Morphology engineering of basidiomycetes for improved laccase biosynthesis

Objective

This work is the first application of a morphological engineering technique called microparticle-enhanced cultivation (MPEC) aimed at the facilitation of laccase production in the submerged cultures by two basidiomycetes species Cerrena unicolor and Pleurotus sapidus.

Results

The positive effect of the applied 10 μm Al₂O₃ microparticles at concentrations from 5 to 30 g Al₂O₃ l^?1 was shown. Laccase activity increased 3.5-fold for C. unicolor and 2-fold for P. sapidus at 15 g Al₂O₃ l^?1 on 9 and 14 day of the cultivation, respectively, compared to the control culture without microparticles. The increase of laccase activity in the cultivation broths was caused by the action of Al₂O₃ microparticles on the agglomeration of hyphae. It led to the decrease of the size of the pellets, (on average by 2 mm for C. unicolor), the change of their shape (star-shaped pellets for C. unicolor) and the change of their structure (more compact pellets for P. sapidus).

Conclusions

Application of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.

     

Kinetic model to describe the morphological evolution of filamentous fungi during their early stages of growth in the standard submerged and microparticle‐enhanced cultivations

Biosynthesis of metabolites and enzymes by filamentous fungi depends on their morphological form in submerged cultures. However, their early stages of growth lasting approximately 24 h, from the introduction of spores to the medium until the formation of stable morphological forms, such as clumps or pellets, have rarely been the objects of experimental and modeling studies. Microparticle‐enhanced cultivation (MPEC) has been applied only to a few fungal species, mainly Aspergilli. Therefore, the objective of this work was to formulate the kinetic model to describe the early stages of the fungal evolution in the standard cultivation and MPEC for Aspergillus terreus, Chaetomium globosum, Penicillium rubens, and Mucor racemosus. These fungi exhibit various mechanisms of agglomerates formation in submerged cultures. The experiments were performed in batch shake flasks (parameters identification) and a stirred tank bioreactor (model verification). In the balance equation for fungal cells, the mean projected area of hyphal objects measured by the digital analysis of microscopic images was used as the dependent variable. The analysis of the experimental data and model solution revealed that the effect of the microparticles (aluminum oxide at 6 g L^?1) in MPEC toward the studied filamentous fungi was to the high extent species dependent. This effect was most evident in the case of spore coagulative A. terreus and noncoagulative M. racemosus.     

A current approach to the control of filamentous fungal growth in media: microparticle enhanced cultivation technique

Various strategies have been carried out to date in order to overcome the problem of the adverse effects of bulk fungal growth in bioreactors. Nevertheless, previous conventional methods such as modifying the cultivation temperature or pH resulted in limited biomass production and consequently lower yields. In recent years microparticle enhanced cultivation (MPEC) techniques are one of the most remarkable and novel methods employed for submerged fungal production to overcome bulk microbial growth. In addition to low cost advantages, MPEC also provides benefits such as not interfering with fungal metabolism, enhancing final product concentration and improving homogeneity in the fermentation broth. In this review, a comparison of conventional and novel methods to control fungal morphology has been discussed. Additionally, the application of microparticles in fungal fermentations, their benefits to the process in terms of fungal morphology, biomass accumulation, substrate consumption, and product formation also effect mechanisms of microparticle function are discussed in detail.     

Microparticle-enhanced cultivation of filamentous microorganisms: increased chloroperoxidase formation by Caldariomyces fumago as an example

Microparticle-enhanced cultivation (MPEC) was applied as a novel method for improved biomass and product formation during cultivation of filamentous microorganisms. Exemplarily, chloroperoxidase (CPO) formation by Caldariomyces fumago was analyzed in the presence and absence of microparticles of different size. Particles of approximately 500 microm in diameter had no effect on growth morphology or productivity of CPO formation by C. fumago. In contrast particles of < or =42 microm in diameter led to the dispersion of the C. fumago mycelia up to the level of single hyphae. Under these conditions the maximum specific productivity of CPO formation was enhanced about fivefold and an accumulated CPO activity in the culture supernatant of more than 1,000 U mL(-1) was achieved after 10-12 days of cultivation. In addition, the novel cultivation method also showed a positive effect on growth characteristics of other filamentous microorganisms proven by the stimulation of single hyphae/cell formation.     

