Stimulation of pollen tube growthin vitro by dicarboxylic acids

Summary Effect of eight dicarboxylic acids and three monocarboxylic acids on pollen growth ofCamellia japonica was tested. While monocarboxylic acids inhibited pollen germination and pollen tube elongation, dicarboxylic acids, namely oxalic, succinic, suberic, adipic, sebacic, traumatic cis-1,2-cyclohexane dicarboxylic, and 3,3-diethyl glutaric acids stimulated pollen tube elongation stronger than indoleacetic acid.     

The biological origin of ketotic dicarboxylic aciduria. In vivo and in vitro investigations of the omega-oxidation of C6-C16-monocarboxylic acids in unstarved, starved and diabetic rats

The conversion of radioactive C6-C16-monocarboxylic acids to urinary adipic, suberic, sebacic and 3-hydroxybutyric acids was investigated in vivo in unstarved, starved and diabetic ketotic rats. Hexanoic, octanoic and decanoic acids were converted to C6-, C6-C8- and C6-C10-dicarboxylic acids, respectively, in fed and 72-h-starved rats. Lauric acid was converted to C6-C8-dicarboxylic acids in starved rats but not in unstarved rats. Decanoic and lauric acids were converted to relatively high amounts of C6-C8-dicarboxylic acids compared with myristic acid in myristic acid in ketotic diabetic rats, while radioactivity from [1-14C]-and [16-(14)] palmitic acid was not incorporated into C6-C8-dicarboxylic acids in diabetic ketotic rats. C6-C12-monocarboxylic acids in hydrolysed rat adipose tissue wee determined by gas-liquid chromatography-mass spectrometry (selected ion monitoring). Decanoic and lauric acids were found in amounts of 7.6-9.1 and 85.9-137.5 micrometers/100 mg tissue, respectively, whereas the amounts of hexanoic and octanoic acids were negligible. It is concluded that the biological origin of the C6-C8-dicarboxylic aciduria seen in ketotic rats are C10-C14-monocarboxylic acids, which are initially omega-oxidised solely or partly as free acids and subsequently beta-oxidised to adipic and suberic acids. The in vitro omega-oxidation of C6-C16-monocarboxylic acids to corresponding dicarboxylic acids in the 100,000 Xg supernatant fraction of rat liver homogenate was measured by selected ion monitoring. 0.09, 0.14, 16.1, 5.8, 7.0 and -6.9% of, respectively, hexanoic, octanoic, decanoic, lauric, myristic and palmitic acid were omega-oxidised to dicarboxylic acids of corresponding chain lengths after 90 min of incubation, when correction for the production of dicarboxylic acids in control assays was made. An in vitro production of C12-C16-dicarboxylic acids was detected in all assays ()including control assays), probably formed from"endogenous' monocarboxylic acids preexistent in the homogenate. Ths "endogenous' production of dicarboxylic acids was inhibited by C10-C16-monocarboxylic acids, where palmitic acid had the strongest effect. In fact, palmitic acid inhibited its own omega-oxidation when added in concentrations above 0.6 mM. Starvation of rats for 72 h did not alter the "endogenous' in vitro production of hexadecanedioic acid.     

Metabolic engineering for the production of dicarboxylic acids and diamines

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.     

Urinary excretion of dicarboxylic acids from patients with the Zellweger syndrome. Importance of peroxisomes in beta-oxidation of dicarboxylic acids

The urinary excretion of adipic acid, suberic acid and sebacic acid from two patients with the cerebrohepato-renal syndrome of Zellweger was studied. The patients had a complete lack of peroxisomes in the liver as judged by electron microscopy. In the non-ketotic state, the total excretion of free and conjugated adipic acid, suberic acid and sebacic acid was increased by about 100%, 200% and 350%, respectively, as compared to the corresponding excretion from six healthy infants of the same age. The excretion of free dicarboxylic acid was increased to a considerably lesser extent than the free + conjugated dicarboxylic acid. In view of the presence of adipic acid in urine of the Zellweger patients, it is concluded that peroxisomes are not obligatory for beta-oxidation of medium-chain dicarboxylic acids in vivo. The relative accumulation of suberic acid and sebacic acid as compared to adipic acid is, however, consistent with a relative block in the conversion of suberic acid and sebacic acid into adipic acid in patients with the Zellweger syndrome.     

