acetyl-CoA Synthetase Activity Research Articles

Tissue-specific Short Chain Fatty Acid Metabolism and Slow Metabolic Recovery after Ischemia from Hyperpolarized NMR in Vivo

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetyl-carnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.

Metabolic Adaptations During Lactogenesis

1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days postpartum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities ofrelevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and α-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDPglucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (α-lactalbumin) serving a similar function in lactose biosynthesis.

Lysine 88 Acetylation Negatively Regulates Ornithine Carbamoyltransferase Activity in Response to Nutrient Signals

Ornithine carbamoyltransferase (OTC) is a key enzyme in the urea cycle to detoxify ammonium produced from amino acid catabolism. OTC deficiency is an X-linked genetic disorder ranging from fatal in newborns to hyperammonemia and anorexia in adults. Through affinity purification of acetylated peptides and mass spectrometry, we identified that OTC is acetylated on lysine residues, including Lys88, which is also mutated in OTC-deficient patients. OTC acetylation was confirmed to occur under physiological conditions. Biochemical characterizations revealed that OTC Lys88 acetylation decreases the affinity for carbamoyl phosphate, one of the two OTC substrates, and the maximum velocity, whereas the K(m) for ornithine, the other OTC substrate, is not affected. Furthermore, Lys88 acetylation is regulated by both extracellular glucose and amino acid availability, indicating that OTC activity may be regulated by cellular metabolic status. Our results provide an example of the novel mechanism of regulating metabolic enzyme activity through protein acetylation.

A colorimetric assay method to measure acetyl-CoA synthetase activity: Application to woodchuck model of hepatitis virus-induced hepatocellular carcinoma

A new spectrophotometric method for quantitation of acetyl-CoA synthetase (ACAS) activity is developed. It has been applied for ACAS assay in the liver tissues of a woodchuck model of hepatitis virus-induced hepatocellular carcinoma (HCC). The assay is based on the established pyrophosphate (PPi) detection system. ACAS activity is indexed by the amount of PPi, the product of ACAS reaction system of activated form of acetate (acetyl-CoA) with ACAS catalysis. PPi is determined quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and 2-mercaptoethanol. PPi reacts with molybdate reagent to produce phosphomolybdate and PPi-molybdate complexes. 2-mercaptoethanol is responsible for color formation which has the peak absorbance at 580 nm. This method was sensitive from 1 to 20 nmol of PPi in a 380-μl sample (1-cm cuvette). A ten-fold excess of Pi did not interfere with the determination of PPi. To study the major metabolic pathways of imaging tracer [1- 11C]-acetate in tumors for detection of HCC by Positron Emission Tomography (PET), the activity of one of the key enzymes involved in acetate or [1- 11C]-acetate metabolism, ACAS was assayed by this newly developed assay in the tissue samples of woodchuck HCCs. A significant increase of ACAS activity was observed in the liver tissues of woodchuck HCCs as compared with neighboring regions surrounding the tumors ( P < 0.05). The respective ACAS activities in the subcellular locations were also significantly higher in HCCs than in the surrounding tissues ( P < 0.05) (total soluble fraction: 876.61 ± 34.64 vs. 361.62 ± 49.97 mU/g tissue; cytoplasmic fraction: 1122.02 ± 112.39 vs. 732.32 ± 84.44 mU/g tissue; organelle content: 815.79 ± 100.77 vs. 547.91 ± 97.05 mU/ g tissue; sedimentable fragment: 251.92 ± 51.56 vs. 90.94 ± 18.98 mU/ g tissue). The finding suggests an increase in ACAS activity in the liver cancer of woodchuck models of HCC as compared to that in the normal woodchuck liver. The developed assay is rapid, simple and accurate and is suitable for the investigation of ACAS activity under physiologic and pathophysiologic conditions.

Specific features of the synthesis of the exopolysaccharide ethapolan on a mixture of energy-deficient growth substrates

Intensification of the synthesis of the microbial exopolysaccharide ethapolan by Acinetobacter sp. B-7005 was shown to occur on a mixture of energy-deficient growth substrates (acetate + glucose). When the bacterium grew on the substrate mixture, both substrates were utilized simultaneously; acetate was taken up by means of active transport at the expense of the energy of the proton-motive force. When acetate was present in the form of a sodium salt, the activities of acetyl-CoA synthetase and phosphoenolpyruvate synthetase (the key enzyme of gluconeogenesis) were tenfold higher than in the presence of potassium acetate, and the indexes of ethapolan synthesis were two times higher. The positive effect of Na+ on ethapolan synthesis is supposed to consist in the creation of ion gradients on the membrane, necessary for the generation of the proton-motive force. Simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, as well as an increase in the activity of isocitrate lyase, malate synthease, and phosphoenolpyruvate synthetase, provide evidence of increased gluconeogenesis in the presence of the acetate + glucose mixture (as compared to gluconeogenesis on the corresponding monosubstrates).

Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

The Sir2 family of proteins consists of broadly conserved NAD(+)-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD(+) at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with related biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called "C pocket" binding site for the nicotinamide group of NAD(+). These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.

Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2

We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetyl-CoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.

Sir2 regulated deacetylation and activation of mammalian Acetyl CoA Synthetase 1

The Silent Information Regulator 2 (Sir2) enzyme family are conserved NAD-dependant deacetylases that include seven human homologues (SirT1-7). Sir2 homologues in bacteria and yeast have been implicated in the regulation of Acetyl CoA synthetase (AceCS) activity by reversible acetylation. AceCS is evolutionarily conserved and utilizes acetate, ATP, and CoA for production of acetyl CoA. Mammals contain two AceCS homologues, AceCS1 and AceCS2, which produce acetyl CoA for fatty acid metabolism and the TCA cycle, respectively. The acetylation site of bacterial AceCS shows sequence homology to lysine 661 of AceCS1 to which a specific anti-acetyl-AceCS1 antibody was developed from a corresponding 13mer acetylated peptide. This antibody detected acetylated AceCS1 in Cos-7 cells transiently transfected with mammalian AceCS1. To determine which of the seven SIRtuins displayed the highest in vivo specificity for acetylated-AceCS1, we conducted transient co-expression experiments and found that SirT1 had the greatest specificity for acetyl-AceCS1. Recombinant acetyl-AceCS1 displayed negligible activity, whereas deacetylation by Sir2 enzymes restored AceCS1 activity. Using purified proteins, we examined the specificity of certain SIRtuins for stoichiometrically acetylated AceCS1. SirT1 and SirT3 exhibited high catalytic efficiency on purified acetyl-AceCS1, while SirT2 was 3-fold less efficient, and SirT5 and SirT6 displayed no detectable deacetylation. Thus, mammalian AceCS1 activity can be posttranslationally regulated by SirT1, which has important implications in the generation of Acetyl CoA pools and in fatty acid metabolism. NIH grant GM065386

Acetate excretion during growth of Salmonella enterica on ethanolamine requires phosphotransacetylase (EutD) activity, and acetate recapture requires acetyl-CoA synthetase (Acs) and phosphotransacetylase (Pta) activities

This report shows that Salmonella enterica catabolizes ethanolamine to acetyl-CoA (Ac-CoA), which enters the glyoxylate bypass and tricarboxylic acid cycle for the generation of energy and central metabolites. During growth on ethanolamine, S. enterica excreted acetate, whose recapture depended on Ac-CoA synthetase (Acs) and the housekeeping phosphotransacetylase (Pta) enzyme activities. The Pta enzyme did not play a role in acetate excretion during growth of S. enterica on ethanolamine. It is proposed that during growth on ethanolamine, acetate excretion is necessary to maintain a pool of free CoA. Acetate excretion requires the eut operon-encoded phosphotransacetylase (EutD) and acetate kinase (Ack) enzymes. EutD function was not required for growth on ethanolamine, and an eutD strain showed only a slight reduction in growth rate. The existence of an as-yet-unidentified system that releases acetate was revealed during growth of a strain lacking Acs, the housekeeping phosphotransacetylase (Pta), and EutD. The functions of pyruvate oxidase (PoxB), Ack and STM3118 protein [a homologue of the Saccharomyces cerevisiae Ac-CoA hydrolase (Ach1p) enzyme] were not involved in the release of acetate by the acs pta eutD strain.

Peroxisomal and Mitochondrial Oxidation of Fatty Acids in the Heart, Assessed from the 13C Labeling of Malonyl-CoA and the Acetyl Moiety of Citrate