Response and interaction of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and arbuscular mycorrhizal fungi in the soybean rhizosphere

Regulatory response and interaction of Bradyrhizobium and arbuscular mycorrhizal fungi (AMF) play a vital role in rhizospheric soil processes and productivity of soybean (Glycine max L.). Nitrogen (N) and phosphorus (P) are essential nutrients for plant growth and productivity, the synergistic interaction(s) of AMF and Bradyrhizobium along with rhizospheric beneficial microorganisms stimulate soybean growth and development through enhanced mineral nutrient acquisition (N and P) and improved rhizosphere environment. Such interactions are crucial, especially under low-input eco-friendly agricultural cropping systems, which rely on biological processes rather than agrochemicals to maintain soil quality, sustainability, and productivity. Furthermore, enhancement of N-fixation by root nodules along with AMF-mediated synergism improves plant P nutrition and uptake, and proliferation of phosphate-solubilizing fungi. However, the genetic and/or allelic diversity among native strains, their genes/enzymes and many environmental factors (e.g., soil organic matter, fertilizers, light, temperature, soil moisture, and biotic interactors) affect the interactions between AMF and Bradyrhizobium. New information is available regarding the genetic composition of elite soybean inoculant strains in maximizing symbiotic performance, N-fixing capabilities and depending on N and P status the host-mediated regulation of root architecture. Overall, for sustainable soybean production systems, a deeper understanding of the interaction effects of Bradyrhizobium and AMF co-inoculation are expected in the future, so that optimized combinations of microorganisms can be applied as effective soil inoculants for plant growth promotion and fitness. The objective of this review is to offer insights into the mechanistic interactions of AMF and Bradyrhizobium and rhizopheric soil health, and elucidate the role of environmental factors in regulating growth, development and sustainable soybean productivity.     

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.     

Polysaccharide-Degrading Activity in Marine and Terrestrial Strains of Mycelial Fungi

The activity of extracellular polysaccharide-degrading enzymes and glycosidases from mycelial fungi towards various carbohydrates and carbohydrate derivatives from plant and algal cell walls has been screened. Twenty-three strains of mycelial fungi isolated from the marine sediment and dung were grown by submerged cultivation on a plant-based substrate (a by-product of the grain processing industry) for previous screening for their biomass and protein productivity. Molecular identification allowed for the assignment of marine fungal strains to the following species: Sirastachys phyllophila, Ochroconis mirabilis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Beauveria felina, Scopulariopsis brevicaulis, Cladosporium sp., and Trichoderma sp. The terrestrial strains belonged to the species Thermomyces thermophilus, Thermomyces dupontii, Thermomyces lanuginosus, Fusarium avenaceum, Mycothermus thermophilum, and Thermothelomyces thermophila. Seven strains of thermophilic terrestrial fungal species T. thermophila, T. thermophilus, T. dupontii and M. thermophilus and two marine fungal strains of S. brevicaulis and Beauveria felina exhibited the highest protein yields and a wide range of polysaccharide-degrading activity when the cultures were cultivated at 22–25°C. The cellulolytic thermophilic strain M. thermophilus 55 isolated from dung demonstrated unusual specificity, most intensive increase of mycelial biomass, and high activity towards algal polysaccharides after seven days of cultivation. The specific activity of laminarinase was one order of magnitude higher than in the marine strains and amounted to 1180 U/mg, and the alginate lyase, carrageenase, polymannuronate lyase, agarase, and fucoidanase activity levels (from 208 to 500 U/mg) were also higher than in all marine strains. All active polysaccharide-degrading strains of thermophilic terrestrial and marine fungi identified in the present study are of considerable interest, as the potential of these fungi for polysaccharide degradation can be applied in the transformation of various agricultural and maricultural waste of plant origin and in the modification of carbohydrate-containing substances in structural research and biotechnology.     

Antifungal and phytostimulating characteristics of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Ch-13 rhizospheric strain,producer of bioprepations

Bacillus subtilis Ch-13 industrial strain was shown to have a wide spectrum of antagonistic activities against different species of phytopathogenic fungi and bacteria. The B. subtilis Ch-13 strain produces lytic enzymes; cyanide and other antifungal metabolites; stimulates plant growth, producing phytohormones—auxin derivatives. This strain by 2.5 times reduced the quantity of tomato plants infected with phytopathogenic fungus Fusarium oxysporum during inoculation. Fungi abundance on roots with bacterial inoculation was 6.9 times less than in the absence of inoculation. The application of detected antifungal metabolites as biochemical markers for the strain enables to control the stability of physiologic and biochemical characteristics of the producer, and ensures a rapid quality assay of biopreparations with high performance liquid chromatography (HPLC).     