Measurements of urinary adipic acid and suberic acid using high-performance liquid chromatography

A sensitive and specific method was developed for measuring medium-chain dicarboxylic acids (adipic and suberic acid) in urine. These acids were extracted from urine with diethyl ether and converted into fluorescent derivatives with 9-anthryldiazomethane, which can be separated by high-performance liquid chromatography. The reproducibility was high and the recovery from urine was above 90%. Urinary concentrations of adipic acid in streptozotocin-induced diabetic rats were significantly higher than those in control rats. In diabetic patients, both adipic acid and suberic acid tended to be high, but not significantly. This method should be useful for measuring dicarboxylic acids in urine     

Metabolism of deuterium-labeled nonanoic acids in the riboflavin-deficient rat model of multiple acyl-CoA dehydrogenase deficiency

Riboflavin-deficient rats are used to study the metabolism of deuterium-labeled nonanoic acids under conditions mimicking the human disorder of multiple acyl-CoA dehydrogenase deficiency in which large amounts of ethyl-malonic, glutaric, adipic, suberic, 4-octenedioic, sebacic and 4-decenedioic acids are excreted. Both control and deficient rats convert the nonanoic acids to labeled azelaic and pimelic acids. The labeling pattern in pimelic acid is consistent with the omega-oxidation of nonanoic acids to azelaic acid followed by beta-oxidation to pimelic acid.     

Metabolic origin of urinary 3-hydroxy dicarboxylic acids

3-Hydroxy dicarboxylic acids with chain lengths ranging from 6 to 14 carbons are excreted in human urine. The urinary excretion of these acids is increased in conditions of increased mobilization of fatty acids or inhibited fatty acid oxidation. Similar urinary profiles of 3-hydroxy dicarboxylic acids were also observed in fasting rats. The metabolic genesis of these urinary 3-hydroxy dicarboxylic acids was investigated in vitro with rat liver postmitochondrial and mitochondrial fractions. 3-Hydroxy monocarboxylic acids ranging from 3-hydroxyhexanoic acid to 3-hydroxyhexadecanoic acid were synthesized. In the rat liver postmitochondrial fraction fortified with NADPH, these 3-hydroxy fatty acids with carbon chains equal to or longer than 10 were oxidized to (omega - 1)- and omega-hydroxy metabolites as well as to the corresponding 3-hydroxy dicarboxylic acids. 3-Hydroxyhexanoic (3OHMC6) and 3-hydroxyoctanoic (3OHMC8) acids were not metabolized. Upon the addition of mitochondria together with ATP, CoA, carnitine, and MgCl2, the 3-hydroxy dicarboxylic acids were converted to 3-hydroxyoctanedioic, trans-2-hexenedioic, suberic, and adipic acids. In the urine of children with elevated 3-hydroxy dicarboxylic acid levels, 3OHMC6, 3OHMC8, 3-hydroxydecanoic, 3,10-dihydroxydecanoic, 3,9-dihydroxydecanoic, and 3,11-dihydroxydodecanoic acids were identified. On the basis of these data, we propose that the urinary 3-hydroxy dicarboxylic acids are derived from the omega-oxidation of 3-hydroxy fatty acids and the subsequent beta-oxidation of longer chain 3-hydroxy dicarboxylic acids. These urinary 3-hydroxy dicarboxylic acids are not derived from the beta-oxidation of unsubstituted dicarboxylic acids.     

Gas chromatography-mass spectrometry profile of urinary organic acids of Wistar rats orally treated with ozonized unsaturated triglycerides and ozonized sunflower oil

The main products in the ozonolysis of unsaturated triglycerides or vegetable oils are peroxides, aldehydes, Criegee ozonides and carboxylic acids. Some of these compounds are present in different concentrations in the biological fluids. The aim of this work is to study, using gas chromatography-mass spectrometry (GC-MS), the organic acid excretion in urine of rats orally treated with ozonized sunflower oil (OSO), ozonized triolein or ozonized trilinolein. Oral administration of OSO to Wistar rats has produced changes in the urinary content of dicarboxylic organic acids. Among others heptanedioic (pimelic acid) and nonanedioic acids (azelaic acid) were the major increased dicarboxylic acids found. The urinary dicarboxylic acid profiles of rats which received ozonized triolein only showed an increase in heptanedioic and nonanedioic acids. However, when ozonized trilinolein is applied, the profile is similar to that obtained when OSO is administered. A biochemical mechanism is proposed to explain the formation of dicarboxylic acids from ozonated unsaturated triglycerides.     