We previously showed that a fraction of the acetyls used to synthesize malonyl-CoA in rat heart derives from partial peroxisomal oxidation of very long and long-chain fatty acids. The 13C labeling ratio (malonyl-CoA)/(acetyl moiety of citrate) was >1.0 with 13C-fatty acids, which yields [13C]acetyl-CoA in both mitochondria and peroxisomes and < 1.0 with substrates, which yields [13C]acetyl-CoA only in mitochondria. In this study, we tested the influence of 13C-fatty acid concentration and chain length on the labeling of acetyl-CoA formed in mitochondria and/or peroxisomes. Hearts were perfused with increasing concentrations of labeled docosanoate, oleate, octanoate, hexanoate, butyrate, acetate, or dodecanedioate. In contrast to the liver, peroxisomal oxidation of 1-13C-fatty acids in heart does not form [1-13C]acetate. With [1-13C]docosanoate and [1,12-13C2]dodecanedioate, malonyl-CoA enrichment plateaued at 11 and 9%, respectively, with no detectable labeling of the acetyl moiety of citrate. Thus, in the intact rat heart, docosanoate and dodecanedioate appear to be oxidized only in peroxisomes. With [1-13C]oleate or [1-13C]octanoate, the labeling ratio >1 indicates the partial peroxisomal oxidation of oleate and octanoate. In contrast, with [3-13C]octanoate, [1-13C]hexanoate, [1-13C]butyrate, or [1,2-13C2]acetate, the labeling ratio was <0.7 at all concentrations. Therefore, in rat heart, (i) n-fatty acids shorter than 8 carbons do not undergo peroxisomal oxidation, (ii) octanoate undergoes only one cycle of peroxisomal beta-oxidation, (iii) there is no detectable transfer to the mitochondria of acetyl-CoA from the cytosol or the peroxisomes, and (iv) the capacity of C2-C18 fatty acids to generate mitochondrial acetyl-CoA decreases with chain length.

Presence of Acetyl-CoA Synthetase in Ripened Bananas

Acetyl-CoA synthetase (ACS) activity in fully ripened bananas was determined by HPLC. The ACS activity was higher for acetate as a substrate than for butyrate. The higher substrate specificity for acetate than for butyrate evidences that banana produces a great amount of acetate esters and a less amount of butyrate esters. The Km for MgCl2 for butyryl-CoA formation was 5-fold higher than that for acetyl-CoA formation. ACS activity was assayed in mitochondrial, microsomal, and cytosol fractions isolated from ripened banana pulp and the highest total enzyme activity was detected in the cytosol fraction. This is the first report on the presence of this enzyme, ACS, in fruit pulp.

Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production.

To investigate the relationship between the activity of pyruvate dehydrogenase (PDH) bypass and the production of pyruvate of a multi-vitamin auxotrophic yeast Torulopsis glabrata. Torulopsis glabrata CCTCC M202019, a multi-vitamin auxotrophic yeast that requires acetate for complete growth on glucose minimum medium, was selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata WSH-IP303 screened in previous study [Li et al. (2001) Appl. Microbiol. Biotechnol. 55, 680-685]. Strain CCTCC M202019 produced 21% higher pyruvate than the parent strain and was genetically stable in flask cultures. The activities of the pyruvate metabolism-related enzymes in parent and mutant strains were measured. Compared with the parent strain, the activity of pyruvate decarboxylase (PDC) of the mutant strain CCTCC M202019 decreased by roughly 40%, while the activity of acetyl-CoA synthetase (ACS) of the mutant increased by 103.5 or 57.4%, respectively, in the presence or absence of acetate. Pyruvate production by the mutant strain CCTCC M202019 reached 68.7 g l(-1) at 62 h (yield on glucose of 0.651 g g(-1)) in a 7-l jar fermentor. The increased pyruvate yield in T. glabrata CCTCC M202019 was due to a balanced manipulation of the PDH bypass, where the shortage of cytoplasmic acetyl-CoA caused by the decreased activity of PDC was properly compensated by the increased activity of ACS. Manipulating the PDH bypass may provide an alternative approach to enhance the production of glycolysis-related metabolites.

Coordinate metabolism? Yes, Sir2!

The highly conserved Sir2 family of proteins are involved in regulating chromosome stability and cell aging in eukaryotes, as well as gene silencing in both eukaryotes and archea. Sir2 has nicotinamide adenine dinucleotide (NAD + )-dependent deacetylase activity, and its substrates include histones and the tumor suppressor p53. Starai et al. show that the Salmonella enterica acetyl-CoA synthetase is turned off by posttranslational acetylation of a lysine residue in the active site. The Sir2 homolog CobB switches on the activity of acetyl-CoA synthetase by deacetylating this residue. The conservation of these proteins suggests that lysine acetylation is a common regulatory mechanism in prokaryotes and eukaryotes, in the latter case providing a link between the acetylation of histones and the physiological state of the cell. V. J. Starai, I. Celic, R. N. Cole, J. D. Boeke, J. C. Escalante-Semerena, Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298 , 2390-2392 (2002). [Abstract] [Full Text]

Regulation of acetate metabolism in a strain of Acinetobacter sp., growing on ethanol

