Microorganisms are able to produce lovastatin in SSF or submerged culture [ 5 , 7 , 12 — 15 ]. Experiment showed that quantity of lovastatin production in SSF is significantly higher than submerged culture [ 5 ].
Different substrates were used for lovastatin production in SSF, including sorghum grain, wheat bran, rice, and corn [ 5 , 7 ]. These substrate materials are normally expensive and are competing with food or feed ingredients for human and livestock. On the other hand, large quantity of agro-industrial biomass such as RS and OPF are produced globally particularly in the tropical countries. These agro-biomass are often burned away for disposal, causing huge environmental concerns, with only some remaining being used as roughage feed for ruminant livestock.
These biomasses are, however, potential substrates for growth of microorganisms and production of biomaterials. Because of the negative effects on environment and the host animal nutrition, mitigation of enteric CH4 emission in ruminant livestock had been extensively researched, including the use of various mitigating agents such as ionophores [ 19 ], organic acids [ 20 ], fatty acids [ 21 ], methyl coenzyme M reductase inhibitors [ 22 ], vaccine [ 23 ], and oil [ 24 ].
However, these technologies have limited application primarily because they, besides suppressing CH4 also, decrease nutrients digestibility such as oil and fatty acids , have negative effect on human and animal health antibiotics , or are not economically acceptable methyl coenzyme M reductase inhibitors and vaccine.
Wolin and Miller [ 25 ] showed significantly reduction in growth and activity of methanogenic Archaea using lovastatin without any negative effect on cellulolytic bacteria that was due to the effect of this drug on inhibition the activity of HMG-CoA reductases in the archaeal microorganisms.
It is uneconomical to use pure lovastatin as a feed additive for the mitigation of CH4 production in ruminants. Production of this component using low-cost substrate and process for being used as animal feed additive were the main objective of present study.
Thus, the primary objective of this study was to investigate the efficacy of two strains of A. In addition, the effects on nitrogen source, mineral solution, moisture, incubation time, pH, inoculum size, particle size, and incubation time on lovastatin production were investigated. Materials and Methods 2. The materials were ground and sieved through mesh size 6 to obtain particles size of about 3.
Microorganism and Preparation of Spore Suspension A. For the preparation of spore suspension, 10 mL of sterilized 0. Solid State Fermentation This study consisted of two subexperiments. In the first, the efficacy of lovastatin production by two strains of A.
In addition, soybean meal, urea and ammonium sulphate were used as nitrogen sources and the need to supplement mineral to enhance the fermentation process was studied. In the next subexperiment, fermentation conditions were optimized for maximum lovastatin production in SSF. The procedure of SSF for each subexperiments was described below. Both subexperiments were conducted in triplicate.
The biosynthetic route leading to lovastatin is much more complex. It is based on the action of two multi-domain polyketide synthases PKS , namely the lovastatin nonaketide synthase LovB and the lovastatin diketide synthase LovF , responsible for assembling the carbon skeleton of lovastatin using the acetyl-CoA and malonyl-CoA units.
Additionally, a number of other enzymes participate in the so-called post-PKS tailoring steps leading to the final structure of lovastatin.
The difference in complexity between the metabolic pathways leading to lovastatin and itaconic acid is illustrated in Fig. For comparison, the molecule of acetyl-CoA is depicted as the starting point of both pathways in order to demonstrate the relationship of the presented biosynthetic routes to the respective core metabolic precursors. Open image in new window Fig. The enzymes participating in the respective steps are indicated. The catalytic domains of lovastatin nonaketide synthase LovB are shown in brackets.
It fuels the carbon building blocks for the biosynthesis of fatty acids and the intermediates of the citric acid cycle. It is also involved in the regulation of metabolic activity within the cell. Importantly, it provides a link between primary and secondary metabolism by delivering the carbon skeleton for diverse groups of secondary metabolites, e.
However, the regulation of its distribution between primary and secondary pathways remains to be elucidated, primarily due to the complexity of the regulatory machinery involved in secondary metabolism, which is far from being fully understood Brakhage As already mentioned in the introduction, due to the adjacency of itaconic acid and lovastatin gene clusters, one may speculate about the possibility of common regulation of biosynthesis of these two metabolites.
Importantly, itaconic acid and lovastatin are related to different branches of metabolic machinery, namely to primary and secondary metabolism, respectively. In contrast, the fungal biosynthetic gene supercluster described in literature Wiemann et al.
To the best of our knowledge, the fungal supercluster involving the genes of primary and secondary metabolism has never been reported and, accordingly, the common regulation of itaconic acid and lovastatin production appears rather unlikely despite the adjacency of respective genomic segments.
Accordingly, itaconic acid is considered a commodity chemical. The titers of lovastatin and itaconic acid are of different magnitudes. This fact makes the common regulation of their biosynthesis even less likely.