Products of the oxidation of selected alkanes by a gram-negative bacterium

Cultures of a soil pseudomonad grown withn-octane as the sole source of carbon and energy have been shown to accumulate suberic, adipic, acetic and butyric acids. Cultures grown at the expense ofn-octoic acid did not yield either suberic or adipic acids. Whenn-heptane was the growth substrate,n-heptoic acid was detected in the medium. A trace of pimelic acid, the expectedn-alkanedioic acid, also appeared to be present. The principal non-volatile acidic products were recognised to be either hydroxy acids or the lactones of these acids. The formation of suberic and adipic acids fromn-octane is discussed in terms of current views of the biological oxidation ofn-alkanes.     

Formation and degradation of dicarboxylic acids in relation to alterations in fatty acid oxidation in rats.

Dicarboxylic acids are excreted in urine when fatty acid oxidation is increased (ketosis) or inhibited (defects in beta-oxidation) and in Reye's syndrome. omega-Hydroxylation and omega-oxidation of C6-C12 fatty acids were measured by mass spectrometry in rat liver microsomes and homogenates, and beta-oxidation of the dicarboxylic acids in liver homogenates and isolated mitochondria and peroxisomes. Medium-chain fatty acids formed large amounts of medium-chain dicarboxylic acids, which were easily beta-oxidized both in vitro and in vivo, in contrast to the long-chain C16-dicarboxylic acid, which was toxic to starved rats. Increment of fatty acid oxidation in rats by starvation or diabetes increased C6:C10 dicarboxylic acid ratio in rats fed medium-chain triacylglycerols, and increased short-chain dicarboxylic acid excretion in urine in rats fed medium-chain dicarboxylic acids. Valproate, which inhibits fatty acid oxidation and may induce Reye like syndromes, caused the pattern of C6-C10-dicarboxylic aciduria seen in beta-oxidation defects, but only in starved rats. It is suggested, that the origin of urinary short-chain dicarboxylic acids is omega-oxidized medium-chain fatty acids, which after peroxisomal beta-oxidation accumulate as C6-C8-dicarboxylic acids. C10-C12-dicarboxylic acids were also metabolized in the mitochondria, but did not accumulate as C6-C8-dicarboxylic acids, indicating that beta-oxidation was completed beyond the level of adipyl CoA.     

Metabolic conversion of dicarboxylic acids to succinate in rat liver homogenates. A stable isotope tracer study

The metabolic conversion of dicarboxylic acids into succinate and other gluconeogenic intermediates in rat liver homogenates was investigated using [1,2,4-13C4]dodecanedioic acid as tracer. Isotope enrichments in 3-hydroxybutyrate, succinate, fumarate, and malate, as well as dicarboxylates (dodecanedioic, sebacic, suberic, and adipic acids) were measured with selected ion monitoring capillary column gas chromatograph-mass spectrometry. Significant enrichment in the M + 4 (four labeled carbons) ion of succinate (0.4-2.9%) was detected, unequivocally demonstrating the direct conversion of dicarboxylate into succinate. In addition, significant enrichment of the M + 2 ion of succinate was also observed. This labeled species was generated from labeled acetyl-CoA through the tricarboxylic acid cycle. The partition of acetyl-CoA into the tricarboxylic acid cycle relative to ketone body formation was higher in the beta oxidation of dicarboxylate than monocarboxylate. Therefore, in addition to the production of succinate, the beta oxidation of dodecanedioate resulted in the channeling of the acetyl-CoA produced to the tricarboxylic acid cycle instead of to acetoacetate production. The enrichments in lower chain dicarboxylates are consistent with a partial bidirectional beta oxidation of dodecanedioic acid. In addition to the expected M + 0 and M + 4 labels, significant M + 2 species were detected in suberic and adipic acids. These M + 2-labeled species were produced from the released free dicarboxylate intermediates which were then reactivated and metabolized. In these experiments, the overall succinate production was derived 4% from the direct conversion of dodecanedioic acid and 11% from the indirect route via acetyl-CoA through tricarboxylic acid.     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Characterization by high performance liquid chromatography (HPLC) of the solubilization of phosphorus in iron ore by a fungus

