An acetyl-CoA synthetase not encoded by the facA gene is expressed under carbon starvation in Phycomyces blakesleeanus (original) (raw)
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Isolation of an acetyl-CoA synthetase gene(ZbACS2) fromZygosaccharomyces bailii
Yeast, 2004
A gene homologous to Saccharomyces cerevisiae ACS genes, coding for acetyl-CoA synthetase, has been cloned from the yeast Zygosaccharomyces bailii ISA 1307, by using reverse genetic approaches. A probe obtained by PCR amplification from Z. bailii DNA, using primers derived from two conserved regions of yeast ACS proteins, RIGAIHSVVF (ScAcs1p; 210-219) and RVDDVVNVSG (ScAcs1p; 574-583), was used for screening a Z. bailii genomic library. Nine clones with partially overlapping inserts were isolated. The sequenced DNA fragment contains a complete ORF of 2027 bp (ZbACS2 ) and the deduced polypeptide shares significant homologies with the products of ACS2 genes from S. cerevisiae and Kluyveromyces lactis (81% and 82% identity and 84% and 89% similarity, respectively). Phylogenetic analysis shows that the sequence of Zbacs2 is more closely related to the sequences from Acs2 than to those from Acs1 proteins. Moreover, this analysis revealed that the gene duplication producing Acs1 and Acs2 proteins has occurred in the common ancestor of S. cerevisiae, K. lactis, Candida albicans, C. glabrata and Debaryomyces hansenii lineages. Additionally, the cloned gene allowed growth of S. cerevisiae Scacs2 null mutant, in medium containing glucose as the only carbon and energy source, indicating that it encodes a functional acetyl-CoA synthetase. Also, S. cerevisiae cells expressing ZbACS2 have a shorter lag time, in medium containing glucose (2%, w/v) plus acetic acid (0.1-0.35%, v/v). No differences in cell response to acetic acid stress were detected both by specific growth and death rates. The mode of regulation of ZbACS2 appears to be different from ScACS2 and KlACS2, being subject to repression by a glucose pulse in acetic acid-grown cells. The nucleotide sequence of a common 5269 bp fragment has been deposited in the EMBL Data Library under Accession No. AJ314837.
Microbiology, 1998
Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.
AMP-forming acetyl-CoA synthetase (ACS) catalyzes the formation of acetyl-CoA. Here, a cDNA of ACS from Dunaliella tertiolecta (DtACS) was isolated using RACEs. The full-length DtACS cDNA (GenBank: KT692941) is 2,464 bp with a putative ORF of 2,184 bp, which encodes 727 amino acids with a predicted molecular weight of 79.72 kDa. DtACS has a close relationship with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis. ACSs existing in Bacteria, Archaea and Eukaryota share ten conserved motifs (A1–A10) and three signature motifs (I–III) of the acyl-adenylate/thioester forming enzyme superfamily. DtACS was expressed in E. coli BL21 as Trx-His-tagged fusion protein (~100 kDa) and the enzymatic activity was detected. The recombinant DtACS was purified by HisTrap TM HP affinity chromatography to obtain a specific activity of 52.873 U/mg with a yield of 56.26%, which approached the specific activity of ACS isolated from other eukaryotes. Kinetic analysis indicated that the Km of DtACS was 3.59 mM for potassium acetate, and the purified DtACS exhibited a temperature optimum of 37 °C and a pH optimum of 8.0. In addition, the expression levels of DtACS were increased after nitrogen starvation cultivation, indicating that ACS activity may be related to the lipid accumulation under nitrogen deficient condition. Acetyl-CoA is an intermediate metabolite at the intersection of various anabolic and catabolic pathways, and its interconversion with acetate occurs by three distinct mechanisms 1. One pathway consists of the acetate kinase (ACK, EC 2.7.2.1)/phosphotransacetylase (PTA, EC 2.3.1.8) enzymes, which catalyze acetate to acetyl-CoA via acetyl phosphate. Most anaerobic bacteria activate acetate to acetyl-CoA via ACK/PTA pathway. A second pathway of catalyzing acetate to acetyl-CoA is composed of ADP-forming acetyl-CoA synthetase (ADP-forming ACS, EC 6.2.1.13). It has been only existed in some archean halophytes and thermophiles, as well as in anaerobic protists 2,3. A third route is composed of AMP-forming ACS (EC 6.2.1.1), and has a broader distribution and has been found in eubacteria, a few archaea, and eukaryotes 1. In contrast to ACS, ACK and PTA function primarily in the catabolic direction, whereby acetate is excreted and ATP is synthesized. Hence, in bacteria, ACS is the preferred route of acetate assimilation. It seems that the role of ACS is more important in eukaryotes than in prokaryotes, since ACS is the only route for the activation of acetate to acetyl-CoA in eukaryotes. AMP-forming ACS, which catalyzes the formation of acetyl-CoA from acetate, ATP and CoASH (acetate + ATP + CoASH → acetyl-CoA + AMP + PPi), is a member of the acyl-adenylate-forming enzyme super-family that includes nonribosomal peptide synthetases, firefly luciferase, and acyl-and aryl-CoA synthetases 4. ACS carries out an irreversible reaction via two enzymatic steps. The first step is to form acetyl-AMP by the reaction of acetate with ATP. Then acetyl-AMP reacts with CoASH to form acetyl-CoA releasing AMP. It was shown that the overexpression of ACS in E. coli caused significant reduction in acetate during glucose metabolism 5. And the overexpression of ACS2 in Saccharomyces cerevisiae also showed that the capacity of S. cerevisiae to synthesize acetyl-CoA from acetate was increased. It was presumed that increased ACS levels enhanced the formation of acetyl-CoA, which may increase the rate of fatty acid synthesis. Recently, the E. coli ACS gene was introduced
Acetyl-CoA hydrolase involved in acetate utilization in Saccharomyces cerevisiae
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1996
Acetyl-CoA hydrolase, catalyzing the hydrolysis of acetyl-CoA, is presumably involved in regulating the intracellular acetyl-CoA or CoASH pools. The yeast enzyme is encoded by ACH1 (acetyl-CoA hydrolase) and the expression of ACH1 is repressed by glucose (
Expression of a yeast acetyl CoA hydrolase in the mitochondrion
Plant Molecular Biology, 2004
Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.
