This high-throughput genetic technique can be easily applied to rapidly assigning functions to genes of any metabolic pathway. No adipic acid was detected in the 14B3 strain and in the SuperCos1 vector control strain. Small amounts are used as a. Two isolates were able to grow on both cyclopentanol and cyclohexanol. There are three scenarios to explain why a mutant strain could not convert cyclohexanol.
Adipic acid was also produced when cyclohexanone or ɛ-caprolactone was used as the substrate for the E. Polymolybdate species were responsible of the high selectivity to benzene, while on titania alone, only carbon dioxide was formed. Other insertions occurred either on the SuperCos1 vector or on another part of the insert on 8F6. A number of microorganisms are capable of oxidizing cyclic alcohols to dicarboxylic acids. The petri dish was sealed with Parafilm and incubated with the compound on the lid. The aldehyde dehydrogenases usually prevent toxicity of reactive aldehydes that formed as intermediates. Samples 100 μl were injected, and 210 nm was used for detection.
This suggested that the chnA gene could not be turned on in the ChnR null mutant, and the cyclohexanol substrate was not used. After monolayer formation, segregation of MoO 3 crystallites has been observed, with a significant loss in photo-oxidative dehydrogenation activity. The other five genes encode enzymes for the five steps leading from cyclohexanol to adipic acid Fig. Black arrows, six genes shown to be essential for conversion of cyclohexanol to adipic acid; gray arrows, three genes that are not essential for the conversion. Gene organization of the 17-kb cluster required for conversion of cyclohexanol to adipic acid in Acinetobacter sp. The culture was diluted every 2 to 10 days by replacing 10 ml of the culture with the same volume of S12 medium. ChnA is proposed to catalyze the conversion of cyclohexanol to cyclohexanone.
ChnD had 353 amino acid residues and belonged to zinc-dependent long-chain alcohol dehydrogenases. The vertical lines represent the beginning 0 and the end 17417 bp of the reported cluster. These cosmids contained inserts of 35 to 40 kb that were homologous to the cyclohexanone monooxygenase gene from Acinetobacter sp. In most insertion mutants, the pathway intermediate that accumulated was the compound prior to the step blocked by the transposon insertion. These nine genes were arranged in two sets.
The ChnA mutant appears to fit the third scenario since ChnA has homology to alcohol dehydrogenases. Adipic acid was detected after 2 h of incubation and had increased more than 10-fold by 20 h Fig. Identification of adipic acid production. The in vivo substrate specificity of ChnA and ChnD was unambiguously determined by the differently accumulated intermediates in the ChnA and ChnD mutants. The cosmid library of Acinetobacter sp.
Nine full-length genes residing on the 14-kb insert conferred on E. The vertical arrow with the plus sign represents positive regulation of the first step of the reaction by the transcriptional regulator ChnR. The two alcohol dehydrogenases ChnA and ChnD belonged to two different families of dehydrogenases. The cells were lysed by incubating them at 94°C for 5 min and then cycled 25 times at 92°C for 1 min, 50°C for 45 s, and 72°C for 1 min. One possible reason could be the inability of E. Since ChnR regulates both chnA and chnB, the upstream regions of these two genes were compared. Screening of cosmids for the cyclohexanone monooxygenase gene.
Functional analysis of the genes contained in the localized region essential for adipic acid production. Among the six essential genes, chnR encodes a transcriptional activator required for the induction of the cyclohexanol oxidation pathway. Identification of intermediates of cyclohexanol oxidation. Boxes, genes organized on the 14-kb cluster that encode enzymes for adipic acid production; space between the boxes, intergenic region; line, upstream and downstream regions of the genes on the 14-kb cluster in 8F6; horizontal arrows, direction of transcription of the genes; vertical arrows, approximate locations of the transposon insertion sites obtained within the 14-kb region mapped by sequencing 120 mutants. The five steps of the reactions are represented by horizontal arrows.
Five of the essential genes encode enzymes, and one encodes a regulatory protein. Southern hybridization results data not shown indicated that the cosmid clone 5B12 had about 20 kb upstream of the monooxygenase gene and that cosmid clone 8F6 had about 30 kb downstream of the monooxygenase gene. Some proteins are not expressed in soluble forms in E. A control culture consisting of the host strains transformed only with the SuperCos1 vector was grown under the same conditions. The enzymes that catalyze some of the degradative reactions in xenobiotic metabolism can potentially be used for biotransformations. The x axis represents samples prepared from E.
Heating in the presence of acid catalysts converts cyclohexanol to. ChnE has high homology to other aldehyde dehydrogenases in GenBank. Two of them, ChnA and ChnD, were putatively identified as alcohol dehydrogenases. In this paper, we report the genetic analysis of a gene cluster involved in the degradation of cyclohexanol from Acinetobacter sp. Results are summarized in Table. The extraction residues were derivatized with 0.