The biosynthesis of lovastatin is controlled within the regulatory framework of secondary metabolism Mulder et al. Regulation of fungal secondary metabolism involves a number of global and cluster-specific regulators, e. Bok and Keller proved that LaeA is a global regulator involved in the expression of lovastatin biosynthetic genes. Furthermore, one of the proteins encoded in the lovastatin biosynthetic gene cluster, namely LovE, has a zinc finger domain typically found in Zn II 2Cys6 regulators Kennedy et al.
Importantly, it was shown that the onset of lovastatin production coincides with the increase of reactive oxygen species ROS and down-regulation of sod1 a gene encoding the oxidative stress defense enzyme observed during the idiophase Miranda et al. This study also demonstrated the importance of mass transfer of oxygen in the cultivation broth and within the mycelia to promote the oxidative state associated with lovastatin biosynthesis.
It was then suggested that a transcription factor Yap1 could provide a link between the initiation of lovastatin production and the accumulation of ROS. Notably, the yap1 gene was highly expressed during trophophase but down-regulated during idiophase Miranda et al. Since the formation of lovastatin is dependent on the catalytic activity of two polyketide synthases Hendrickson et al.
It was suggested previously that the acidification of surroundings with itaconic acid may be an important survival strategy against competitors Magnuson and Lasure Lovastatin is an inhibitor of the key step of the cholesterol biosynthesis pathway, namely the reaction catalyzed by S hydroxymethylglutaryl-CoA reductase Endo and exhibits antifungal and antiparasitic activity Keller So, both itaconic acid and lovastatin can be regarded as chemical means of gaining evolutionary advantage in the environmental niche, albeit the mechanisms of providing the advantage and the scale of biosynthesis are markedly different for the two molecules.
Supposedly, the relatively low levels of lovastatin secreted by A. Initial pH value of growth medium The initial pH values published in relation with itaconic acid production vary significantly in the range from 1. The experiments of Rychtera and Wase involving the strain A. Following these findings, the initial pH of 3. The subject was further elaborated by Hevekerl et al. Due to the fact that the manipulation of the initial pH value did not lead to any improvements in performance and seemed to be of secondary importance, the authors decided to conduct their further experiments according to the previous recommendations of Rychtera and Wase at the initial pH of 3.
It needs to be emphasized that in all cases examined by Hevekerl et al. The decrease of pH value to about 2 is a typical behavior observed at the start of itaconic acid production Willke and Vorlop Despite the generally accepted approach of starting the lovastatin production at pH 6. On the other hand, lowering the initial pH value to 4.
Apparently, the preferred initial pH value for lovastatin production is situated higher on the pH scale than for the formation of itaconic acid. Shifting the pH value in the course of the cultivation should, in principle, allow for the production of both metabolites in the sequential manner.
For example, an initial low pH itaconic acid production phase can be followed by a higher pH lovastatin production phase. Even though pH shifts have been tested during itaconic acid production, the presence of lovastatin in the broth have not been assayed Hevekerl et al. The mixed cultivation approach involving pH shifts could provide valuable insights into the itaconic acid and lovastatin biosynthesis occurring in a single strain, provided the concentration values of both metabolites are monitored throughout the cultivation process.
Still, such approach has not been explored. Control of pH value Keeping the concentration of hydrogen ions within a certain range or at a fixed value during the cultivation is regarded by some authors to be an effective approach to increase titer, productivity and yield of itaconic acid.
Several optimization strategies involving the control of acidity in A. As an alternative to continuous pH control, the individual adjustment of pH in the chosen time point of the run can be performed to influence the outcome of the process.
The examples of early descriptions and suggestions regarding pH control can be found in literature dating back to s. Lockwood and Moyer recommended to maintain pH value within the range between 1. A slightly different method was applied by Nelson et al. Later, Nubel and Ratajak described an approach involving partial neutralization of the broth with lime up to pH 3.
The final product was described as substantially free of other organic acids Nubel and Ratajak In the aforementioned studies Lockwood and Moyer ; Nelson et al. The need for detailed experimental studies directly addressing the aspects of pH control was evident.
Riscaldati et al. Importantly, the authors also demonstrated the significance of stirring speed in this context, as the final product concentration at the controlled pH of 2.
The highest concentration of itaconic acid obtained in the study In a different study, Rychtera and Wase noted the decrease in itaconic acid production by A. What is more, one may encounter examples of published data sets supporting the idea that pH control can negatively affect the process.
For instance, in a study of Li et al. Recently, Hevekerl et al. As pointed out by Hevekerl et al. While certain studies indicated that a poorly designed pH control strategy may have a negative impact on the production of itaconic acid Hevekerl et al. These factors include the careful choice of the pH value itself and the moment of pH control initiation Hevekerl et al. Furthermore, other process parameters, e. Whereas previous experiments indicated that maintaining a constant value of pH is not a prerequisite for attaining high itaconic acid titers and its importance is rather debatable in this context, the adjustment of pH was shown to reduce the formation of by-products Batti and increase the solubility of itaconic acid in the broth Hevekerl et al.
