Citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 and purification of citric acid

In this study, citric acid production from extract of Jerusalem artichoke tubers by the genetically engineered yeast Yarrowia lipolytica strain 30 was investigated. After the compositions of the extract of Jerusalem artichoke tubers for citric acid production were optimized, the results showed that natural components of extract of Jerusalem artichoke tubers without addition of any other components were suitable for citric acid production by the yeast strain. During 10 L fermentation using the extract containing 84.3 g L^?1 total sugars, 68.3 g L^?1 citric acid was produced and the yield of citric acid was 0.91 g g^?1 within 336 h. At the end of the fermentation, 9.2 g L^?1 of residual total sugar and 2.1 g L^?1 of reducing sugar were left in the fermented medium. At the same time, citric acid in the supernatant of the culture was purified. It was found that 67.2 % of the citric acid in the supernatant of the culture was recovered and purity of citric acid in the crystal was 96 %.     

Enhanced citric acid production by a yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> over-expressing a pyruvate carboxylase gene

In this study, after the expression of a pyruvate carboxylase gene (PYC) cloned from Meyerozyma guilliermondii in a marine-derived yeast Yarrowia lipolytica SWJ-1b, a transformant PG86 obtained had much higher PYC activity than Y. lipolytica SWJ-1b. At the same time, the PYC gene expression and citric acid (CA) production by the transformant PG86 were also greatly enhanced. When glucose concentration in the medium was 60.0 g L^?1, CA concentration formed by the transformant PG86 was 34.02 g L^?1, leading to a CA yield of 0.57 g g^?1 of glucose. During a 10-L fed-batch fermentation, the final concentration of CA was 101.0 ± 1.3 g L^?1, the yield was 0.89 g g^?1 of glucose, the productivity was 0.42 g L^?1 h^?1 and only 5.93 g L^?1 reducing sugar was left in the fermented medium within 240 h of the fed-batch fermentation. HPLC analysis showed that most of the fermentation products were CA.     

Physiological characterization of ATP-citrate lyase in Aspergillus niger

Acetyl-CoA, an important molecule in cellular metabolism, is generated in multiple subcellular compartments and mainly used for energy production, biosynthesis of a diverse set of molecules, and protein acetylation. In eukaryotes, cytosolic acetyl-CoA is derived mainly from the conversion of citrate and CoA by ATP-citrate lyase. Here, we describe the targeted deletions of acl1 and acl2, two tandem divergently transcribed genes encoding subunits of ATP-citrate lyase in Aspergillus niger. We show that loss of acl1 or/and acl2 results in a significant decrease of acetyl-CoA and citric acid levels in these mutants, concomitant with diminished vegetative growth, decreased pigmentation, reduced asexual conidiogenesis, and delayed conidial germination. Exogenous addition of acetate repaired the defects of acl-deficient strains in growth and conidial germination but not pigmentation and conidiogenesis. We demonstrate that both Acl1 and Acl2 subunits are required to form a functional ATP-citrate lyase in A. niger. First, deletion of acl1 or/and acl2 resulted in similar defects in growth and development. Second, enzyme activity assays revealed that loss of either acl1 or acl2 gene resulted in loss of ATP-citrate lyase activity. Third, in vitro enzyme assays using bacterially expressed 6His-tagged Acl protein revealed that only the complex of Acl1 and Acl2 showed ATP-citrate lyase activity, no enzyme activities were detected with the individual protein. Fourth, EGFP-Acl1 and mCherry-Acl2 proteins were co-localized in the cytosol. Thus, acl1 and acl2 coordinately modulate the cytoplasmic acetyl-CoA levels to regulate growth, development, and citric acid synthesis in A. niger.     

Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.     

Expression of TPS1 Gene from Saccharomycopsis fibuligera A11 in Saccharomyces sp. W0 Enhances Trehalose Accumulation,Ethanol Tolerance,and Ethanol Production

It has been reported that trehalose plays an important role in stress tolerance in yeasts. Therefore, in order to construct a stably recombinant Saccharomyces sp. W0 with higher ethanol tolerance, the TPS1 gene encoding 6-phosphate-trehalose synthase cloned from Saccharomycopsis fibuligera A11 was ligated into the 18S rDNA integration vector pMIRSC11 and integrated into chromosomal DNA of Saccharomyces sp. W0. The transformant Z8 obtained had the content of 6.23 g of trehalose/100 g of cell dry weight, while Saccharomyces sp. W0 only contained 4.05 g of trehalose/100 g of cell dry weight. The transformant Z8 also had higher ethanol tolerance (cell survival was 25.1 % at 18 ml of ethanol/100 ml of solution) and trehalose-6-phosphate synthase (Tps1) activity (1.3 U/mg) and produced more ethanol (16.4 ml of ethanol/100 ml of medium) than Saccharomyces sp. W0 (cell survival was 12.1 % at 18 ml of ethanol/100 ml of solution, Tps1 activity was 0.8 U/mg and the produced ethanol concentration was 14.2 ml of ethanol/100 ml of medium) under the same conditions. The results show that trehalose indeed can play an important role in ethanol tolerance and ethanol production by Saccharomyces sp. W0.     

Rapid Purification of Enzymes of Fatty Acid Biosynthesis from Rat Adipose Tissue

Abstract

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protien kinases.     