Ethanol metabolism in Acinetobacter sp. is limited by the rate of acetate assimilation in a reaction catalyzed by acetyl-CoA synthetase (EC 6.2.1.1). Effects of ions (sodium, potassium, and magnesium), byproducts of ethanol and acetaldehyde oxidation (NADH and NADPH), and pantothenic acid on this enzyme have been studied (sodium, NADH, and NADPH inhibit acetyl-CoA synthetase; pantothenic acid, potassium, and magnesium act as the enzyme activators). Conditions of culturing were developed, under which ethanol, acetaldehyde, and acetate in Acinetobacter cells were oxidized at the same rates, producing a threefold increase in the activity of acetyl-CoA synthetase in the cell-free extract. The results of studies of acetyl-CoA synthetase regulation in a mutant strain of Acinetobacter sp., which is incapable of forming exopolysaccharides, provide a basis for refining the technology of ethapolan production, involving the use of C2 substrates.

Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine.

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes.

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri.

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.

The Pseudomonas aeruginosa acsA gene, encoding an acetyl-CoA synthetase, is essential for growth on ethanol.

Pseudomonas aeruginosa ATCC 17933 uses a pyrroloquinoline quinone-dependent ethanol oxidation system. Two mutants of P. aeruginosa, unable to grow on ethanol and showing no acetyl-CoA synthetase (ACS) activity under standard test conditions, were complemented by cosmid pTB3018. Subcloning led to the isolation of a gene which encodes a protein with high similarity to acetyl-CoA synthetases. Interruption of the putative acsA gene by a kanamycin-resistance cassette resulted in a mutant also unable to grow on ethanol and with very low residual acetyl-CoA-forming activity. Complementation by the wild-type allele of the acsA gene restored growth and led to the expression of ACS activity in excess of that of wild-type cells. In wild-type P. aeruginosa, ACS activity was induced upon growth on ethanol, 2,3-butanediol, malonate and acetate. The wild-type and mutants defective in ACS activity showed an active acetate kinase (ACK) under the growth conditions used; however, phosphotransacetylase (PTA) could not be detected. The data indicate that P. aeruginosa requires active acsA gene product for growth on ethanol.

13] Acetyl-CoA Synthetases I and II from Pyrococcus furiosus

Publisher Summary Acetate and acetyl-CoA are important intermediates in microbial metabolism. Fatty acids, polysaccharides, and proteins are all broken down into acetyl-CoA units, a requisite step prior to energy generation. Acetyl CoA is used as a building block in the synthesis of various cell components or it is transformed to acetate as an end-product of certain fermentative pathways. The conversion of acetyl-CoA to acetate is, therefore, a key step in general metabolism. In most bacteria, this transformation is catalyzed by two enzymes: phosphoacetyltransferase and acetate kinase. Two distinct enzymes with ADP-dependent acetyl-CoA synthetase activity have been purified from cell-free extracts of P. furiosus , an organism that grows by fermenting both sugars and peptides. These enzymes convert the acetyl-CoA produced by the fermentative pathways into acetate with the concomitant production of ATP. Acetyl-CoA is produced by the oxidative decarboxylation of pyruvate by pyruvate ferredoxin oxidoreductase (POR). However, the two enzymes, which are termed acetyl-CoA synthetase (ACS) I and II, differ in their substrate specificity. This chapter describes the methods used to assay and purify ACS I and ACS II from P. furiosus , along with some of their molecular and catalytic properties.

セルレニン耐性による酢酸低生産性清酒酵母の分離

Low acetic acid-producing strains were isolated from cerulenin-resistant mutants of the sake yeast K 7 (Saccharomyces cerevisiae). Acetic acid is considered to be an intermediate metabolite of the cytosolic acetyl CoA synthetic pathway from acetaldehyde.Cytosolic acetyl CoA is used for the biosynthesis of sterols, fatty acids, etc. Cerulenin is an inhibitor of fatty acid synthase.Thus, a mutant producing lower levels of acetic acid and higher levels of acetyl CoA is supposed to show cerulenin resistance. Out of 80 cerulenin-resistant mutants, four were selected as low acetic acid-producing mutants using a fermentation test with a liquid synthetic medium and a small scale sake fermentation test. K 7 and one mutant, K 7-163, produced 136.6mg/l and 16.4mg/l of acetic acid, respectively, in pilot scale sake making. Three of the four mutants showed higher acetyl CoA synthetase (ACS) activity than K 7 during the late period of growth, which appeared to be the reason for their low acetic acid productivity. However, all four mutants showed multi-drug resistance, and/or high ethyl caproate productivity, which was probably caused by FAS 2 mutation. Thus, further studies are needed to confirm the relationship between low acetic acid productivity and cerulenin resistance.

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans.

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.