Effect of restricted aeration on catabolism of cholic acid by two Pseudomonas species.

Examination of some previously isolated bile acid-utilizing Pseudomonas strains showed that Pseudomonas sp. ATCC 31752, together with other fluorescent strains, can be assigned to Pseudomonas putida biotype B, whereas Pseudomonas sp. ATCC 31753, like most other nonfluorescent strains, is an unrecognized phenotype. A study was made of the growth of these two species at 25 degrees C and pH 7.0 in a fermentor with 2.5 g of sodium cholate liter-1 as sole carbon source, and the catabolism of the cholate and its products was followed by high-pressure liquid chromatographic and thin-layer chromatographic examination. At aeration rates of either 150 or 5 ml min-1 liter-1, growth of each species followed the same catabolic pathway. 7 alpha, 12 beta-Dihydroxy-1,4-androstadiene-3,17-dione was the major catabolite formed, with 0.3 g liter-1 being the maximum concentration that accumulated at the higher aeration rate, whereas 1.4 g liter-1 accumulated at the lower aeration rate, irrespective of the species used. The latter yield is sufficiently high to be of potential commercial value if such a catabolite were found to be economically useful for steroid drug manufacture. It is postulated that the rate-limiting step in cholic acid catabolism by these species at the lower aeration rate is 9 alpha-hydroxylation, a step requiring molecular oxygen, hence, the marked effect of oxygen limitation on catabolite accumulation. Another consequence of oxygen limitation is the production of a red pigment in the culture medium, which, however, does not affect catabolite recovery.     

Aerobic catabolism of bile acids.

Seventy-eight stable cultures obtained by enrichment on media containing ox bile or a single bile acid were able to utilize one or more bile acids, as well as components of ox bile, as primary carbon sources for growth. All isolates were obligate aerobes, and most (70) were typical (48) or atypical (22) Pseudomonas strains, the remainder (8) being gram-positive actinomycetes. Of six Pseudomonas isolates selected for further study, five produced predominantly acidic catabolites after growth on glycocholic acid, but the sixth, Pseudomonas sp. ATCC 31752, accumulated as the principal product a neutral steroid catabolite. Optimum growth of Pseudomonas sp. ATCC 31752 on ox bile occurred at pH 7 to 8 and from 25 to 30 degrees C. No additional nutrients were required to sustain good growth, but growth was stimulated by the addition of ammonium sulfate and yeast extract. Good growth was obtained with a bile solids content of 40 g/liter in shaken flasks. A near-theoretical yield of neutral steroid catabolites, comprising a major (greater than 50%) and three minor products, was obtained from fermentor growth of ATCC 31752 in 6.7 g of ox bile solids per liter. The possible commercial exploitation of these findings to produce steroid drug intermediates for the pharmaceutical industry is discussed.     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel Pseudomonas sp. that mineralizes high concentrations of pentachlorophenol.

A pentachlorophenol (PCP)-mineralizing bacterium was isolated from polluted soil and identified as Pseudomonas sp. strain RA2. In batch cultures, Pseudomonas sp. strain RA2 used PCP as its sole source of carbon and energy and was capable of completely degrading this compound as indicated by radiotracer studies, stoichiometric release of chloride, and biomass formation. Pseudomonas sp. strain RA2 was able to mineralize a higher concentration of PCP (160 mg liter-1) than any previously reported PCP-degrading pseudomonad. At a PCP concentration of 200 mg liter-1, cell growth was completely inhibited and PCP was not degraded, although an active population of Pseudomonas sp. RA2 was still present in these cultures after 2 weeks. The inhibitory effect of PCP was partially attributable to its effect on the growth rate of Pseudomonas sp. strain RA2. The highest specific growth rate (mu = 0.09 h-1) was reached at a PCP concentration of 40 mg liter-1 but decreased at higher or lower PCP concentrations, with the lowest mu (0.05 h-1) occurring at 150 mg liter-1. Despite this reduction in growth rate, total biomass production was proportional to PCP concentration at all PCP concentrations degraded by Pseudomonas sp. RA2. In contrast, final cell density was reduced to below expected values at PCP concentrations greater than 100 mg liter-1. These results indicate that, in addition to its effect as an uncoupler of oxidative phosphorylation, PCP may also inhibit cell division in Pseudomonas sp. strain RA2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment

A Spirillum sp. and a Pseudomonas sp. possessing crossing substrate saturation curves for L-lactate were isolated from fresh water by chemostat enrichment. Their Ks and mumax values for L-lactate were: Spirillum sp., 23 micrometer and 0.35 h-1, respectively; Pseudomonas sp., 91 micrometer and 0.64 h-1, respectively. Under L-lactate limitation, pseudomonas sp. outgrew Spirillum s. at dilution rates (D) above 0.29 h-1, but the converse occurred at lower D values. The advantage of Spirillum sp. increased with decreasing D until, at D = 0.05 h-1 (i.e. L-lactate concentration of approximately 1 micrometer), Pseudomonas sp. was eliminated from the culture essentially as a non-growing population. In Spirillum sp. the Km for L-lactate transport (5.8 micrometer) was threefold lower than in Pseudomonas sp. (20 micrometer); Spirillum sp. also possessed a higher Vmax for the transport of this substrate. The surface to volume ratio was higher in Spirillum sp. and increased more markedly than in Pseudomonas sp. in response to decreasing D. Thus, a more efficient scavenging capacity contributes to the advantage of Spirillum sp. at low concentrations of the carbon source. Although most of the enzymes of L-lactate catabolism were more active in Pseudomonas sp., NADH oxidase activity was about twice as high in Spirillum sp.; and, unlike Pseudomonas sp., the cytochrome c content of this bacterium increased markedly with decreasing D. A more active and/or more efficient respiratory chain may therefore also play a role in the advantage of Spirillum sp. The other factors which appear to be involved include a lower energy of maintenance of Spirillum sp. [0.016 g L-lactate (g cell dry wt)-1 h-1 compared with 0.066 in Pseudomonas sp.] and a lower minimal growth rate.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Pilot plant production of rhamnolipid biosurfactant by Pseudomonas aeruginosa.

Rhamnolipid biosurfactants were continuously produced with Pseudomonas aeruginosa on the pilot plant scale. Production and downstream processing elaborated on the laboratory scale were adapted to the larger scale. Differences in performance resulting from the scale-up are discussed. A biosurfactant concentration of approximately 2.25 g liter-1 was achieved. The biosurfactant yield on glucose was 77 mg g-1 h-1, and the productivity was 147 mg liter-1 h-1, corresponding to a daily production of 80 g of biosurfactant. The first enrichment step consisted of an adsorption chromatography which was followed by an anion-exchange chromatography. The resulting product was 90% pure, and the overall recovery of active material was above 60% with the downstream processing used.     

Pilot plant production of rhamnolipid biosurfactant by Pseudomonas aeruginosa

Rhamnolipid biosurfactants were continuously produced with Pseudomonas aeruginosa on the pilot plant scale. Production and downstream processing elaborated on the laboratory scale were adapted to the larger scale. Differences in performance resulting from the scale-up are discussed. A biosurfactant concentration of approximately 2.25 g liter-1 was achieved. The biosurfactant yield on glucose was 77 mg g-1 h-1, and the productivity was 147 mg liter-1 h-1, corresponding to a daily production of 80 g of biosurfactant. The first enrichment step consisted of an adsorption chromatography which was followed by an anion-exchange chromatography. The resulting product was 90% pure, and the overall recovery of active material was above 60% with the downstream processing used.     

