Production of lipid compounds in the yeast Saccharomyces cerevisiae

This review describes progress using the yeast Saccharomyces cerevisiae as a model organism for the fast and efficient analysis of genes and enzyme activities involved in the lipid biosynthetic pathways of several donor organisms. Furthermore, we assess the impact of bakers yeast on the production of novel, high-value lipid compounds. Yeast can be genetically modified to produce selected substances in relatively high amounts. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the lipid pathways in this organism have been described in detail and are well characterized. We focus on the de novo production of three major families of lipid products. These are: (1) sterols, providing some previously known and some novel applications as examples of the lipid pathway enhancement that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega-3 and omega-6 fatty acids which do not occur naturally in yeast. We utilize the current knowledge and point out perspectives and problems for future biotechnological applications in the field of lipid compounds.     

Sphingosine 1-phosphate is a key metabolite linking sphingolipids to glycerophospholipids

The sphingolipid metabolite sphingosine 1-phosphate (S1P) is a well-known lipid mediator. As a lipid mediator, S1P must be present in extracellular space and bind to its cell surface receptors (S1P_1–5). However, most S1P, synthesized intracellularly, is metabolized without being released into extracellular space, in other words, without functioning as a lipid mediator in the vast majority of cells except those supplying plasma and lymph S1P such as blood cells and endothelial cells. Instead, intracellular S1P plays an important role as an intermediate of the sole sphingolipid-to-glycerophospholipid metabolic pathway. The degradation of S1P by S1P lyase is the first irreversible reaction (committed step) of this pathway. This metabolic pathway is conserved in eukaryotes from yeast to human, indicating its much older origin than the function of S1P as a lipid mediator, which is found to be present only in vertebrates and chordates. The sphingolipid-to-glycerophospholipid metabolism takes place ubiquitously in mammalian tissues, and its defect causes an aberration of several tissue functions as well as abnormal lipid metabolism. Although this metabolic pathway has been known for over four decades, only recently the precise reactions and enzymes involved in this pathway have been revealed. This review will focus on the recent advances in our understanding of the sphingolipid metabolic pathway via S1P and its physiological and pathological roles. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

The Current Waist Circumference Cut Point Used for the Diagnosis of Metabolic Syndrome in Sub-Saharan African Women Is Not Appropriate

The waist circumference cut point for diagnosing the metabolic syndrome in sub-Saharan African subjects is based on that obtained from studies in European populations. The aim of this study was to measure the prevalence of obesity and related metabolic disorders in an urban population of African females, a group at high risk for such diseases, and to determine the appropriate waist cut point for diagnosing the metabolic syndrome. Anthropometry and fasting lipid, glucose and insulin levels were measured in a cohort of 1251 African females participating in the Birth to Twenty cohort study in Soweto, Johannesburg. The waist circumference cut points for diagnosing metabolic syndrome (as defined using the new harmonised guidelines), insulin resistance, dysglycaemia, hypertension and dyslipidaemia were obtained using receiver operator characteristic curve analysis. The prevalence of obesity, type 2 diabetes and metabolic syndrome were 50.1%, 14.3% and 42.1%, respectively. The appropriate waist cut point for diagnosing metabolic syndrome was found to be 91.5 cm and was similar to the cuts points obtained for detecting increased risk of insulin resistance (89.0 cm), dysglycaemia (88.4 cm), hypertension (90.1 cm), hypo-high density lipoproteinaemia (87.6 cm) and hyper-low density lipoproteinaemia (90.5 cm). The present data demonstrates that urban, African females have a high prevalence of obesity and related disorders and the waist cut point currently recommended for the diagnosis of the metabolic syndrome (80.0 cm) in this population should be increased to 91.5 cm. This latter finding demonstrates a clear ethnic difference in the relationship between abdominal adiposity and metabolic disease risk. The similar waist cut points identified for the detection of the individual components of the metabolic syndrome and related cardiovascular risk factors demonstrates that the risk for different metabolic diseases increases at the same level of abdominal adiposity suggesting a common aetiological pathway.     