Use of autochthonous lactic acid bacteria starters to ferment mango juice for promoting its probiotic roles

Strains of Leuconostoc mesenteroides, Pediococcus pentosaceus, and Lactobacillus brevis were identified from mango fruits by partial 16S rDNA gene sequence. Based on the ability of producing mannitol and diacetyl, Leuconostoc mesenteroides MPL18 and MPL39 were selected within the lactic acid bacteria isolates, and used as mixed starters to ferment mango juice (MJ). Both the autochthonous strains grew well in fermented mango juice (FMJ) and remained viable at 9.81 log cfu mL^?1 during 30 days of storage at 4°C. The content of total sugar of FMJ was lower than that of MJ, while the concentration of mannitol was higher than that of MJ, and the concentration of diacetyl was 3.29 ± 0.12 mg L^?1. Among detected organic acids including citric acid, gallic acid, lactic acid, and acetic acid, only citric acid and gallic acid were found in MJ, while all detected organic acids were found in FMJ. The concentration of lactic acid of FMJ was the highest (78.62 ± 13.66 mM) among all detected organic acids. The DPPH radical scavenging capacity of FMJ was higher than that of MJ. Total phenolic compounds were better preserved in FMJ. The acidity and sweetness had a noticeable impact on the overall acceptance of the treated sample.     

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg^?1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g^?1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L^?1 citric acid and 5.3 g L^?1 iso-citric acid from inulin while 68.9 g L^?1 of citric acid and 4.1 g L^?1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.     

Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS

Mineral phosphate solubilization by bacteria is mediated through secretion of organic acids, among which citrate is one of the most effective. To overproduce citrate in bacterial systems, an artificial citrate operon comprising of genes encoding NADH-insensitive citrate synthase of E. coli and Salmonella typhimurium sodium-dependent citrate transporter was constructed. In order to improve its mineral phosphate solubilizing (MPS) ability, the citrate operon was incorporated into E. hormaechei DHRSS. The artificial citrate operon transformant secreted 7.2 mM citric acid whereas in the native strain, it was undetectable. The transformant released 0.82 mM phosphate in flask studies in buffered medium containing rock phosphate as sole P source. In fermenter studies, similar phenotype was observed under aerobic conditions. However, under microaerobic conditions, no citrate was detected and P release was not observed. Therefore, an artificial citrate gene cluster containing Vitreoscilla hemoglobin (vgb) gene under its native promoter, along with artificial citrate operon under constitutive tac promoter, was constructed and transformed into E. hormaechei DHRSS. This transformant secreted 9 mM citric acid under microaerobic conditions and released 1.0 mM P. Thus, incorporation of citrate operon along with vgb gene improves MPS ability of E. hormaechei DHRSS under buffered, microaerobic conditions mimicking rhizospheric environment.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Inhibitors of the Glyoxylate Cycle Enzyme ICL1 in Candida albicans for Potential Use as Antifungal Agents

Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinski''s rule-of-five and ADMET analysis.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Cloning, Characterization, and Expression of a New cry1Ab Gene from DOR Bt-1, an Indigenous Isolate of Bacillus thuringiensis

A new cry1Ab gene was cloned from the promising local isolate, DOR Bt-1, a Bacillus thuringiensis strain isolated from castor semilooper (Achaea janata L.) cadavers from castor bean (Ricinus communis L.) field. The nucleotide sequence of the cloned cry1Ab gene indicated that the open reading frame consisted of 3,465 bases encoding a protein of 1,155 amino acid residues with an estimated molecular weight of 130 kDa. Homology comparisons revealed that the deduced amino acid sequence of cry1Ab had a variation of seven amino acid residues compared to those of the known Cry1Ab proteins in the NCBI database and this gene has been designated as cry1Ab26 by the B. thuringiensis δ-endotoxin Nomenclature Committee. cry1Ab26 was cloned into pET 29a(+) vector and expressed in E. coli strain BL21 (DE3) under the control of T7 promoter with IPTG induction. ELISA, SDS-PAGE, and Western blot analysis confirmed the expression of 130-kDa protein. Insect bioassays with neonate larvae of Helicoverpa armigera showed that the partially purified Cry1Ab26 caused 97 % mortality within 5 days of feeding.     

Effects of 20 Standard Amino Acids on the Growth,Total Fatty Acids Production,and γ-Linolenic Acid Yield in Mucor circinelloides

Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.     

Rapid purification of enzymes of fatty acid biosynthesis from rat adipose tissue

Acetyl CoA carboxylase, ATP-citrate lyase and fatty acid synthetase were purified to homogeneity by a simple procedure. The purification method consists of polymerization of acetyl CoA carboxylase with citrate followed by avidin-Sepharose affinity chromatography. ATP-citrate lyase and fatty acid synthetase were isolated as by-products of acetyl CoA carboxylase purification and are separated from each other by chromatography on DE-52. ATP-citrate lyase was further purified by CoA-agarose affinity chromatography and fatty acid synthetase was purified on Bio-Gel A-1.5m. Purified ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase had specific activities of 9.9, 2.8 and 1.8 U/mg respectively with an over all recovery of 30, 25 and 50% respectively. Using these purified enzymes, we found that ATP-citrate lyase and acetyl CoA carboxylase were phosphorylated in vitro by both cAMP-dependent protein kinase and ATP-citrate lyase kinase whereas fatty acid synthetase was not phosphorylated by these protein kinases.