Fungal Protein Production Research Articles

The F-box protein gene exo-1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Fungal Protein Production: Design and Production of Chimeric Proteins

For more than a century, filamentous fungi have been used for the production of a wide variety of endogenous enzymes of industrial interest. More recently, with the use of genetic engineering tools developed for these organisms, this use has expanded for the production of nonnative heterologous proteins. In this review, an overview is given of examples describing the production of a special class of these proteins, namely chimeric proteins. The production of two types of chimeric proteins have been explored: (a) proteins grafted for a specific substrate-binding domain and (b) fusion proteins containing two separate enzymatic activities. Various application areas for the use of these chimeric proteins are described.

Green processing of tropical banagrass into biofuel and biobased products: An innovative biorefinery approach

Banagrass (Pennisetum purpureum) approximately 4 months old was hand-harvested and shredded. Half of the sample was dewatered using a screw-press, whereby the extracted juice was used for cultivating an edible fungus, Rhizopus microsporus, for aquaculture feed supplementation. The remaining biomass was divided into four separate streams: (1) wet, juiced; (2) dry, juiced; (3) wet, unjuiced; and (4) dry, unjuiced. Each stream was pretreated with dilute sulfuric acid and compared on the basis of sugar release at varying acid concentrations, temperatures, and residence times. Wet, juiced banagrass released the most soluble sugars (theoretical xylose and ∼85% glucose). Ultrasonication (20 kHz) was applied to further increase monomeric sugar release but demonstrated little improvement on total sugar yields. Fungal biomass generated from banagrass juice exhibited potential as a fungal-protein production medium producing 1.16±0.34 g biomass increase/g initial biomass.

Investigation of the use of ram horn hydrolysate for fungal protein production

Production of the fungus Aspergillus niger NRRL 330 was studied in submerged fermentation with ram horn hydrolysate (RHH) as substrate. The characteristics of RHH have been reported previously. The RHH was enriched by addition of glucose and KH2PO4. The effects of kinetic parameters on the biomass yield of the fungus were investigated. The optimal conditions for growth of A niger on RHH were initial pH 6.5, temperature 30 °C, fermentation time 96 h and agitation speed 150 rpm. Under these optimal conditions the initial carbohydrate content of RHH was reduced from 1.52 to 0.2% and the biomass yield was 8.9 g l−1. The biomass contained about 48.1% protein, 5.2% fat and 9.4% ash (on a dry weight basis). The amino acid content of the biomass was compared with Food and Agricultural Organisation (FAO) and animal feed standards. The protein produced contained all the essential amino acids for animal feed, but the amounts of these amino acids were somewhat lower than those of FAO and soybean reference protein. However, the amino acid composition of the biomass was better than that of animal feed. The results with RHH were also compared with previously reported data on fungal mycelium grown on waste liquor substrate. In conclusion, it was found that RHH could be used as a substrate in the production of fungal protein for use as animal feed. Copyright © 2003 Society of Chemical Industry

Mycelial morphology and fungal protein production from starch processing wastewater in submerged cultures of Aspergillus oryzae

Three strains of Aspergillus oryzae were used for fungal protein production from starch processing wastewater (SPW) as part of a comprehensive SPW utilization and treatment program. The mycelial morphologies of the three strains varied from fluffy, clumpy mycelia to compact pellets with various strains and cultivation conditions of growth pH, inoculum quantity and superficial air velocity in an air lift bioreactor process. An inoculum technique was developed to ensure a desirable morphology with an improved fungal protein yield. Experimental relationship between the morphology and the yield revealed that the formation of small compact pellets under designed cultivation conditions favoured higher fungal protein yields, easier product separation and better process operation.