Effect of various factors on the biosynthesis of piscarinines,secondary metabolites of the fungus <Emphasis Type="Italic">Penicillium piscarium</Emphasis> Westling

Piscarinines A and B were synthesized most actively during the surface cultivation of the fungus Penicillium piscarium in a complex medium (5.5 mg/l). Under conditions of submerged cultivation in a mineral medium, the yield of piscarinines was two times lower. An increase in the inoculum quantity of conidia treated with Tween-80 increased the culture productivity. The biosynthesis of the alkaloid was completely suppressed when mannitol was replaced with glucose or when zinc, iron, or copper ions were added to the culture medium. The metabolites were active against the prostate cancer cell line LNCAP (IC₅₀ were 2.195 and 1.914 μg/ml for piscarinines A and B, respectively).     

Microparticle based morphology engineering of filamentous microorganisms for industrial bio-production

Filamentous microorganisms are important work horses in industrial biotechnology and supply enzymes, antibiotics, pharmaceuticals, bulk and fine chemicals. Here we highlight recent findings on the use of microparticles in the cultivation of filamentous bacteria and fungi, with the aim of enabling a more precise control of their morphology towards better production performance. First examples reveal a broad application range of microparticle based processes, since multiple filamentous organisms are controllable in their growth characteristics and respond by enhanced product formation.     

Biotechnological advancement in genetic improvement of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

With the advent of molecular biotechnology, plant genetic engineering techniques have opened an avenue for the genetic improvement of important vegetable crops. Vegetable crop productivity and quality are seriously affected by various biotic and abiotic stresses which destabilize rural economies in many countries. Moreover, absence of proper post-harvest storage and processing facilities leads to qualitative and quantitative losses. In the past four decades, conventional breeding has significantly contributed to the improvement of vegetable yields, quality, post-harvest life, and resistance to biotic and abiotic stresses. However, there are many constraints in conventional breeding, which can only be overcome by advancements made in modern biology. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop, of the family Brassicaceae; however, various biotic and abiotic stresses cause enormous crop yield losses during the commercial cultivation of broccoli. Thus, genetic engineering can be used as a tool to add specific characteristics to existing cultivars. However, a pre-requisite for transferring genes into plants is the availability of efficient regeneration and transformation techniques. Recent advances in plant genetic engineering provide an opportunity to improve broccoli in many aspects. The goal of this review is to summarize genetic transformation studies on broccoli to draw the attention of researchers and scientists for its further genetic advancement.     

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O₂, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.     

Controlling filamentous fungi morphology with microparticles to enhanced β-mannanase production

β-mannanase was produced mainly by Aspergillus species and can degrade the β-1,4-mannose linkages of galactomannans. This study was undertaken to enhance mannanase production using talcum and aluminum oxide as the microparticles, which control cell morphology of recombinant Aspergillus sojae in glucose and carob extract medium. Both microparticles improved fungal growth in glucose and carob pod extract medium. Aluminum oxide (1 g/L) was the best agent for glucose medium which resulted in 514.0 U/ml. However, the highest mannanase activity was found as 568.7 U/ml with 5 g/L of talcum in carob extract medium. Increase in microparticle concentration resulted in decreasing the pellet size diameter. Furthermore, more than 10 g/L of talcum addition changed the filamentous fungi growth type from pellet to pellet/mycelium mixture. Results showed that right type and concentration of microparticle in fermentation media improved the mannanase activity and production rate by controlling the growth morphology.     