Summary The value of iron ore is adversely affected by phosphorus in concentrations over 0.03% by weight. The present research concerns the use of metabolic products of aPenicillium-like fungus to leach insoluble phosphates (hydroxyapatite) from ores. Ion chromatography was used to measure metabolism of glucose into acidic fragments. The rate and products of glucose degradation depended on both the chemical composition of the growth medium (buffered or not) and incubation conditions (shaken or quiescent). The principal products were identified as oxalic acid and isomers of propylene dicarboxylic acid, mainly itaconic acid. Continued, slow metabolism of itaconic acid generates more oxalic acid. Aliphatic acids were not detected. Both iron ore phosphate and calcium phosphate were partially solubilized by either the spent broth or aqueous oxalic acid. Solubilization of ore phosphorus was greatly assisted by hydrochloric acid added to the spent broth in small increments. The data suggest biological alternatives to costly leaching procedures that use only mineral acids.     

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.     

Metabolism of dicarboxylic acids in rat hepatocytes

[carboxyl-14C]Dodecanedioic acid (DC12) is metabolized in hepatocytes at a rate about two thirds that of [1-14C]palmitate. Shorter dicarboxylates (sebacic (DC10), suberic (DC8), and adipic (DC6) acid) are formed, mainly DC6, less DC8 and only a little DC10. In hepatocytes from clofibrate-treated rats, more polar products account for most of the breakdown products, presumably because the beta-oxidation proceeds all the way to succinate and acetyl-CoA. [carboxyl-14C]Suberic acid (DC8) is oxidized at a rate only one fifth that of dodecanedioic acid. (+)-Decanoylcarnitine inhibits palmitate oxidation but not the oxidation of dodecanedioic acid. At low concentrations of [carboxyl-14C]dodecanedioic acid or of [1-14C]palmitate, acetylsulfanilamide is more efficiently labeled by the former. High concentrations of dodecanedioic acid inhibit palmitate oxidation and the acetylation of sulfanilamide, presumably because their CoA-esters accumulate in the cytosol. These results indicate that medium-chain dicarboxylic acids are beta-oxidized mainly in the peroxisomes.     

Effect of treatment with very dilute acids on the wet tensile strength and chemical properties of paper

Filter paper (α-cellulose content 99·3%) was soaked in very dilute solutions of several mineral and aliphatic dicarboxylic acids and heated for various times at temperatures ranging from 100°C to 140°C. The wet strength of the paper, measured with an Instron tester using a jaw span of 15 cm, was greatly increased by these treatments. The largest increase was by nearly 1000%; this was achieved by heating with oxalic acid at pH 2·7 for 1 h at 140°C. Increases of nearly 750% and 850% were obtained with hydrochloric and sulphuric acids, respectively, under similar conditions. Homologues of oxalic acid, which were too weak to furnish solutions of pH 2·7, caused only small increases in wet strength.The increases in wet strength have been attributed to the formation of inter-fibre cross-links. A small loss of dry strength was observed and this, together with increases in copper number and fluidity, was taken as evidence that some hydrolytic degradation occurred simultaneously with cross-linking. Short-span breaking-load tests also confirmed this conclusion. The zero-span breaking load, which is a measure of the strength of the fibres rather than of the paper itself, fell markedly as a result of the acid treatments. However, as the span was increased the breaking load of the treated paper levelled out at a finite value whereas that of the untreated paper fell close to zero.The nature of the cross-links was studied by chemical tests. The effect of chlorous acid and borohydride treatment on the tensile strength of the acid-treated paper showed that a small proportion of the cross-links were hemiacetals. However, the main proportion appeared to be ethers. This was demonstrated by measuring the effects of the acid treatments on the proportions of ‘available’ primary and secondary hydroxyl groups by tosylation followed by iodination.     

Expression, purification, and characterization of BioI: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI. We have subcloned biol and overexpressed the encoded protein, Biol. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography. Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coli acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production.     

The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase

Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.     

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C₆–C₁₀ carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C₆–C₁₀ dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C₆–C₁₀ ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.     

Properties of an inducible C 4 -dicarboxylic acid transport system in Bacillus subtilis

The transport of the tricarboxylic acid cycle C(4)-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C(4)-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C(4)-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor alpha-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (K(m) approximately 10(-4) M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of alpha-ketoglutarate dehydrogenase were shown to accumulate both alpha-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C(4)-dicarboxylic acids, suggesting a regulatory role.