Production of fungal protein and glucoamylase by Rhizopus oligosporus from starch processing wastewater

A simple, non-aseptic, low-cost single process had been developed for the treatment of starch processing wastewater (SPW) with the production of fungal protein and glucoamylase enzyme. The selected fungus Rhizopus oligosporus DAR 2710 has the ability to covert more than 95% starch materials in SPW to produce 4.5–5.2 g of dry fungal biomass from a litre of SPW in 14 h cultivation at 35°C and initial pH 4.0. The fungal biomass contained 46% protein and was safe for human and animal consumption. The process using an air lift bioreactor was successfully carried out in a batch system without sterilization and/or preliminary hydrolysis of SPW. In addition to the production of fungal protein and glucoamylase, the removal of 95% COD and total suspended solids would lead to a potential benefit to the environment.

Investigation of the use of agricultural byproducts for fungal protein production

Cellulose and nutrient salts as well as potato pulp and potato protein liquor (PPL), were used as substrates for the cultivation of Chaetomium cellulolyticum in batch and repeated-batch operations. Using cellulose as the substrate a linear relationship existed between the rates of cell mass formation and acid production. The repeated-batch process was controlled by NaOH consumption using a simple computer model. When the production of cell mass and acid stopped because of a lack of substrate cellulose was fed into the reactor. This occurred within 10 min at which point no NaOH-feed was needed to maintain a constant pH. Repeated-batch operations yielded higher cell concentrations and productivities than batch operations. The relationship between the NaOH and H 2SO 4 consumed, and the fungal mass concentration was complex in cultivation media containing potato pulp and PPL, because various substrates were consumed by the fungus simultaneously and successively. Therefore, for repeated-batch cultivation a constant time interval was used. Repeated-batch cultivation of the fungus on potato pulp and PPL did not yield higher cell concentrations and productivities than did batch cultivation. With the optimal pulp-to-PPL ratio a maximum specific growth rate of 0.61 h 1 was obtained. These investigations indicate, that potato pulp and PPL are well suited to fungal protein production by Chaetomium cellulolyticum for fodder supplement.

Studies on the effect of particle size on solid-state fermentation of sugarcane bagasse into animal feed using white-rot fungi

Sugarcane bagasse, a complex substrate, when differentiated into four fractions of particle sizes (<1 mm, 1–3 mm, 3–5 mm, 5–10 mm) with a view to enhancing its nutritive value as animal feed rather than production of fungal protein for human consumption, showed a varying pattern of degradation by white-rot fungi and varying in vitro rumen digestions. The fractions of bagasse were assessed for their bioconversion efficiencies in terms of disappearance of lignin and change in digestibility using Pleurotus sp. P7, Agrocybe aegarita A1, Pleurotus eryngii, Pleurotus sp. P1 and Kuehneromyces mutabilis. The P. eryngii improved the digestibility of all the bagasse fractions. The mechanical separation of a substrate can be of significance when it is utilized as a material to be fermented by white-rot fungi to produce enriched animal feed.

Production of fungal protein by Aspergillus awamori var. kawachi grown in Shochu distillery wastewater

The possibility of producing single cell protein (SCP) from shochu distillery wastewater by Aspergillus awamori var. kawachi has been examined. A yield of about 40 g/ l of biomass, about 40% of which was protein, was obtained from a 24-h jar-fermentor culture. The yield of biomass from shochu distillery wastewater, calculated on the basis of organic matter content, was about 40%. Digestibility, as determined by digestion with pepsin, was only about 52% without pretreatment, but this value increased to 62% after heating the biomass at 100°C for 20 min. Cultivation of the fungus was also suitable as a treatment for shochu distillery wastewater since solid matter was degraded and the separation of liquids and solids was facilitated.

Effect of Different Nitrogen Sources and Cultural Conditions on Fermentation of Bagasse byAspergillus terreus

Abstract Effect of different nitrogen supplements and cultural conditions viz., pH, temperature and shaking on fungal protein production by Aspergillus terreus was investigated using alkali- treated bagasse as substrate. The efficiency in terms of bagasse degradation and protein production was maximum at pH 4.0 and temperature 30C with 5% (V/V) inoculum in submerged culture fermentation. Cornsteep liquor was found to be the best nitrogen supplement.

