Production of lovastatin by a wild strain of Aspergillus terreus

Of 68 Aspergillus terreus, three produced lovastatin with equivalent or better yield than strain ATCC 20542 originally described for lovastatin production. Medium optimization experiments with the best isolate (TUB F-514) indicated that lactose, rapeseed meal and KNO3 were the best carbon, organic nitrogen and inorganic nitrogen sources, respectively. In shake-flasks with optimized medium containing 4 % (w/v) lactose, 400 g lovastatin/ml was produced, with a yield of 10 mg/g lactose. In solid substrate fermentation on extracted sweet sorghum pulp supplemented with cheese whey 1500 g lovastatin/g dry weight was produced with a yield of 37.5 mg/g lactose. © Rapid Science Ltd. 1998     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

The influence of culturing environments on lovastatin production by Aspergillus terreus in submerged cultures

The effect of the changes of culturing environments of Aspergillus terreus on lovastatin production was investigated in the study. A relatively low supplement of dissolved O₂ (DO) by the fungus almost stopped performing product formation. With the DO controlled at 20%, lovastatin production using a 5-l fermenter enhanced by 38%, biomass production decreased by 25% and sugar utilization increased by 18%, as compared with the shaking-flask culture. Meanwhile, an average diameter 0.95 mm of compact pellets was found. We thus concluded that pellet formation with a narrow size distribution dominated lovastatin production by A. terreus, which was closely affected by the relatively saturated level of DO. Nevertheless, manipulating the broth pH at 5.5–7.5 starting from 48 h provided no benefit to product formation although biomass production was reduced largely. In the part of work, a pH/DO interaction was also confirmed.A simple temperature-shift method (28–23 °C) was proved surprisingly valuable to the fermentation process. Such experiments showed that the maximum of lovastatin production was further enhanced by 25% (572 mg/l at day 10) in comparison with that when the fungus was cultured at 28 °C. The timing to initiate the temperature-shift (96 h) corresponded to that of pellet formation and the subsequent core compactness. Hence, it was found that lovastatin production by A. terreus favored sub-optimal growth conditions.     

Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process

Lovastatin biosynthesis with Aspergillus terreus in batch fermentation reached 160 U/l in 161 h at pH 6.8 and a dissolved O tension maintained at 70%. At the end of repeated fed batch fermentations, the yield of lovastatin was increased by 37% though this took over twice as long as in the batch fermentation.     

Pellet morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus

Pellet growth of Aspergillus terreus ATCC 20542 in submerged batch fermentations in stirred bioreactors was used to examine the effects of agitation (impeller tip speed u(t) of 1.01-2.71 ms(-1)) and aeration regimens (air or an oxygen-enriched mixture containing 80% oxygen and 20% nitrogen by volume) on the fungal pellet morphology, broth rheology and lovastatin production. The agitation speed and aeration methods used did not affect the biomass production profiles, but significantly influenced pellet morphology, broth rheology and the lovastatin titers. Pellets of approximately 1200 microm initial diameter were reduced to a final stable size of approximately 900 microm when the agitation intensity was >/=600 rpm (u(t)>/=2.03 ms(-1)). A stable pellet diameter of approximately 2500 microm could be attained in less intensely agitated cultures. These large fluffy pellets produced high lovastatin titers when aerated with oxygen-enriched gas but not with air. Much smaller pellets obtained under highly agitated conditions did not attain high lovastatin productivity even in an oxygen-enriched atmosphere. This suggests that both an upper limit on agitation intensity and a high level of dissolved oxygen are essential for attaining high titers of lovastatin. Pellet size in the bioreactor correlated equally well with the specific energy dissipation rate and the energy dissipation circulation function. The latter took into account the frequency of passage of the pellets through the high shear regions of the impellers. Pellets that gave high lovastatin titers produced highly shear thinning cultivation broths.     

A macrokinetic modelling of the biosynthesis of lovastatin by Aspergillus terreus

In this work a simple kinetic model to describe the biosynthesis of lovastatin by Aspergillus terreus ATCC 20542 was proposed. Several series of experiments were conducted at different media compositions. The concentrations of C- and N-sources were changed over a wide range and so were the initial biomass concentrations. From these runs the relationships ruling the substrates uptake, biomass and product formation were learnt. Lovastatin biosynthesis appeared to be partly growth associated. The inhibitive effect of organic nitrogen on lovastatin biosynthesis was found and lactose appeared to be an important limiting substrate in the formation of lovastatin. The parameters of the model were evaluated on the basis of the kinetic data obtained in the separate experiments made in triplicate at two chosen media compositions. Other results obtained at different media compositions were independent of the ones mentioned above and used for the verification of the model. The validity of the model was also examined for the lactose-fed fed-batch run. Finally, a sensitivity analysis of the model parameters was performed. The formulated model, although relatively simplified, described the experimental data quite well and could be regarded as the background for further attempts to mathematically describe the process of lovastatin biosynthesis.     

