve the rate of benzylpenicillin biosynthesis in Penicillium chrysogenum

The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of the pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defined medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in the quantities of benzylpenicillin accumulated in the broths (between 1.8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.

Phenylacetyl-coenzyme A is the true inducer of the phenylacetic acid catabolism pathway in Pseudomonas putida U

ABSTRACT Aerobic degradation of phenylacetic acid in Pseudomonas putida U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A [CoA] catabolon core). Induction of this route was analyzed by using different mutants specifically designed for this objective. Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds.

Genetic analyses and molecular characterization of the pathways involved in the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid in Pseudomonas putida U.

In Pseudomonas putida U two different pathways (Pea, Ped) are required for the conversion of 2-phenylethylamine and 2-phenylethanol into phenylacetic acid. The 2-phenylethylamine pathway (PeaABCDEFGHR) catalyses the transport of this amine, its deamination to phenylacetaldehyde by a quinohaemoprotein amine dehydrogenase and the oxidation of this compound through a reaction catalysed by a phenylacetaldehyde dehydrogenase. Another catabolic route (PedS(1)R(1)ABCS(2)R(2)DEFGHI) is needed for the uptake of 2-phenylethanol and for its oxidation to phenylacetic acid via phenylacetaldehyde. This implies the participation of two different two-component signal-transducing systems, two quinoprotein alcohol dehydrogenases, a cytochrome c, a periplasmic binding protein, an aldehyde dehydrogenase, a pentapeptide repeat protein and an ABC efflux system. Additionally, two accessory sets of elements (PqqABCDEF and CcmABCDEFGHI) are necessary for the operation of the main pathways (Pea and Ped). PqqABCDEF is required for the biosynthesis of pyrroloquinoline quinone (PQQ), a prosthetic group of certain alcohol dehydrogenases that transfers electrons to an independent cytochrome c; whereas CcmABCDEFGHI is required for cytochrome c maturation. Our data show that the degradation of phenylethylamine and phenylethanol in P. putida U is quite different from that reported in Escherichia coli, and they demonstrate that PeaABCDEFGHR and PedS(1)R(1)ABCS(2)R(2)DEFGHI are two upper routes belonging to the phenylacetyl-CoA catabolon.

Unusual PHA Biosynthesis

Unusual polyhydroxyalkanoates (UnPHAs) constitute a particular group of polyoxo(thio)esters belonging to the PHA family, which are tailored with uncommon monomers. Thus, unusual PHAs include (1) polyhydroxyalkanoates (PHAs) of microbial origin that have been synthesized either from natural monomers bearing different chemical functions, or from chemical derivatives of the natural ones and (2) PHAs obtained either by chemical synthesis or by physical modifications of naturally occurring polymers. Regarding their chemical structure, UnPHAs can be grouped in four different classes. Class 1 includes PHAs whose lateral chains contain double or triple bounds or/and different functional groups (methyl, methoxy, ethoxy, acetoxy, hydroxyl, epoxy, carbonyl, cyano, phenyl, nitrophenyl, phenoxy, cyanophenoxy, benzoyl, halogen atoms, etc.). Classes 2 and 3 have been established regarding the nature of the PHA backbone; whereas class 2 includes PHAs in which the length of the monomer participating in the oxoester linkage has been modified (the hydroxyl group to be esterified is not located at C-3), class 3 groups those polymers in which some oxoester linkages have been replaced by thioester functions (thioester-containing PHAs). Finally, class 4 includes those PHAs that have been manipulated chemically or physically. In this chapter we shall describe the chemical structure of unusual PHAs belonging to these four classes; we shall analyse their biosynthetic particularities (if any), and we shall discuss some of their characteristics and biotechnological applications.

Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway

Nineteen different steroid-degrading bacteria were isolated from soil samples by using selective media containing either cholesterol or deoxycholate as sole carbon source. Strains that assimilated cholesterol (17 COL strains) were gram-positive, belonging to the genera Gordonia, Tsukamurella, and Rhodococcus, and grew on media containing other steroids but were unable to use deoxycholate as sole carbon source. Surprisingly, some of the COL strains unable to grow using deoxycholate as sole carbon source were able to catabolize other bile salts (e.g., cholate). Conversely, strains able to grow using deoxycholate as the sole carbon source (two DOC isolates) were gram-negative, belonging to the genus Pseudomonas, and were unable to catabolize cholesterol and other sterols. COL and DOC were included into the corresponding taxonomic groups based on their morphology (cells and colonies), metabolic properties (kind of substrates that support bacterial growth), and genetic sequences (16S rDNA and rpoB). Additionally, different DOC21 Tn5 insertion mutants have been obtained. These mutants have been classified into two different groups: (1) those affected in the catabolism of bile salts but that, as wild type, can grow in other steroids and (2) those unable to grow in media containing any of the steroids tested. The identification of the insertion point of Tn5 in one of the mutants belonging to the second group (DOC21 Mut1) revealed that the gene knocked-out encodes an A-ring meta-cleavage dioxygenase needed for steroid catabolism.

Strategy for cloning large gene assemblages as illustrated using the phenylacetate and polyhydroxyalkanoate gene clusters

ABSTRACT We report an easy procedure for isolating chromosome-clustered genes. By following this methodology, the entire set of genes belonging to the phenylacetic acid (PhAc; 18-kb) pathway as well as those required for the synthesis and mobilization of different polyhydroxyalkanoates (PHAs; 6.4 kb) in Pseudomonas putida U were recovered directly from the bacterial chromosome and cloned into a plasmid for the first time. The transformation of different bacteria with these genetic constructions conferred on them the ability to either degrade PhAc or synthesize bioplastics (PHAs).