Xylanase ofChaetomium cellulolyticum: Its nature of production and hydrolytic potential

Maximum xylanase production byChaetomiumcellulolyticum was obtained in the culture supernatant after 30 h of growth at 37°C in basal medium containing 1% xylan at pH maintained between 6.5 and 7.5. Addition of 0.05% Tween 80 to the medium increased the enzyme production considerably. Xylanase production was found to be growth associated. The optimal conditions for enzymatic hydrolysis of xylan were found to be pH 6.0 and 50°C. During enzymatic hydrolysis, xylose, xylobiose and other xylooligosaccharides were liberated from xylan. The pH values for xylanase production and for xylan hydrolysis were closely related to the utilization of hemicelluloses of aspen wood for fungal protein production by this organism as reported in our earlier work.

Bioconversion of hemicelluloses into fungal protein

The utilization of cellulose from one ton of lignocellulose for ethanol production would yield 150–250 kg of hemicelluloses. The total soluble solids in the hemicellulose fraction (HF) obtained with the Universite de Sherbrooke (UdeS) process contained about 56% carbohydrates. These carbohydrates were present in the form of oligomers of various sugars, predominantly xylose. All the test fungi,Chaetomium cellulolyticum, C. cellulolyticum (asporogenous mutant) andPleurotus sajor-caju, were capable of utilizing all the carbohydrates present in HF.C. cellulolyticum gave the highest amount of protein (7 g/l) from 19 g carbohydrates/l. The yield of protein was higher than expected, indicating that carbon compounds other than reducing sugars present in HF might have been consumed for fungal growth. The inhibitory effect of toxic compounds on protein production increased with an increase in concentration of soluble solids in HF. The inhibitory effect was overcome by increasing the pH of the medium to 6.0 or 7.0. Fungal protein production from hemicelluloses will give extra revenue in our integrated approach for ethanol production from lignocelluloses.

Production of fungal protein from rasped fresh cassava roots using 200- and 3000-litre fermentors

The results are given of experiments on the nonaseptic, high-temperature production of a fungal biomass from cassava ( Manihot esculenta Crantz). The experiments were conducted at the pilot plant level at CIAT, using the fungus Aspergillus fumigatus I-21A as a second stage in the evaluation of the technology developed and tested previously on a laboratory scale at the University of Guelph. A brief description of the process and a schematic representation of the different steps required at this level are included. The substrate employed was grated fresh cassava roots from several low-cyanide cultivars with a total quantity utilized of approximately 425 kg of cassava per fermentation. The initial concentration of carbohydrates in the culture medium was adjusted to 40 g l −1. The fungus used is characterized by its ability to grow at very low pH (3.5) and reasonably high temperature (45°C), selective growth conditions which appear to obviate the need for sterilization and maintenance of aseptic conditions. The protein yields reported were not as high as obtained in previous small-scale fermentations, with the total crude protein content of the final product averaging < 35% of dry matter. This was partially due to an incomplete utilization of carbohydrates. The quality of the tap water as related to pH of the culture medium might also have had some adverse effect on the growth of the micro-organism. While the protein of the final product was low in sulphur-containing amino acids, particularly methionine, its amino acid profile compared favorably with that of other protein sources. The results obtained to date suggest that single-cell protein (SCP) production from cassava may have potential for practical application in cassava producing areas provided safe operational procedures can be established.

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h−1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l−1 for lactose and 0.14 mg l−1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g−1 biomass per lactose and the maintenance coefficient 0.005 g g−1 h−1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l−1 h−1 biomass at dilution rates of 0.16–0.17 h−1 with a lactose concentration of 20 g l−1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h−1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.