Pyrimidine biosynthesis in Pseudomonas veronii and its regulation by pyrimidines

Pyrimidine biosynthesis in the nutritionally versatile bacterium Pseudomonas veronii ATCC 700474 appeared to be controlled by pyrimidines. When wild type cells were grown on glucose in the presence of uracil, four enzyme activities were depressed while all five enzyme activities increased in succinate-grown cells supplemented with uracil. Independent of carbon source, orotic acid-grown cells elevated aspartate transcarbamoylase, dihydroorotase, orotate phosphoribosyltransferase or OMP decarboxylase activity. Pyrimidine limitation of glucose-grown pyrimidine auxotrophic cells lacking OMP decarboxylase activity resulted in at least a doubling of the enzyme activities relative to their activities in uracil-grown cells. Less derepression of the enzyme activities was observed after pyrimidine limitation of succinate-grown mutant cells possibly due to catabolite repression. Aspartate transcarbamoylase activity in Ps. veronii was regulated at the level of enzyme activity since the enzyme was strongly inhibited by pyrophosphate, UDP, UTP, ADP, ATP and GTP. Overall, the regulation of pyrimidine biosynthesis in Ps. veronii could be used to differentiate it from other taxonomically related species of Pseudomonas.     

Enrichment of linear alkylbenzenesulphonate (LAS) degrading bacteria in continuous culture

Enrichment experiments were carried out in continuous-flow units using a mineral medium with commercial linear alkylbenzenesulphonate (LAS) as the limiting carbon- and energy-source. The mixed bacterial culture originating from the waste water of a detergent plant consisted of five strains belonging to the genus Pseudomonas and two strains each of the genera Achromobacter and Acinetobacter. The cultivation conditions corresponding to dilution rates of 0.025-0.1 h^-1 and LAS concentrations of 20–50 mg/1 were examined. During the experiments the composition of mixed cultures and the kinetics of LAS biodegradation were followed. Continuous-flow enrichment experiments resulted in the selection of six bacterial cultures with different compositions of individual species and capability to utilize LAS. From the original seven strains at lower dilution rates (0.025 and 0.05 h^-1) six were selected, excluding Pseudomonas sp. 3, while at the highest dilution rate (0.1/h^-1) five strains were selected after eliminating Pseudomonas sp. 5 and Achromobacter sp. 1. All enriched mixed cultures were more efficient in primary than in ultimate LAS degradation. Two of the culture strains were able to achieve primary LAS degradation ( Pseudomonas sp. 1 in mineral medium with LAS as the sole carbon- and energy-source and Acinetobacter sp. 3 in medium supplemented by yeast extract and nutrient broth).
None of the strains could degrade LAS completely, which indicates that many types of interactions based on combined metabolic attack as well as those based on provision of specific nutrients, may exist between culture members during the complete LAS bio-oxidation.     

Molecular cloning, functional expression and characterisation of RCC reductase involved in chlorophyll catabolism

Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Biodegradation of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) by purified pentachlorophenol hydroxylase and whole cells of Flavobacterium sp. strain ATCC 39723 is accompanied by cyanogenesis.

A pentachlorophenol (PCP)-degrading Flavobacterium sp. (strain ATCC 39723) degraded bromoxynil with the production of bromide and cyanide. No aromatic intermediates were detected in the spent culture fluid. The cyanide produced upon bromoxynil metabolism was inhibitory to the Flavobacterium sp. Whole cells degraded PCP more rapidly than they did bromoxynil. Bromoxynil metabolism and PCP metabolism were coinduced, either substrate serving as the inducer. Purified PCP hydroxylase degraded bromoxynil with stoichiometric accumulation of cyanide and without bromide production. A product accumulated which was more hydrophilic than bromoxynil upon high-pressure liquid chromatographic analysis and which, when analyzed by gas chromatography-mass spectrometry, had a mass spectrum consistent with that expected for dibromohydroquinone. PCP hydroxylase consumed NADPH, oxygen, and bromoxynil in a 2:1:1 molar ratio, producing 1 mol of cyanide per mol of bromoxynil degraded. We propose a pathway by which bromoxynil is metabolized by the same enzymes which degrade PCP. The initial step in the pathway is the conversion of bromoxynil to 2,6-dibromohydroquinone by PCP hydroxylase. In addition to its utility for decontaminating PCP-polluted sites, the Flavobacterium sp. may be useful for decontaminating bromoxynil spills. This is the first report of cyanide production accompanying the metabolism of a benzonitrile derivative.     

Oxygen transfer rate and sophorose lipid production by Candida bombicola.

