Pseudomonas putida, mutant strain

A recent experiment compared for the first time pollutant degradation by chemotactic bacteria and nonchemotactic mutants [54]. The result suggested an important role of chemotaxis in the bioremediation of contaminated soils. In a heterogeneous system, in which naphthalene was supplied from a microcapillary, a 90% reduction in the initial amount of naphthalene took six hours with the chemotactic wild-type Pseudomonas putida PpG7, while a similar reduction with either a chemotaxis-negative or a nonmotile mutant strain took about five times longer. Only the systems inoculated with the chemotactic strain exhibited degradation rates in excess of the rate of naphthalene diffusion from the... 

Cyclohexadienol was prepared by Rickborn in 1970 from reaction of the epoxide of 1,4-cyclohexadiene with methyl lithium.100 A hydrate of naphthalene, 1-hydroxy-1,2-dihydro-naphthalene was prepared by Bamberger in 1895 by allylic bromination of O-acylated tetralol (1-hydroxy-l,2,3,4-tetrahydronaphthalene) followed by reaction with base.101 Hydrates of naphthalene and other polycylic aromatics are also available from oxidative fermentation of dihydroaromatic molecules, which occurs particularly efficiently with a mutant strain (UV4) of Pseudomonas putida.102,103 The hydrates are alcohols and they undergo acid-catalyzed dehydration to form the aromatic molecule by the same mechanism as other alcohols, except that the thermodynamic driving force provided by the aromatic product makes deprotonation of the carbocation (arenonium ion) a fast reaction, so that in contrast to simple alcohols, formation of the carbocation is rate-determining (Scheme 6).104,105... 

Strains of Pseudomonas putida are very versatile in metabolizing aromatic compounds, particularly to the corresponding 1,2-dihydro-l,2-diols. The hydroxylating enzyme of the P. putida mutant is not strongly substrate specific and alkyl, aryl and halogen functionalities are usually readily tolerated380. Thus, 4-bromobenzoic acid (1, R = Br) is converted to a. v-4-bro-mo-5,6-dihydroxy-l, 3-cyclohexadiene-l-carboxylic acid (2, R = Br) in 80% yield with 98% cc (determined by chiral NMR shift experiments on the 4-nitrobenzyl ester) 375. The absolute stereochemistry, (5R,6R), was determined by a single crystal X-ray analysis. 

Another example of microbial oxidation of aromatic systems, which provides chiral building blocks, is the formation of three optically active difluorinated m-l,6-dihydroxy-2.4-cyclohcxa-diene-1-carboxylates, 4, 5 and 6, from difluorobenzoates with a mutant strain of Pseudomonas putida JT 103381. 3 has been quantitatively converted to 4 and 5 in a ratio of 3 1. 

Transformation of 6,7-dihydro-5//-benzocycloheptene (10) by growing cultures of a mutant strain of Pseudomonas putida yielded optically active benzylic monohydroxylation 11 and dihydroxylation 12 products383. 

Quinazoline is oxidized rapidly by xanthine oxidase to quinazolin-4(3//)-one which subsequently is oxidized more slowly to quinazoline-2,4(l//,3//)-dione. Quinazoline is also oxidized to quinazolin-4(3//)-one by rabbit liver aldehyde oxidase but the reaction ceases within a short time it appears that quinazoline is able to inactivate the aldehyde oxida.se, Quinazolin-4(3//)-one is rapidly oxidized by aldehyde oxidase to quinazoline-2,4(l//,3//)-dione. Various 6-substituted and 6,7-disubstituted quinazolin-4(3//)-ones are quantitatively oxidized to the respective quinazoline-2,4(l//,3//)-diones by buttermilk xanthine o.xidase, ° Biotransformation of quinazoline by a mutant strain of the bacterium Pseudomonas putida gives quinazolin-4(3//)-one, c(s-5,6-dihydroquinazoline-5,6-diol, and cis-5,6,7,8-tetrahydroquinazoline-5,6-di-... 