Physiological,morphological and kinetic aspects of lovastatin biosynthesis by Aspergillus terreus

This review focuses on selected aspects of lovastatin biosynthesis by Aspergillus terreus. Biochemical issues concerning this process are presented to introduce polyketide metabolites, in particular lovastatin. The formation of other than lovastatin polyketide metabolites by A. terreus is also shown, with special attention to (+)-geodin and sulochrin. The core of this review discusses the physiology of A. terreus with regard to the influence of carbon and nitrogen sources, cultivation broth aeration and pH control strategies on fungal growth and product formation. Attention is paid to the supplementation of cultivation media with various compounds, namely vitamins, methionine, butyrolactone I. Next, the analysis of fungal morphology and differentiation of A. terreus mycelium in relation to both lovastatin and to (+)-geodin formation is conferred. Finally, the kinetics of the process, in terms of associated metabolite formation with biomass growth is discussed in relation to published kinetic models. The review concludes with a list of the most important factors affecting lovastatin and (+)-geodin biosynthesis.     

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.     

响应面法优化黑曲霉产异淀粉酶的培养条件

在液态发酵条件下,采用单因素实验确定了Aspergillus niger PZ331产异淀粉酶的最适碳源和氮源,分别为蔗糖和硝酸铵。在上述基础上利用Plackett-Burman设计对影响产异淀粉酶的因素进行评价,并筛选出硝酸铵、接种量、培养温度3个主要因素;继而利用响应面设计优化了最佳硝酸铵浓度、接种量和培养温度。最终确定了最优培养条件为：蔗糖10 g/L,硝酸铵10 g/L,磷酸氢二钾3 g/L,硫酸亚铁0.01 g/L,硫酸镁1 g/L,起始p H值4.2;接种量2%（孢子浓度为107cfu/m L）,30℃培养72 h,酶活达137.3μ/m L;比基础培养基的提高了1.71倍左右。     

Supplementation of the cultivation media with B-group vitamins enhances lovastatin biosynthesis by Aspergillus terreus

The impact of the supplementation of cultivation media with B-group vitamins on the biosynthesis of lovastatin (mevinolinic acid) by Aspergillus terreus ATCC20542 was investigated. A hypothesis was formulated that as the biosynthesis of lovastatin requires a high throughput of coenzymes in the cells, the application of its precursors in the form of B-group vitamins might positively influence the process. In a nitrogen-deficient medium the B-group vitamins, both single, especially nicotinamide, pyridoxine and calcium D-pantothenate, and a mixture of thiamine, riboflavin, pyridoxine, calcium d-pantothenate and nicotinamide increased the efficiency of lovastatin biosynthesis. The vitamin supplementation also increased both volumetric and specific production rates of mevinolinic acid, especially before 80 h of the process, when no lactose limitation had been observed yet.     

Itaconic acid production from wheat chaff by Aspergillus terreus

Biotechnologically produced itaconic acid is an important building block for the chemical industry and still based on pure carbon sources, detoxified molasses or starch hydrolysates. Changing these first generation feedstocks to alternative renewable resources of a second generation implies new challenges for the cultivation process of the industrial itaconic acid producer Aspergillus terreus, which is known to be very sensitive towards impurities. To select a suitable pretreatment method of a second generation feedstock, the influences of different hydrolysate components, like monosaccharides and sugar degradation products, were tested. Particular the impact of those components on itaconic acid yield, productivity, titer and morphology was investigated in detail. Wheat chaff was used as lignocellulosic biomass, which is an agricultural residue. An alkaline pretreatment method with sodium hydroxide at room temperature and a subsequent enzymatic saccharification at pH 4.8 at 50 °C with 10 FPU/g_Biomass Biogazyme 2x proved to be very suitable for a subsequent biotechnological production of itaconic acid. A purification by a cation exchanger of the wheat chaff hydrolysate resulted in a final titer of 27.7 g/L itaconic acid with a yield of 0.41 g/g_{total sugar}.     

Regulation of the production of polygalacturonase by Aspergillus terreus

The synthesis of polygalacturonase (PG) (EC 3.2.1.15) by a strain of Aspergillus terreus was induced by polygalacturonic acid and repressed by glucose, galactose or fructose even in the presence of the inducer. The production of PG increased when the mycelium was washed free of glucose and incubated in a glucose-free medium containing the inducer, a fact that indicated the reversibility of the repression mechanism. When Actinomycin D and cycloheximide were added to the culture medium, the synthesis of PG ceased. PG synthesis increased 43% with the addition of methionine and 64% both with leucine and with tyrosine. Specific productivity with leucine was 210% higher than that of the control as against 149% with methionine and 70% with tyrosine. The results obtained suggest that PG synthesis is regulated by leucine.     