The genus <Emphasis Type="Italic">Diaporthe</Emphasis>: a rich source of diverse and bioactive metabolites

The genus Diaporthe (asexual state: Phomopsis) comprises pathogenic, endophytic and saprobic species with both temperate and tropical distributions. Although species of Diaporthe have in the past chiefly been distinguished based on host association, studies have confirmed several taxa to have wide host ranges, suggesting that they move freely between hosts, frequently co-colonizing diseased or dead tissue, while some species are known to be host-specific. They are also very frequently isolated as endophytes of seed plants. Due to their importance as plant pathogens, the genus has been thoroughly investigated for secondary metabolites, including during screening programs aimed at the discovery of novel bioactive natural products, but the respective information has never been compiled. Therefore, we have examined the relevant literature to explore and highlight the major classes of metabolites of Diaporthe and their Phomopsis conidial states. These fungi predominantly produce a large number of polyketides, but cytochalasins and other types of commonly encountered fungal secondary metabolites are also predominant in some species. Interestingly, not a single metabolite which is also known from the host plant has ever been isolated as a major component from an endophytic Diaporthe strain, despite the fact that many of the recent studies were targeting endophytic fungi of medicinal plants.     

Slowed Biogeochemical Cycling in Sub-arctic Birch Forest Linked to Reduced Mycorrhizal Growth and Community Change after a Defoliation Event

Sub-arctic birch forests (Betula pubescens Ehrh. ssp. czerepanovii) periodically suffer large-scale defoliation events caused by the caterpillars of the geometrid moths Epirrita autumnata and Operophtera brumata. Despite their obvious influence on ecosystem primary productivity, little is known about how the associated reduction in belowground C allocation affects soil processes. We quantified the soil response following a natural defoliation event in sub-arctic Sweden by measuring soil respiration, nitrogen availability and ectomycorrhizal fungi (EMF) hyphal production and root tip community composition. There was a reduction in soil respiration and an accumulation of soil inorganic N in defoliated plots, symptomatic of a slowdown of soil processes. This coincided with a reduction of EMF hyphal production and a shift in the EMF community to lower autotrophic C-demanding lineages (for example, /russula-lactarius). We show that microbial and nutrient cycling processes shift to a slower, less C-demanding state in response to canopy defoliation. We speculate that, amongst other factors, a reduction in the potential of EMF biomass to immobilise excess mineral nitrogen resulted in its build-up in the soil. These defoliation events are becoming more geographically widespread with climate warming, and could result in a fundamental shift in sub-arctic ecosystem processes and properties. EMF fungi may be important in mediating the response of soil cycles to defoliation and their role merits further investigation.     

Variations of leaf morphology,photosynthetic traits and water-use efficiency in Western-Mediterranean tomato landraces

Modern tomato (Solanum lycopersicum L.) breeding has mainly focused on increasing productivity under unlimited watering. In contrast, some Mediterranean accessions have been traditionally cultivated under water shortage and selected on the basis of their water-use efficiency (WUE). Ramellet and Penjar landraces were planted with other traditional, old and modern inbreeds, under full irrigation. In order to found differences between the tomato accessions, gas-exchange and leaf morphology measurements were performed. Despite high variability, Ramellet and Penjar presented clear differences compared to modern cultivars, mostly related to leaf morphology and photosynthetic traits, while no differences were found in WUE. Results highlighted that better leaf CO₂ conductance might be a main factor determining the improvement of net CO₂ assimilation and WUE.     

Microtiter plate‐based cultivation to investigate the growth of filamentous fungi

Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL‐scale. In this paper, the application of a microtiter plate‐based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose‐like aroma compound 2‐phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms.     

Enhancement of Pyruvate Productivity in <Emphasis Type="Italic">Candida glabrata</Emphasis> by Deleting the <Emphasis Type="Italic">CgADE13</Emphasis> Gene to Improve Acid Tolerance

Acid tolerance is one of the critical factors to evaluate the quality of the industrial production strains, especially organic acid producing microorganisms. To circumvent this problem, we investigated the physiological function of adenylosuccinate lyase in AMP metabolism from Candida glabrata by deleting the corresponding gene, CgADE13. At pH 4.0, CgADE13 deletion resulted in a 68.3% and 112.0% increase in biomass and cell viability compared to those of wild type strain (wt), respectively. In addition, CgADE13 deletion also protected cell morphology and counteracted ROS production. Further, the intracellular ATP level of strain Cgade13Δ was decreased by 25.0%, and its H+-ATPase activity was increased by 15.0%. Finally, pyruvate production with strain Cgade13Δ in a 30-L batch bioreactor at pH 4.0 reached 53.9 g/L, and pyruvate productivity was increased by 166.7% compared to that of wt. This is the first report regarding tolerance engineering of C. glabrata for enhancing pyruvate productivity, which provides a good starting point for metabolic engineering to achieve the industrial production of other chemicals.