Although the product from the transformation of toluene by mutants of Pseudomonas putida lacking dehydrogenase activity is the cis-2R,3S dihydrodiol, the cis-2S,3R dihydrodiol has been synthesized from 4-iodotoluene by a combination of microbiological and chemical reactions. P. putida strain UV4 was used to prepare both enantiomers of the ds-dihydrodiol, and iodine chemically removed with H2-Pd/C. Incubation of the mixture of enantiomers with P. putida NCIMB 8859 selectively degraded the 2R,3S compound to produce toluene cis-2S,3R dihydrodiol (Allen et al. 1995). A few illustrative syntheses using benzene and toluene cis-dihydrodiols are given. 

Using in vivo protein engineering not only mutant strains of Pseudomonas putida... 

The c/s-dihydroxylation reaction catalyzed by these dioxygenases is typically highly enantioselective (often >98% ee) and, as a result, has proven particularly useful as a source of chiral synthetic intermediates (2,4). Chiral cis-dihydrodiols have been made available commercially and a practical laboratory procedure for the oxidation of chlorobenzene to IS, 2S)-3-chlorocyclohexa-3,5-diene-l,2-c diol by a mutant strain of Pseudomonas putida has been published (6). Transformation with whole cells can be achieved either by mutant strains that lack the second enzyme in the aromatic catabolic pathway, cw-dihydrodiol dehydrogenase (E.C. 1.3.1.19), or by recombinant strains expressing the cloned dioxygenase. This biocatalytic process is scalable, and has been used to synthesize polymer precursors such as 3-hydroxyphenylacetylene, an intermediate in the production of acetylene-terminated resins (7). A synthesis of polyphenylene was developed by ICI whereby ftie product of enzymatic benzene dioxygenation, c/s-cyclohexa-3,5-diene-1,2-diol, was acetylated and polymerized as shown in Scheme 2 (8). 

P. putida is an appropriate host strain for DHCD production. The strain mt-2 (ATCC 33015) was isolated from soils in the early 1960s by Hosokawa and others (Nozaki et al. 1963). It is able to grow on mcfa-toluate as the sole carbon source owing to its pWWO plasmid. KT2440 is a mutant strain of mt-2, widely used as a host for Pseudomonas gene cloning and expression. The most important... 

Nakanishi et al obtained a purified 2,3-CTD from an aniline-assimilating bacterium, Pseudomonas sp. FK-8-2 [136]. The relative activity is different from that by Nozaki et al. [133] as shown by the activity order (relative activity) R = 3-Me- (1.1) > H (1) > 4-Me- (0.53) 4-Cl (0.03) > 3-F (0.02) 4-COOH (0). Kohler et al. studied the metapyrocatechase activity of the partially purified enzyme from a mutant Pseudomonas sp. strain HBPl Prep and found a specifically high activity to 2,3-dihydroxybiphenyl [137]. The order of the activity is 2,3-dihydroxybiphenyl (62.2) 3-propyl (3.24) > 3-Me- (1.59) > H- (1.0) 4-Me-catechol (0.08). Catalytic activity of modified enzymes by molecular cloning has been also studied by Kobatake et al. [138], Keil [139], Chang et al. [140, 141], Nishihara et al. [142], and Cerdan et al. [143]. Cerdan et al. reported that 2,3-CTD encoded by TOL Plasmid pWWO of Pseudomonas putida catalyzes ring cleavage of catechol, 3-Me- and 4-Me-catechols but shows only weak activity toward 4-Et-catechol. Two mutants of 2,3-CTD are able to oxidize 4-Et-catechol and another mutant exhibits only weak activity toward 3-Me-catechol but retains the ability to cleave catechol and 4-Me-catechol [143]. 

A BDS patent [106] was awarded for the use of biocatalysts belonging to the group of Pseudomonas, Flavobacterium, Enterobacter, Aeromonas, Bacillus, or Corynebac-terium. One of the strains P. putida was further developed by mutation of the parent strain to obtain organic solvent-resistant mutants [107], The mutated strains were screened by selective cultivation in the presence of 0.1% to 10% by volume (v/v) of concentrations of a toxic organic solvent. The specific mutated strains obtained were P. putida No. 69-1 (PERM BP-4519), P. putida No. 69-2 (PERM BP-4520), and P. putida No. 69-3 (PERM BP-4521). 

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