Adding talc microparticles to Aspergillus terreus ATCC 20542 preculture decreases fungal pellet size and improves lovastatin production

Changing fungal morphology with the use of morphological engineering techniques leads to improving the production of metabolites by filamentous fungi in the submerged culture. Adding mineral microparticles is one such simple method to change fungal pellet size. Here, it was studied for a lovastatin producer, Aspergillus terreus ATCC 20542. The experiments were conducted in shake flasks and 10 μm talc microparticles were added to the preculture. Intrapellet oxygen concentration profiles were determined by an oxygen microprobe. Talc microparticles caused a decrease of A. terreus pellets diameter from about 2000 to 900 μm, dependent on their concentration in the preculture. Smaller pellets produced more lovastatin, whose titre exceeded then 120 mg L^?1, utilising more lactose. The decrease in pellet size resulted in changes of oxygen concentration profiles in the pellets. The estimated critical pellet diameter, at which the non‐oxygenated zone was observed in the centre of the pellets, was 1700 μm. Smaller pellets were fully penetrated by oxygen. To conclude, facilitated diffusion of oxygen into the pellets of smaller diameter and their less dense structure made lactose utilisation by A. terreus more efficient, which ultimately increased lovastatin production in the runs with talc microparticles added, compared to the control runs.     

Influence of the construction of porous spargers on lovastatin production by Aspergillus terreus ATCC 20,542 in a laboratory bubble column

Bioprocess and Biosystems Engineering - In bubble column bioreactors, the hydrodynamic behavior like mixing time, bubble size and morphology of filamentous fungi are influenced by the construction...     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Rational elimination of Aspergillus terreus sulochrin production

Elimination of undesirable co-metabolites from industrial fermentations is often required due to the toxicities associated with the contaminants and/or due to difficulties in removing the contaminants during downstream processing. Sulochrin is a co-metabolite produced during the Aspergillus terreus lovastatin fermentation. Examination of the sulochrin biosynthetic pathway identifies the emodin anthrone polyketide synthase (PKS) at the origin. Thus, genetically disrupting the emodin anthrone PKS gene was expected to result in the elimination of sulochrin biosynthesis. To perform the disruption by homologous recombination, a fragment of the emodin anthrone PKS gene first needed to be isolated. Analysis of several reported fungal PKS amino acid sequences has identified three subfamilies of related sequences (called the Patulin subfamily, the Pigment subfamily, and the Reduction subfamily). PCR primers specific for the Pigment subfamily (of which the emodin anthrone PKS is expected to belong) were used to isolate a fragment of a novel PKS gene from A. terreus. Targeted gene disruption identifies the novel gene fragment as that from the emodin anthrone PKS. Consequently, the gene disruption event eliminated the production of metabolites from the sulochrin biosynthetic pathway.     

Response surface methodology for optimization of medium components in submerged culture of Aspergillus flavus for enhanced heparinase production

Aims: Aim of the study was to develop a medium for optimal heparinase production with a strain of Aspergillus flavus (MTCC‐8654) by using a multidimensional statistical approach. Methods and Results: Statistical optimization of intracellular heparinase production by A. flavus, a new isolate, was investigated. Plackett–Burman design was used to evaluate the affect of medium constituents on heparinase yield. The experimental results showed that the production of heparinase was dependent upon heparin, the inducer; chitin, structurally similar to heparin and NH₄NO_3, the nitrogen source. A central composite design was applied to derive a statistical model for optimizing the composition of the fermentation medium for the production of heparinase enzyme. The optimum fermentation medium consisted of (g l^?1) Mannitol, 8·0; NH₄NO₃, 2·5; K₂HPO₄, 2·5; Na₂HPO₄, 2·5; MgSO₄.7H₂O, 0·5; Chitin, 17·1; Heparin, 0·6; trace salt solution (NaMoO₄.2H₂O, CoCl₂.6H₂O, CuSO₄.5H₂O, FeSO₄.7H₂O, CaCl₂), 10^?4 mol l^?1. Conclusions: A 2·37‐fold increase in heparinase production was achieved in economic and effective manner by the application of statistical designs in medium optimization. Significance and Impact of the Study: Heparinase production was doubled by statistical optimization in a cost‐effective manner. This heparinase can find application in pharmaceutical industry and for the generation of low‐molecular‐weight heparins, active as antithrombotic and antitumour agents.