Metabolic engineering of Saccharomyces cerevisiae for production of novel lipid compounds

The yeast Saccharomyces cerevisiae has been modified successfully for production of numerous metabolites and therapeutic proteins through metabolic engineering, but has not been utilized to date for the production of lipid-derived compounds. We developed a lipid metabolic engineering strategy in S. cerevisiae based upon culturing techniques that are typically employed for studies of peroxisomal biogenesis; cells were grown in media containing fatty acids as a sole carbon source, which promotes peroxisomal proliferation and induction of enzymes associated with fatty acid beta-oxidation. Our results indicate that growth of yeast on fatty acids such as oleate results in extensive uptake of these fatty acids from the media and a subsequent increase in total cellular lipid content from 2% to 15% dry cell weight. We also show that co-expression of plant fatty acid desaturases 2 and 3 ( FAD2 and FAD3), using a fatty acid-inducible peroxisomal gene promoter, coupled the processes of fatty acid uptake with the induction of a new metabolic pathway leading from oleic acid (18:1) to linolenic acid (18:3). Finally, we show that cultivation of yeast cells in the presence of triacylglycerols and exogenously supplied lipase promotes extensive incorporation of triglyceride fatty acids into yeast cells. Collectively, these results provide a framework for bioconversion of low-cost oils into value-added lipid products.     

A novel high-throughput screening assay for putative antidiabetic agents through PPARalpha interactions

As natural peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands, high levels of fatty acids and glucose could lead to hyperactivation of PPARalpha, like that seen in diabetes. Important diabetes research goals are to uncover new metabolic or signaling pathways involved in hyperglycemic cellular injury and to develop therapeutics for preventing or reversing this injury. Consequently, 1040 putative antidiabetic agents were screened for their ability to 1) affect PPARalpha lipid binding, 2) directly bind PPARalpha, and 3) alter PPARalpha transactivation in the presence of high glucose. A high-throughput fluorescent binding assay was developed to examine each compound's ability to restore fatty acyl-CoA binding to PPARalpha in the presence of high glucose concentrations. Approximately 1% of the compounds restored acyl-CoA binding by 60% or more. These compounds directly interacted with PPARalpha with high affinity (nM K(d)s), validating the primary screen. Furthermore, these compounds altered PPARalpha transactivation, and 1 strongly reversed the hyperactivation of PPARalpha found in the presence of clofibrate and high glucose levels.     

Production of 1-octanol in Escherichia coli by a high flux thioesterase route

1-octanol is a valuable molecule in the chemical industry, where it is used as a plasticizer, as a precursor in the production of linear low-density polyethylene (LLDPE), and as a growth inhibitor of tobacco plant suckers. Due to the low availability of eight-carbon acyl chains in natural lipid feedstocks and the selectivity challenges in petrochemical routes to medium-chain fatty alcohols,1-octanol sells for the highest price among the fatty alcohol products. As an alternative, metabolic engineers have pursued sustainable 1-octanol production via engineered microbes. Here, we report demonstration of gram per liter titers in the model bacterium Escherichia coli via the development of a pathway composed of a thioesterase, an acyl-CoA synthetase, and an acyl-CoA reductase. In addition, the impact of deleting fermentative pathways was explored E. coli K12 MG1655 strain for production of octanoic acid, a key octanol precursor. In order to overcome metabolic flux barriers, bioprospecting experiments were performed to identify acyl-CoA synthetases with high activity towards octanoic acid and acyl-CoA reductases with high activity to produce 1-octanol from octanoyl-CoA. Titration of expression of key pathway enzymes was performed and a strain with the full pathway integrated on the chromosome was created. The final strain produced 1-octanol at 1.3 g/L titer and a >90% C₈ specificity from glycerol. In addition to the metabolic engineering efforts, this work addressed some of the technical challenges that arise when quantifying 1-octanol produced from cultures grown under fully aerobic conditions where evaporation and stripping are prevalent.     

Morphofunctional characteristics of the development of strains of Fusidium coccineum differing in antibiotic activity in stab cultivation

The object of the work was to study the morphological and functional characteristics of Fusidium coccineum strains producing fusidic acid and differing in the antibiotic activity. The high metabolic activity of the culture is accompanied by the following morphological characteristics: the cells are rich in ribosomes and mitochondria, they have early vacuolisation, are filled with lipid granules, and then the mycelium undergoes autolysis. As strains with a high activity grow, the structure of the cells changes, the number of ribosomes and mitochondria falls down, and the latter are destroyed. For a long time, the cells contain electron-dense granular structures limited with the membrane and capable of transformation into lipid granules and membranous structures. As was shown by cytochemical studies, the structures have not merely proteins and lipids, but also phosphorus compounds. Their functional role in the fungal metabolism is discussed. As soon as super-synthesis of fusidic acid commences, the cells of the highly active strains are filled with lipid granules associated possibly with the steroid antibiotic. These formations are released from the cell during local lysis of the cell wall and the cytoplasmic membrane.     

