Bacterial formation of hydroxylated aromatic compounds

As stated in the introduction of this thesis, hydroxylated aromatic compounds in general are of great importance for various industries as for instance pharmaceutical, agrochemical and petrochemical industries. Since these compounds can not be isolated in sufficient amounts from natural resources, they have to be synthesized. Chemical synthesis of hydroxylated aromatics is often a difficult task. Direct hydroxylation methods can only be achieved under extreme conditions, while indirect methods often are laborious multi-step processes. Biotechnological formation methods for hydroxylated aromatic compounds are a promissing alternative to the cumbersome organic chemical endeavours. The bioformation of hydroxylated aromatics in principle can be accomplished in four different ways: along biosynthetic routes, by means of direct hydroxylation methods, by replacement of substituents by hydroxyl groups, and by addition and/or modification reactions of side-chains. This research was done to investigate the potential of bacteria or enzymes thereof to form hydroxylated aromatics.To obtain a microorganism which hydroxylates D-phenyIglycine regio- and stereospecifically yielding D-4-hydroxyphenyIglycine, various bacteria were isolated on D-phenyIglycine as sole carbon and energy source. Unfortunately, however, none of the isolates was able to hydroxylate phenylglycine (chapters 1 and 2). Experiments with whole cells and cell extracts showed that the side chain was modified before hydroxylation of the aromatic ring occurred. One of the isolates, Pseudomonas putida LW-4, was also able to grow on D-4-hydroxyphenyIglycine and it was shown that this compound was initially degraded by means of an enantioselective transaminase. Preliminary experiments with partially purified extracts have demonstrated that this reversible enzyme can be used to form D-4-hydroxyphenylglycine from 4-hydroxyphenylglyoxylate (chapter 4). To investigate D-4-hydroxyphenylglycine degradation in general, also other bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. One of these isolates, Pseudomonas putida MW27, possessed a D-selective as well as a L-selective 4-hydroxyphenylglycine transaminase (chapter 5). Evidently some microorganisms transaminate both enantiomers of 4-hydroxyphenylglycine and thus are less suitable for the formation of D-4-hydroxyphenylglycine by means of a trans amination.To apply bacteria or enzymes thereof for the hydroxylation of phenylacetate and/or certain hydroxyphenylacetates a thorough knowledge concerning the bacterial metabolism of these compounds is needed. In chapter 6 the degradation of 4-hydroxyphenylacetate by a Xanthobacter species is described and it is shown that this strain can convert 4-hydroxyphenylacetate to 2,5-dihydroxyphenylacetate (homogentisate). To accomplish a formation of homogentisate by whole cells, further degradation of homogentisate had to be blocked by metalchelators. In chapter 7 the degradation of DL-phenylhydracrylic acid and metabolites thereof, by a Flavobacterium species is described. In the presence of dipyridyl these cells converted both 3- and 4-hydroxyphenylacetate to homogentisate. As stated in chapter 7, the internal regeneration of reduction equivalents by using starting compounds which are more reduced than the compound to be hydroxylated, might be an interesting alternative to the simple addition of cosubstrates.The bioformation of cis- ,2-dihydroxycyclohexa-3,5-diene (cis-benzeneglycol) from benzene illustrates the potential of biotransformations. The chemical synthesis of cis-benzeneglycol consists of several steps with a very low yield, whereas the biological formation is a one step process with a high yield. Continuous bioformation of cis-benzeneglycol from benzene by mutant cells growing on succinate under nitrogen-limited conditions in a chemostat, was easily achieved (chapter 8). In order to predict the cis-benzeneglycol concentration at various times, a mathematical model was developed that fitted rather well for both benzene-transport-limited and kinetically limited production conditions. This continuous process, however, resulted in two products: cis-benzeneglycol and cells. In order to make the continuous process economically more attractive, it is necessary to reuse the produced cells. Another problem encountered during the bioproduction of cis-benzeneglycol was the toxicity of benzene; a low benzene concentration was a prerequisite for good performance of the bioconversion process. Incubation experiments with the cis-benzeneglycol-producing mutant showed that hexadecane is a suitable solvent to circumvent benzene toxicity (chapter 9). Moreover, the addition of hexadecane did not significantly effect the rate of cis-benzeneglycol formation.Chapters 10, 11 and 12 deal with the bioformation of 4-hydroxybenzoate from various 4-halobenzoates. Bioformation of 4-hydroxybenzoate was only achieved when whole cells were incubated with the specified 4-halobenzoates under conditions of low and controlled oxygen concentrations. Surprisingly no formation of 4- hydroxybenzoate occurred under anaerobic conditions, this in spite of the fact that such dehalogenases have been demonstrated to be hydrolytic. 4-Hydroxybenzoate was also formed from 2,4-dichlorobenzoate. This latter compound was initially reductively dechlorinated to 4-chlorobenzoate which in turn was converted to 4-hydroxybenzoate (chapter 11). In order to study the feasibility of continuous bioproduction of hydroxyaromatics from haloaromatics, the bioconversion of 4-chlorobenzoate to 4-hydroxybenzoate by cells immobilized in carrageenan was used as a model system. At air saturation the rate of dechlorination was rapidly limited by internal oxygen transport. However, high oxygen concentration resulted in maximal 4-chlorobenzoate dehalogenation, while 4-hydroxybenzoate formation under these conditions was negligible. Consequently, the oxygen concentration has to be strictly controlled to obtain a good production of 4-hydroxybenzoate at an acceptable rate