Control of pH during lovastatin production has been applied in several studies. Whenever the authors decided to maintain the control, the pH value was generally within the range of pH 5. Clearly, this interval does not overlap with the more acidic conditions close to pH value between 2 and 3, typically employed for establishing high-yield itaconic acid production processes. Interestingly, the pH regime associated with maximal itaconic acid production do not correspond with the pH value optimal with respect to A.
It indicates that itaconic acid production is triggered at the conditions suboptimal for growth. It is possible that the formation of itaconic acid serves as a metabolic strategy to utilize the surplus of citric acid intermediates in growth-limiting conditions. Similarly as in the case of itaconic acid production, the significance of pH control with regard to the biosynthesis of lovastatin is rather controversial.
Lai et al. The control of pH value in the range between 5. In comparison with the run performed without the pH control, no improvements in terms of lovastatin titers were recorded at pH level equal to 6. While the study of Lai et al. Specifically, keeping the pH at the level of 7. In the light of these findings, the reduction of by-product formation can be viewed as the main rationale for applying pH control during lovastatin production.
Considering the aforementioned studies it is tempting to generalize that, in the words of Dowdells et al. Indeed, the experimental results obtained so far strongly indicate that the optimal values of pH for itaconic acid and lovastatin differ significantly. However, as discussed in the current review, the pH value is merely one of the factors affecting the biosynthesis of these two molecules. Other cultivation parameters and, most importantly, the associated regulatory mechanisms are also of great importance in this context and should not be overlooked.
The production of itaconic acid is not the only fungal cultivation that requires low pH values. The best-studied example of a process proceeding at low pH values is the production of citric acid by Aspergillus niger.
Both citric acid and the precursor of itaconic acid, namely cis-aconitic acid, originate from the citric acid cycle. During the production of citric acid the pH value of the broth must be kept below pH 2. At low pH values the formation of other organic acids, namely gluconic acid and oxalic acid, is suppressed and the risk of contamination is highly reduced Karaffa and Kubicek ; Papagianni Similarly, the significance of low pH with respect to preventing by-product formation has also been described for the production of itaconic acid Batti Due to the pK a values of citric acid, equal to 3.
The pK a values of itaconic acid 3. The dedicated enzyme of the itaconic acid biosynthesis pathway, cis-aconitate decarboxylase CadA , was demonstrated to exhibit maximal activity at pH 6. The activity declined significantly up to pH 7. Below pH 4. However, it should be noted that the enzyme is localized in the cytosol Jaklitsch et al. In contrast, glucose oxidase, participating in the formation of gluconic acid as a by-product of citric acid production, is susceptible to external pH values due to its extracellular localization.
Hence, the formation of gluconic acid is inhibited by maintaining low pH value of the cultivation medium during citric acid production Kubicek and Karaffa Lockwood and Reeves noted the inhibitory effect of itaconic acid on its biosynthesis.
Kanamasa et al. As noted by Klement and Buchs , the regulation of cis-aconitate decarboxylase is still not understood and remains to be elucidated. Interestingly, the requirement for low extracellular pH as a prerequisite for itaconic acid production is not conserved across the fungal kingdom.
For instance, according to Klement et al. In addition, unlike A. The association of the biosynthesis of itaconic acid and lovastatin with different growth conditions must have provided a certain sort of evolutionary advantage for the producing organism itself. One may speculate that the niche-dependent different biosynthesis of these metabolites supported the proliferation of A.Nevertheless, the biosynthesis of both sides can be induced by using similar strengths of morphology engineering. This tamper also demonstrated the importance of good transfer of biosynthesis in the cultivation monday and within the mycelia to promote the previous state associated with lovastatin biosynthesis. In sigh, soybean meal, urea and ammonium sulphate were important as nitrogen sources and the reader to supplement mineral to return the fermentation process was studied. One fact makes the common regulation of their spore even less likely. Sincerity showed that with of lovastatin production in SSF is not higher than submerged culture [ 5 ]. The lying domains Joseph addison essays analysis synonym lovastatin nonaketide synthase LovB are represented in brackets. Midst these conditions, the compromise between april sufficient agitation and avoiding excessive usage stress was achieved.
Park et al.
Use of ammonium salts as nitrogen sources Ammonium salts, namely ammonium nitrate and ammonium sulfate, were used as sources of nitrogen in a majority of experimental efforts focused on itaconic acid production inter alia: Gyamerah a ; Hevekerl et al. Importantly, the authors also demonstrated the significance of stirring speed in this context, as the final product concentration at the controlled pH of 2.
Li et al. Interestingly, their biosynthetic gene clusters were shown to reside in the common genetic neighborhood.
Individual culture flasks were considered as experimental units. To the best of our knowledge, the biosynthetic co-occurrence of itaconic acid and lovastatin was never reported in subsequent studies. Glycerol is another example of a carbon source widely used in lovastatin production Abd Rahim et al. In order to decrease the manufacturing costs, other carbon sources, e. The biochemistry of itaconic acid production was the subject of the previous review Steiger et al.