PTP1B inhibitor improves both insulin resistance and lipid abnormalities in vivo and in vitro

PTP1B is a negative regulator of insulin signaling pathway. This study investigated the effects of compound CCF06240, a PTP1B inhibitor, on insulin sensitivity and lipid metabolic abnormalities in vivo and in vitro, respectively. The insulin resistant IRM mouse model was induced by HFD. The responses to insulin were determined by OGTT, ITT, and hyperinsulinemic-euglycemic clamp test. The body weight and the levels of serum TC and TG were measured to estimate the lipid metabolism in vivo. Recombinant human GST-PTP1B protein was used to measure the inhibition of CCF06240 on PTP1B activity. The hepatocyte lipid accumulation was induced by high concentrations of FFA and insulin in HepG(2) cells, and evaluated by the Oil Red O method. In IRM mice, the insulin resistance was improved; the body weight and the levels of TC and TG were also reduced by oral CCF06240 administration. In lipid accumulated model cells, CCF06240 was found to reverse the increased PTP1B activity, enhance the insulin-induced tyrosine phosphorylation in insulin signaling pathway, attenuate the FFA-insulin-induced cellular lipid accumulation, and down-regulate the expressions of genes related fatty acid synthesis. These results demonstrated that the PTP1B inhibitor, compound CCF06240, could increase insulin sensitivity through the regulation of insulin signaling pathway, and decrease FFA-insulin-induced hepatocytes lipid accumulation by reducing fatty acid syntheses.     

Multiple lipid transport pathways to the plasma membrane in yeast

The plasma membrane of the yeast Saccharomyces cerevisiae is devoid of lipid-synthesizing enzymes, but contains all classes of bilayer-forming lipids. As the lipid composition of the plasma membrane does not match any of the intracellular membranes, specific trafficking of lipids from internal membranes, especially the endoplasmic reticulum and the Golgi, to the cell periphery is required. Although the secretory pathway is an obvious route to translocate glycerophospholipids, sphingolipids and sterols to the plasma membrane, experimental evidence for the role of this pathway in lipid transport is rare. Addressing this issue in a systematic way, we labeled temperature-sensitive secretory yeast mutants (sec mutants) with appropriate lipid precursors, isolated the plasma membranes at high purity and quantified labeled lipids of this compartment. Shifting sec mutants to the restrictive temperature reduced transport of both proteins and lipids to the plasma membrane, indicating that the latter compounds are also trafficked to the cell periphery through the protein secretory pathway. However, efficient sec blocks did not abrogate protein and lipid transport, suggesting that parallel pathway(s) for the translocation of membrane components to the plasma membrane of yeast must exist.     

Proteome analysis of Pseudomonas sp. K82 biodegradation pathways

Pseudomonas sp. K82 is a soil bacterium that can degrade and use monocyclic aromatic compounds including aniline, 3-methylaniline, 4-methylaniline, benzoate and p-hydroxybenzoate as its sole carbon and energy sources. In order to understand the impact of these aromatic compounds on metabolic pathways in Pseudomonas sp. K82, proteomes obtained from cultures exposed to different substrates were displayed by two-dimensional gel electrophoresis and were compared to search for differentially induced metabolic enzymes. Column separations of active fractions were performed to identify major biodegradation enzymes. More than thirty proteins involved in biodegradation and other types of metabolism were identified by electrospray ionization-quadrupole time of flight mass spectrometry. The proteome analysis suggested that Pseudomonas sp. K82 has three main metabolic pathways to degrade these aromatic compounds and induces specific metabolic pathways for each compound. The catechol 2,3-dioxygenase (CD2,3) pathway was the major pathway and the catechol 1,2-dioxygenase (beta-ketoadipate) pathway was the secondary pathway induced by aniline (aniline analogues) exposure. On the other hand, the catechol 1,2-dioxygenase pathway was the major pathway induced by benzoate exposure. For the degradation of p-hydroxybenzoate, the protocatechuate 4,5-dioxygenase pathway was the major degradation pathway induced. The nuclear magnetic resonance analysis of substrates demonstrated that Pseudomonas sp. K82 metabolizes some aromatic compounds more rapidly than others (benzoate > p-hydroxybenzoate > aniline) and that when combined, p-hydroxybenzoate metabolism is repressed by the presence of benzoate or aniline. These results suggest that proteome analysis can be useful in the high throughput study of bacterial metabolic pathways, including that of biodegradation, and that inter-relationships exist with respect to the metabolic pathways of aromatic compounds in Pseudomonas sp. K82.     