Improvement of Production of Fungal Protein by Solid Substrate Fermentation

An improved solid substrate fermentation process was studied for fungal protein production, using unutilized cellulose and protein as carbon and nitrogen source from water-hyacinth with Trichoderma viride 1662 as organism. A preliminary study with different concentrations of hydrochloric acid, water to substrate ratios, and swelling of straw in hydrochloric acid was carried out to increase the protein value of the fungus. It was observed that a HCl concentration (15% v/w) in substrate: water (ratio 1: 2) after 24 hours immersion, when autoclaved at 121°C for an hour, increased mycelium and protein content. This process was compared with other methods, developed for solid fermentation and almost similar or little increase in yields of soluble protein was observed with this simple and economical method. Es wurde ein verbesserter Fermentierungsprozeß unter Verwendung eines festen Substrates zur Erzeugung von pilzlichem Eiweiß getestet. Als Kohlenstoff- und Stickstoffquelle dienten die Zellulose- und Eiweißbestandteile der bisher wenig verwerteten Wasserhyazinthe ( Eickhofnia crassipes ). Testorganismus war Trichoderma viride . Untersucht wurden unterschiedliche Salzsäurekonzentrationen, Wasser: Substrat-Verhältnisse und Vorquellung des Strohs mit Salzsäure. Es wurde beobachtet, daß 24stündiges Vorquellen, eine HCl-Konzentration von 15% und ein Substrat: Wasser-Verhältnis von 1: 2 den Myzel- und Eiweißgehalt erhöhten. Verglichen mit anderen Methoden zeigte diese einfache und ökonomische Methode gleiche oder bessere Ergebnisse.

Effect of growth rate on the amino acid composition of Fusarium moniliforme cultivated continuously for fungal protein production

AbstractFusarium moniliforme was shown to be a promising fungus for microbial protein production. 1−3The fungus grows well on the aqueous extracts of carob pods, a low value agricultural product well known in the Mediterranean areas. In a previous paper2quantitative data were presented on the production of fungal protein by growing F. moniliforme on a carob aqueous extract in a continuous culture. In the present paper the amino acid profile of the biomass was determined and the resulting essential amino acid index was calculated.

Technical note: Utilization of citrus wastes for the production of fungal protein

Technical note: Utilization of citrus wastes for the production of fungal protein

Production of fungal protein from oat hulls

Production of fungal protein from oat hulls

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

AbstractFusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr−1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr−1 were obtained at μ = 0.205 hr−1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr−1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr−1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr−1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr−1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr−1 in order to maximize protein production.

Barley grain as carbon substrate for fungal protein production in a farm process

AbstractA simple, non‐aseptic, submerged aerobic fermentation was sought in which microbial protein could be synthesised from non‐protein nitrogen, at the expense of barley carbohydrate, to supplement the protein content of barley grain for non‐ruminant feeding. Screening of 23 isolates of fungi in shake flask cultures showed that microbial protein could be successfully produced by a number of different species in a medium containing barley as a source of carbohydrate. Protein synthesis was markedly affected by the addition of different sources of non‐protein N to the fermentation; and (NH4)2SO4 or urea proved to be good N sources in this respect. Aspergillus oryzae was selected as the most suitable species for protein synthesis, based on its performance when supplemented with the various N sources. The growth rate of A. oryzae in stirred culture was such that high yields of mycelial protein could be achieved within 24 h from a culture inoculated with a spore suspension. Under the conditions of growth used in these experiments there appeared to be little advantage in adding more than 2% barley to the medium. Higher yields of protein could be achieved at barley concentrations in excess of 2%, but only if the incubation period was extended. The tolerance of A. oryzae to low pH values was exploited to enable fungal cultures to be grown under non‐aseptic conditions. Good yields of protein were obtained in 40 litre cultures grown at pH 3.5 in a modified domestic washing machine. Although protein yields were poor, non‐aseptic growth was also sucesssful at pH 3.5 in 1000 litre cultures grown in an agricultural feed‐mixing vessel.