Sophorose lipids (SLs) have applications as surfactants and are produced at high levels by several yeasts. We developed a fed-batch shake-flask method for the production of SLs by Candida bombicola ATCC 22214. Optimal aeration, expressed in terms of oxygen transfer rate, was between 50 and 80 mM O(2)/L h(-1) and resulted in maximum values for both volumetric product formation (1-1.5 g/L h(-1)) and SL yield (350 g/L). The lowest aeration levels resulted in the enrichment in saturated fatty acid SLs at the expense of unsaturated fatty acid SLs.     

抑氨菌筛选鉴定、培养条件优化及在鸡粪除臭中的应用

目的筛选能抑制鸡粪中恶臭气体NH3释放的亚硝化、硝化细菌菌株。方法以亚硝化、硝化细菌培养基为筛选培养基筛选菌株,然后将菌株分别以10%(v/m,下同)的接种量接种到鸡粪中,测定其对鸡粪中NH3释放量的影响,从中筛选出可减少NH3释放的菌株。根据菌株的形态特征和16S rDNA序列分析对其进行鉴定。通过自动发酵系统对菌株培养温度、pH、通气量及转速四个因素进行正交优化。结果通过筛选得到两株细菌YF1和YS2,经鉴定分别为假单胞菌属(Pseudomonas sp.)和中华根瘤菌属(Sinorhizobium sp.)。菌株YF1最适培养条件为温度28℃,pH 7.0,通气量5 L/min,转速200 r/min;菌株YS2最适培养条件为温度30℃,pH6.5,通气量5 L/min,转速300 r/min。温度、pH、接种量和通气量对YF1、YS2影响均极显著(P0.01)。YF1和YS2单独按10%剂量接种分别使鸡粪中NH3的释放量降低26.0%和28.4%,而两菌1∶1混合接种可使NH3释放量降低75.6%。结论 YF1和YS2是抑制鸡粪中NH3释放的优良菌株。     

Bioconversion of cinnamic Acid to acetophenone by a pseudomonad: microbial production of a natural flavor compound

A mutant derivative of a novel pseudomonad isolated from the soil accumulated acetophenone when supplied with cinnamic acid. The microorganism has been identified as an unclassified Pseudomonas sp., similar to Pseudomonas acidovorans. Mass spectrum analysis of the product acetophenone derived from catabolism of cinnamic acid in the presence of O(2) or H(2)O supported the conclusion that cinnamic acid degradation is initiated by addition of water to the double bond of the side chain, followed by dehydrogenation to generate 3-keto-3-phenylpropionic acid. The intermediate 3-keto-3-phenylpropionic acid is accumulated in cultures of the mutant during active cinnamic acid catabolism. However, this intermediate is unstable so a portion of it spontaneously decarboxylates to form acetophenone. Neither 3-keto-3-phenylpropionic acid nor acetophenone is a precedented intermediate in cinnamic acid degradation. Isolation of the novel strain and mutant provide the rudiments for a process to produce natural acetophenone by biotransformation of natural cinnamic acid.     

Microbial transformation of quinoline by a Pseudomonas sp.

A Pseudomonas sp. isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. During early growth of the organism on quinoline, 2-hydroxyquinoline accumulated as the intermediate; 8-hydroxycoumarin accumulated as the major metabolite on further incubation. 2,8-Dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid were identified as the other intermediates. Inhibition of quinoline metabolism by 1 mM sodium arsenite led to the accumulation of pyruvate, whereas inhibition by 5 mM arsenite resulted in the accumulation of 2-hydroxyquinoline as the major metabolite and 2,8-dihydroxyquinoline as the minor metabolite. Coumarin was not utilized as a growth substrate by this bacterium, but quinoline-grown cells converted it to 2-hydroxyphenylpropionic acid, which was not further metabolized. Quinoline, 2-hydroxyquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid were rapidly oxidized by quinoline-adapted cells, whereas 2,8-dihydroxyquinoline was oxidized very slowly. Quinoline catabolism in this Pseudomonas sp. is therefore initiated by hydroxylation(s) of the molecule followed by cleavage of the pyridine ring to yield 8-hydroxycoumarin, which is further metabolized via 2,3-dihydroxyphenylpropionic acid.