Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.     

Effect of deletion of chitin synthase genes on mycelial morphology and culture viscosity in Aspergillus oryzae

The objective of this study was to quantify the effect of disrupting two chitin synthases, chsB and csmA, on the morphology and rheology during batch cultivation of Aspergillus oryzae. The rheological properties were characterized in batch cultivations at different biomass concentrations (from 3.4-22.5 g kg(-1) biomass) and the power-law model adequately described the rheological properties. In the cultivations there were pellets, clumps, and freely dispersed hyphal elements. The different morphological fractions were quantified using image analysis. The apparent viscosity of the fermentation broth was significantly affected by the biomass concentration, the morphology, and also by pH. The chsB disruption strain had lower consistency index K values for all biomass concentrations investigated, which is a desirable trait for industrial Aspergillus fermentations.     

Morphology engineering of Aspergillus oryzae for l-malate production

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l -malate production. Specifically, correlation between A. oryzae morphology and l -malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l -malate production was 120–130 mm³/ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l -malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l -malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.     

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.     

Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8–2.9 up to 4.0–4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.     

Aspergillus oryzae laeA regulates kojic acid synthesis genes

Kojic acid synthesis genes regulation was investigated in Aspergillus oryzae. Our results indicate that kojic acid production was lost in the laeA disruption strain, but was recovered in the LaeA complement strain. Real-time PCR also confirmed that expression of kojic acid biosynthesis genes decreased in the laeA disruption strain, indicating that these genes are under the control of LaeA.     

Differential repression of arylsulphatase synthesis in Aspergillus oryzae.

1. The activities of the three arylsulphatases (arylsulphate sulphohydrolase, EC 3.1.6.1) of Aspergillus oryzae produced under a variety of repressing and non-repressing conditions were determined. 2. These enzymes exhibit different sensitivities to repression by inorganic sulphate. 3. Arylsulphatase I, but not arylsulphatases II and III, exhibits a transient de-repression in the early growth phase in sulphate media. 4. When the fungus is cultured in repressing media and subsequently transferred to non-repressing media, the synthesis of the three enzymes is non-co-ordinate. 5. Growth of the fungus in media containing choline O-sulphate or tyrosine O-sulphate as the sole source of sulphur results in complete de-repression of arylsulphatase I, But the synthesis of arylsulphatases II and III is essentially fully repressed. 6. The marked similarities between the repression characteristics of arylsulphatases II and III, contrasted with those of arylsulphatase I, indicate that the genetic locus of arylsulphatase I is distinct from that of arylsulphatases II and III, suggesting that there are distinct physiological roles for the enzyme.     

Biosynthesis of L-DOPA by Aspergillus oryzae

The present investigation deals with the biosynthesis of L-DOPA by parental (GCB-6) and mutant (UV-7) strains of Aspergillus oryzae. There was a marked difference between the mycelial morphology and pellet type of parental and UV-irradiated mutant culture. The mutant strain of A. oryzae UV-6 exhibited pellet-like mycelial morphology and improved tyrosinase activity. Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinase is an intracellular enzyme. The mutant was found to yield 3.72 fold higher production of L-DOPA than the parental strain. The mutant strain is stable and D-glc-resistant. The comparison of kinetic parameters was also done which showed the greater ability of the mutant to yield L-DOPA (i.e., Yp/x 40.00+/-0.01 d mg/mg with parent and 182.86+/-0.02a mg/mg in case of mutant). When cultures grown for various incubation periods, were monitored for Qp, Qs and q(p), there was significant enhancement (p < 0.0025-0.005) in these variables by the mutant strain of A. oryzae UV-7 over GCB-6 on all the rates. L-DOPA (3,4-dihydroxy phenyl L-alanine) is a drug of choice in the treatment of Parkinson's disease and myocardium following neurogenic injury.     

Protease and alpha-amylase synthesis by washed cells of Aspergillus oryzae 251-90

Regularities of protease and alpha-amylase production by washed cells of Aspergillus oryzae 251-90 were being studied. The results obtained enabled us to assume a constitutive character of the both enzymes synthesis by the given producer. Sources of carbon, nitrogen and sulphur take part in regulation of the protease production, whereas in the case of the alpha-amylase synthesis only carbon sources that are important. Elimination of phosphorus from the medium affects the synthesis of both enzymes. Celatin stimulates the production of the two enzymes, being a supplier of amino acids.     