Algal Research, 2018
Ge netic en gi neer ing can be the so lu tion to achieve the eco nom i cally fea si ble pro duc tion of mi croal gal based bio fu els and other bulk ma te ri als. A good num ber of mi croal gal species can grow mixotroph i cally us ing ac etate as car bon source. More over, ex per i men tal ev i dence sug gests that the biosyn the sis of acetyl-CoA could be a lim it ing step in the com plex mul ti fac tor-de pen dent biosyn the sis of acyl glyc erides and point to acetyl-CoA syn thetase (ACS) as a key en zyme in the process. In or der to test this hy poth e sis we have en gi neered the model chloro phyte Chlamy domonas rein hardtii to over ex press the en doge nous chloro plas tic acetyl-CoA syn thetase, ACS2. Ex pres sion of the ACS2 en cod ing gene un der the con trol of the strong con sti tu tive RbcS2 pro moter in ni tro gen-re plete cul tures re sulted in a 2-fold in crease in starch con tent and 60% higher acyl-CoA pool com pared to the parental line. Un der ni tro gen de pri va tion, the Cr-acs2 trans for mant shows 6-fold higher lev els of ACS2 tran script and a 2.4-fold higher ac cu mu la tion of tri a cyl glyc erol (TAG) than the un trans formed con trol. Analy sis of lipid species and fatty acid pro files in the Cr-acs2 trans for mant re vealed a higher con tent than the parental strain in the ma jor gly col ipids and sug gests that the en hanced syn the sis of tri a cyl glyc erol in the trans for mant is not achieved at ex pense of mem brane lipids, but is due to an in crease in the car bon flux to wards the syn the sis of acetyl-CoA in the chloro plast. This data demon strates the po ten tial of en gi neer ing the chloro plas tic ACS to in crease the car bon flux to wards the syn the sis of fatty acids as an al ter na tive strat egy to en hance the biosyn the sis of lipids in mi croal gae.
Microbial Cell Factories
Background Oleaginous yeasts are able to accumulate very high levels of neutral lipids especially under condition of excess of carbon and nitrogen limitation (medium with high C/N ratio). This makes necessary the use of two-steps processes in order to achieve high level of biomass and lipid. To simplify the process, the decoupling of lipid synthesis from nitrogen starvation, by establishing a cytosolic acetyl-CoA formation pathway alternative to the one catalysed by ATP-citrate lyase, can be useful. Results In this work, we introduced a new cytoplasmic route for acetyl-CoA (AcCoA) formation in Rhodosporidium azoricum by overexpressing genes encoding for homologous phosphoketolase (Xfpk) and heterologous phosphotransacetylase (Pta). The engineered strain PTAPK4 exhibits higher lipid content and produces higher lipid concentration than the wild type strain when it was cultivated in media containing different C/N ratios. In a bioreactor process performed on glucose/xylose mixture, to s...
The evolution of acetyl-CoA synthase
Origins of life and evolution of the biosphere : the journal of the International Society for the Study of the Origin of Life
Acetyl-coenzyme A synthases (ACS) are Ni-Fe-S containing enzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACS's catalyze the synthesis of acetyl-CoA from CO2 + 2e-, CoA, and a methyl group, and contain 5 types of subunits (alpha, beta, gamma, delta, and epsilon). Class II enzymes catalyze essentially the reverse reaction and have similar subunit composition. Class III ACS's catalyze the same reaction as Class I enzymes, but use pyruvate as a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an alpha 2 beta 2 tetramer and a gamma delta heterodimer. Class IV enzymes catabolize CO to CO2 and are alpha-subunit monomers. Phylogenetic analyses were performed on all five subunits. ACS alpha sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residues that may function as ligands to the B- and C-clusters were identified. Other residues exclusively conserved in Class I/II seq...