Engineering metabolic systems for production of advanced fuels

The depleting petroleum storage and increasing environmental deterioration are threatening the sustainable development of human societies. As such, biofuels and chemical feedstocks generated from renewable sources are becoming increasingly important. Although previous efforts led to great success in bio-ethanol production, higher alcohols, fatty acid derivatives including biodiesels, alkanes, and alkenes offer additional advantages because of their compatibility with existing infrastructure. In addition, some of these compounds are useful chemical feedstocks. Since native organisms do not naturally produce these compounds in high quantities, metabolic engineering becomes essential in constructing producing organisms. In this article, we briefly review the four major metabolic systems, the coenzyme-A mediated pathways, the keto acid pathways, the fatty acid pathway, and the isoprenoid pathways, that allow production of these fuel-grade chemicals.     

Mechanisms of protein degradation in mantle muscle and proposed gill remodeling in starved Sepia officinalis

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.     

Metabolomic analysis of livers and serum from high-fat diet induced obese mice

Liver and serum metabolites of obese and lean mice fed on high fat or normal diets were analyzed using ultraperformance liquid chromatography-quadrupole-time-of-flight mass spectrometry, gas chromatography-mass spectrometry, and partial least-squares-discriminant analysis (PLS-DA). Obese and lean groups were clearly discriminated from each other on PLS-DA score plot and major metabolites contributing to the discrimination were assigned as lipid metabolites (fatty acids, phosphatidylcholines (PCs), and lysophosphatidylcholines (lysoPCs)), lipid metabolism intermediates (betaine, carnitine, and acylcarnitines), amino acids, acidic compounds, monosaccharides, and serotonin. A high-fat diet increased lipid metabolites but decreased lipid metabolism intermediates and the NAD/NADH ratio, indicating that abnormal lipid and energy metabolism induced by a high-fat diet resulted in fat accumulation via decreased β-oxidation. In addition, this study revealed that the levels of many metabolites, including serotonin, betaine, pipecolic acid, and uric acid, were positively or negatively related to obesity-associated diseases. On the basis of these metabolites, we proposed a metabolic pathway related to high-fat diet-induced obesity. These metabolites can be used to better understand obesity and related diseases induced by a hyperlipidic diet. Furthermore, the level changes of these metabolites can be used to assess the risk of obesity and the therapeutic effect of obesity management.     

Predicting biological functions of compounds based on chemical-chemical interactions

Given a compound, how can we effectively predict its biological function? It is a fundamentally important problem because the information thus obtained may benefit the understanding of many basic biological processes and provide useful clues for drug design. In this study, based on the information of chemical-chemical interactions, a novel method was developed that can be used to identify which of the following eleven metabolic pathway classes a query compound may be involved with: (1) Carbohydrate Metabolism, (2) Energy Metabolism, (3) Lipid Metabolism, (4) Nucleotide Metabolism, (5) Amino Acid Metabolism, (6) Metabolism of Other Amino Acids, (7) Glycan Biosynthesis and Metabolism, (8) Metabolism of Cofactors and Vitamins, (9) Metabolism of Terpenoids and Polyketides, (10) Biosynthesis of Other Secondary Metabolites, (11) Xenobiotics Biodegradation and Metabolism. It was observed that the overall success rate obtained by the method via the 5-fold cross-validation test on a benchmark dataset consisting of 3,137 compounds was 77.97%, which is much higher than 10.45%, the corresponding success rate obtained by the random guesses. Besides, to deal with the situation that some compounds may be involved with more than one metabolic pathway class, the method presented here is featured by the capacity able to provide a series of potential metabolic pathway classes ranked according to the descending order of their likelihood for each of the query compounds concerned. Furthermore, our method was also applied to predict 5,549 compounds whose metabolic pathway classes are unknown. Interestingly, the results thus obtained are quite consistent with the deductions from the reports by other investigators. It is anticipated that, with the continuous increase of the chemical-chemical interaction data, the current method will be further enhanced in its power and accuracy, so as to become a useful complementary vehicle in annotating uncharacterized compounds for their biological functions.     