Microbial degradation of monocrotophos by Aspergillus oryzae

Organophosphorus pesticides are widely used in India for protection of agricultural yields. However, these pesticides pose various threats to organisms, including humans, and hamper soil microbial activity; thus, they are a cause for concern. As a measure of bioremediation, soil fungi capable of degrading monocrotophos (MCP) were isolated from various geographical and ecological sites. Twenty-five strains were isolated by an enrichment method using MCP as a carbon and phosphorus source. On the basis of MCP tolerance capacity exhibited in gradient agar plate assay the isolate M-4, identified as Aspergillus oryzae ARIFCC 1054, was selected for further studies. The ability of the isolate to mineralize MCP was investigated under different culture conditions. The isolate was found to possess phosphatase activity. The course of the degradation process was studied using HPTLC and FTIR analyses. The results suggest that this organism could be used for bioaugmentation of soil contaminated with MCP and for treatment of aqueous wastes.     

米曲霉发酵纯化无患子皂苷的工艺研究

采用微生物发酵法对无患子皂苷水提取液进行纯化.比较了采用自然发酵、接种酵母菌发酵和接种米曲霉发酵纯化无患子皂苷的效果.结果表明,提取液不灭菌,接种米曲霉发酵纯化效果较为明显,优化后的发酵条件为:温度30℃、接种龄12 h、接种量为3％、摇床转速150 r/min,发酵7d后,皂苷含量稍有下降,但皂苷纯度可从48.71％提高到82.47％.米曲霉发酵法明显优于水提醇沉法、絮凝法和正丁醇萃取法.     

Invasion of paranasal sinuses by Aspergillus oryzae

A 24-year-old male patient receiving chemotherapy for acute promyelocytic leukemia developed fever, right periorbital swelling and mild right proptosis. A head scan showed opacification of the right maxillary and ethmoid sinuses with adjacent soft tissue swelling. Biopsy of the nasal mucosa demonstrated the typical septate hyphae of Aspergillus species which was later shown on culture to be Aspergillus oryzae. A. oryzae has only rarely been reported in human disease and there is confusion as to its precise identification and role. We would like to confirm the pathogenicity of A. oryzae with this uncommon presentation of aspergillosis and also emphasize the need to take adequate and multiple cultures in suspected cases so that the possibility of species identification will be maximized.     

Metabolic engineering of monoterpene synthesis in yeast

Terpenoids are one of the largest and most diverse families of natural compounds. They are heavily used in industry, and the trend is toward engineering modified microorganisms that produce high levels of specific terpenoids. Most studies have focused on creating specific heterologous pathways for sesquiterpenes in Escherichia coli or yeast. We subjected the Saccharomyces cerevisiae ERG20 gene (encoding farnesyl diphosphate synthase) to a set of amino acid mutations in the catalytic site at position K197. Mutated strains have been shown to exhibit various growth rate, sterol amount, and monoterpenol-producing capacities. These results are discussed in the context of the potential use of these mutated strains for heterologous expression of monoterpenoid synthases, which was investigated using Ocimum basilicum geraniol synthase. The results obtained with up to 5 mg/L geraniol suggest a major improvement compared with previous available expression systems like Escherichia coli or yeast strains with an unmodified ERG20 gene that respectively delivered amounts in the 10 and 500 μg/L range or even a previously characterized K197E mutation that delivered amounts in the 1 mg/L range.     

Metabolic engineering of plants for artemisinin synthesis

Artemisinin, a natural compound from Artemisia annua, is highly effective in treating drug-resistant malaria. Because chemical synthesis of this natural terpenoid is not economically feasible, its only source remains as the native plant which produces only small quantities of it, resulting in a supply that is far short of demand. Extensive efforts have been invested in metabolic engineering for the biosynthesis of artemisinin precursors in microbes. However, the production of artemisinin itself has only been achieved in plants. Since, A. annua possesses only poorly developed genetic resources for traditional breeders, molecular breeding is the best alternative. In this review, we describe the efforts taken to enhance artemisinin production in A. annua via transgenesis and advocate metabolic engineering of the complete functional artemisinin metabolic pathway in heterologous plants. In both cases, we emphasize the need to apply state-of-the-art synthetic biology approaches to ensure successful biosynthesis of the drug.     

Repetitive attack by Aspergillus oryzae alpha amylase

The action of Aspergillus oryzae alpha amylase on reducing-end, and uniformly radiolabeled maltotriose through maltodecaose has been studied. The enzyme is found to hydrolyze more than a single glycosidic bond during some enzyme-substrate encounters. The extent of this repetitive attack is quantitated.