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Enhancing itaconic acid production by Aspergillus terreus

Aspergillus terreus is successfully used for industrial production of itaconic acid. The acid is formed from cis-aconitate, an intermediate of the tricarboxylic (TCA) cycle, by catalytic action of cis-aconitate decarboxylase. It could be assumed that strong anaplerotic reactions that replenish the pool of the TCA cycle intermediates would enhance the synthesis and excretion rate of itaconic acid. In the phylogenetic close relative Aspergillus niger, upregulated metabolic flux through glycolysis has been described that acted as a strong anaplerotic reaction. Deregulated glycolytic flux was caused by posttranslational modification of 6-phosphofructo-1-kinase (PFK1) that resulted in formation of a highly active, citrate inhibition-resistant shorter form of the enzyme. In order to avoid complex posttranslational modification, the native A. niger pfkA gene has been modified to encode for an active shorter PFK1 fragment. By the insertion of the modified A. niger pfkA genes into the A. terreus strain, increased specific productivities of itaconic acid and final yields were documented by transformants in respect to the parental strain. On the other hand, growth rate of all transformants remained suppressed which is due to the low initial pH value of the medium, one of the prerequisites for the accumulation of itaconic acid by A. terreus mycelium.

Characterization and overexpression of the Aspergillus niger gene encoding the cAMP-dependent protein kinase catalytic subunit

The gene pkaC encoding the catalytic subunit of cAMP-dependent protein kinase has been isolated from the industrially important filamentous fungus Aspergillus niger. A probe for screening A. niger phage libraries was generated by a polymerase chain reaction using degenerate primers. cDNA and genomic DNA clones were isolated and sequenced. An open reading frame of 1440 bp, interrupted by three short introns, encodes a polypeptide of 480 amino acids with a calculated molecular mass of 53813 Da. The cAMP-dependent protein kinase catalytic subunit (PKA-C) from A. niger has a 126 amino acid extension at the N-terminus compared to the PKA-C of higher eukaryotes that-except for the first 15 amino acids, which are homologous to the Magnaporthe grisea PKA-C-shows no significant similarity to the N-terminal extension of PKA-C of other lower eukaryotes. The catalytic core of PKA-C of A. niger shows extensive homology with the PKA-C isolated from all other eukaryotes. Low-stringency hybridization did not reveal any other pkaC homologue in A. niger. The cloned pkaC was used for transformation of A. niger, leading to increased levels of pkaC mRNA and PKA-C activity. Transformants overexpressing pkaC were phenotypically different with respect to growth, showing a more compact colony morphology, accompanied by a more dense sporulation, especially on media containing trehalose and glycerol. A number of transformants also showed a strongly reduced or complete absence of sporulation. This phenotype was quickly lost upon propagation of the strains.

Cross-talk between cAMP and calcium signalling in Aspergillus niger

Very little is known about cross‐talk between cAMP and calcium signalling in filamentous fungi. The aim of this study was to analyse the influence of cAMP and protein kinase A (PKA)‐dependent phosphorylation on calcium signalling in Aspergillus niger. For this purpose, cytosolic free calcium ([Ca2+]c) was measured in living hyphae expressing codon‐optimized aequorin. The calcium signature following mechanical perturbation was analysed after applying dibutryl‐cAMP or IBMX which increased intracellular cAMP, or H7 which inhibited phosphorylation by PKA. Calcium signatures were also measured in mutant strains in which phosphorylation by PKA was increased or lacking. The results indicated that calcium channels were activated by cAMP‐mediated, PKA‐dependent phosphorylation. Further evidence for cross‐talk between cAMP and calcium signalling came from the analysis of a mutant in which the catalytic subunit of PKA was under the control of an inducible promoter. The consequence of PKA induction was a transient increase in [Ca2+]c which correlated with a polar–apolar transition in hyphal morphology. A transient increase in [Ca2+]c was not observed in this mutant when the morphological shift was in the opposite direction. The [Ca2+]c signatures in response to mechanical perturbation by polarized and unpolarized cells were markedly different indicating that these two cell types possessed different calcium signalling capabilities. These results were consistent with PKA‐dependent phosphorylation increasing [Ca2+]c to induce a polar to apolar shift in hyphal morphology.

Highly active, citrate inhibition resistant form of Aspergillus niger 6-phosphofructo-1-kinase encoded by a modified pfkA gene

In Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85kDa) is first cleaved to an inactive fragment (49kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native pfkA genes were available, Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.

Increased mannitol production in Lactobacillus reuteri ATCC 55730 production strain with a modified 6-phosphofructo-1-kinase

Based on established knowledge of the simultaneous use of the phosphoketolase pathway (PKP) and the Embden-Meyerhof pathway (EMP) - as a secondary pathway with a smaller flux - by mannitol producer Lactobacillus reuteri ATCC 55730, we demonstrated the hypothesis that by enhancing the flux through the EMP the ability of the microorganism to handle elevated glucose concentrations will be improved, in addition to its growth rate and biomass yield. NADH availability will be increased and its demand will be satisfied, allowing the electron acceptor fructose to be more efficiently transformed into mannitol. A truncated version of the gene encoding 6-phospho-1-fructokinase (tpfkA) from the NRRL 2270 strain of Aspergillus niger along with its activator pkaC were introduced into the microorganism by plasmid transformation. Growth of the transformants at elevated glucose concentrations in the presence of fructose resulted in improved assimilation of the provided carbohydrates and a significant increase in the overall fermentation productivities. At the highest tested levels of glucose and fructose (75g/l each), the transformant strain experienced a 4-fold increase in PFK activity and a 2.3-fold increase in the glycolytic flux while the biomass yield reached 7g/l (1.6g/l in the parental strain), the mannitol yield was 56g/l (10g/l in the parental strain) and the lactate yield was 21g/l (3.5g/l in the parental strain). A high NADH/NAD(+) ratio occurred under increased glycolytic flux conditions and facilitated the efficient conversion of fructose to mannitol. A direct effect of deregulated PFK activity on the glycolytic flux is therefore demonstrated in the present case suggesting an alternative approach of metabolic engineering in L. reuteri for increased mannitol production.

Stress mediated changes in expression of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase, in Aspergillus niger.

The transcriptional regulation of the pkaC gene, encoding the catalytic subunit of cAMP-dependent protein kinase from Aspergillus niger was analysed under different environmental conditions. Quantitative determination of pkaC transcript showed a significant decrease in concentration of specific mRNA immediately after a temperature, hypoosmotic and hyperosmotic shock followed by stimulated synthesis. The amount of pkaC mRNA as well as PKA enzymatic activity steadily decreased during the initial phase of growth in 15 % sucrose medium while a slight increase was observed at the time of a change in morphology from bulbous cells to filamentous growth. Transcriptional alternation might be mediated by multiple putative stress elements in the promoter region of pkaC gene.

Non-radioactive multiple reverse transcription-PCR method used for low abundance mRNA quantification

A non-radioactive multiple reverse transcription-PCR method for the relative quantitative analysis of low abundance mRNA is described. Chemiluminescent detection of cDNA products amplified by PCR and incorporating biotin-labelled dCTP was used for analysis of specific gene expression. After determining the optimal concentrations of RNA and cDNA, the tube-to-tube variations were minimised by transcription and amplification of the endogenous standard mRNA coding for protease C from Aspergillus niger, together with the transcript of interest. All the obtained data were relative values comparing to the internal standard pepC. By the method the difference in mRNA level between the two transformants with different pkaC gene copy number coding for cAMP-dependent protein kinase (PKA) catalytic subunit from A. niger was detected which was in accordance with the differences in specific PKA enzymatic activity between both strains.