链霉菌基因组及次生代谢研究进展

链霉菌属革兰氏阳性放线菌,具有复杂的生活周期和次生代谢途径,并产生大量具有重要价值的天然代谢物。本文概述了链霉菌基因组染色体的独特结构与次生代谢途径的研究进展,重点论述了利用基因组信息改造和调控链霉菌次生代谢途径的研究成果。后基因组时代的功能基因组研究使人类能深入了解链霉菌家族,对链霉菌进行更加合理高效的遗传操作,为提高具有重要价值的天然代谢物的产量和获得新代谢物创造更有利的条件。     

Brain uptake and metabolism of ketone bodies in animal models

As a consequence of the high fat content of maternal milk, the brain metabolism of the suckling rat represents a model of naturally occurring ketosis. During the period of lactation, the rate of uptake and metabolism of the two ketone bodies, beta-hydroxybutyrate and acetoacetate is high. The ketone bodies enter the brain via monocarboxylate transporters whose expression and activity is much higher in the brain of the suckling than the mature rat. beta-Hydroxybutyrate and acetoacetate taken up by the brain are efficiently used as substrates for energy metabolism, and for amino acid and lipid biosynthesis, two pathways that are important for this period of active brain growth. Ketone bodies can represent about 30-70% of the total energy metabolism balance of the immature rat brain. The active metabolism of ketone bodies in the immature brain is related to the high activity of the enzymes of ketone body metabolism. Thus, the use of ketone bodies by the immature rodent brain serves to spare glucose for metabolic pathways that cannot be fulfilled by ketones such as the pentose phosphate pathway mainly. The latter pathway leads to the biosynthesis of ribose mandatory for DNA synthesis and NADPH which is not formed during ketone body metabolism and is a key cofactor in lipid biosynthesis. Finally, ketone bodies by serving mainly biosynthetic purposes spare glucose for the emergence of various functions such as audition, vision as well as more integrated and adapted behaviors whose appearance during brain maturation seems to critically relate upon active glucose supply and specific regional increased use.     

Induced Pluripotent Stem Cells Show Metabolomic Differences to Embryonic Stem Cells in Polyunsaturated Phosphatidylcholines and Primary Metabolism

Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS), gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs) compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs). With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.     

Adequate S supply protects barley plants from adverse effects of salinity stress by increasing thiol contents

As the salt-affected areas are expected to increase substantially in subsequent years, the impact of salinity on plant growth and yield is likely to increase. One of the first consequences of plant exposure to high saline concentrations is the formation of reactive oxygen species (ROS). In order to allow adjustment of the cellular redox state, plant antioxidative system has to be activated. This system involves several enzymes and compounds, as the sulphur-containing metabolite glutathione (GSH). Therefore, our aim was to determine whether adequate sulphur nutrition might alleviate the adverse effects of salt stress on barley plants grown in the presence of different sulphate application rate and exposed to 100 mM NaCl, by studying differences in growth parameters, lipid peroxidation, sulphate and thiol accumulation and sulphur assimilation pathway. In salt-treated plants, an adequate sulphur supply allows adequate GSH synthesis (high-thiol concentration) thus avoiding the effects of ROS on photosynthetic functions (no effect on both chlorophyll and protein content), whereas in S-deficient plants, salt stress leads to excess ROS production that induces stress and plants showed reduction of photosynthetic efficiency (loss of chlorophyll and protein contents). As thiol levels are more abundant in S-sufficient plants than in those S-deficient, one might expect that S-sufficient plants are more able to remove the harmful effects of high salinity. The comparison of malondialdehyde levels between +S and ?S salt-treated plants strongly supports this idea. In conclusion, we found that plant sulphur nutritional status plays a key role in the metabolic modifications necessary to cope with salt stress.     

Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus.

Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated (2H) medium were used to inoculate cultures in which natural-abundance 1H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the beta-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the beta-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol degradation, pathway mutants were constructed. Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis-muconate, inhibited the utilization of p-hydroxybenzoate in the presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.