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Lovastatin is an anti-cholesterol medicine that is commonly prescribed to manage cholesterol levels, and minimise the risk of suffering from heart-related diseases. Aspergillus terreus (ATCC 20542) supplied with carbohydrates or sugar alcohols can produce lovastatin. The present work explored the application of metabolic engineering in A. terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor flux, and eliminate ( +)-geodin biosynthesis (a competing secondary metabolite) by removing the gene for emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. The gedCΔ*accox strain, when cultivated with a mixture of glycerol and lactose, significantly (p

In a commercial oyster mushroom farm, from 300 g of the total harvest, only the cap and stem of the fruiting body parts are harvested (200 g) while the unused lower section called fruiting-body-base (FBB) is discarded (50 g). A new antioxidative FBB flour (FBBF) conversion to mixed-ratio chicken patty was recently developed which converts 16.67% of FBB into an edible flour. At the initial stage, pretreatments of FBBF were optimized at particle size (106 µm) and citric acid concentration (0.5 g/100 mL) to improve flour antioxidant responses. Such pretreatments boosted total phenolic content (2.31 ± 0.53 mg GAE/g) and DPPH (51.53 ± 1.51%) of pretreated FBBF. Mixed-ratio chicken patty containing FBBF (10%, 20%, 30%) significantly (P < 0.05) influenced the hardness, cohesiveness, springiness, and chewiness of the patties. However, only the hardness and chewiness increased proportionally with the increase FBBF in concentration. Notably, 60 panellists considered that 10% FBBF-chicken patty sensory attributes, including lightness, redness, and yellowness, is acceptable to consumers. This information could be used to market any type of commercial mushroom farm waste as alternative food products. Practical Application: This study shows that unused harvested mushroom waste from a local farm can be used to make an antioxidative chicken patty that is acceptable to consumer panellists. The converted mushroom waste into flour suggests that smaller particles and citric acid pretreatment can increase its nutritional value. This information can be used for waste conversion into new product development from any type of mushroom farm. © 2020 Institute of Food Technologists®

Soy sauce fermentation was improved using the trio of A. oryzae (AO), B. cereus (BC), and the newly identified Tetragenococcus halophilus KBC (TH) in liquid-state fermentation. The effect of molasses (5%, 10%, and 20%) and type of wheat flour (organic-WFO and commercial-WFC) were tested on the koji-moromi formulation for efficient ɣ-aminobutyric acid (GABA) production. The koji-WFO produced higher GABA (1290 mg/L) compared to koji-WFC (13 mg/L). In the initial moromi mixture, 2 × 10−4 mL of BC inoculum containing 5–20% of molasses was added and replaced the same amount with TH in the second trial. Subsequently, the moromi was mixed with 1 × 10−4 mL of BC and TH, which shortened the 60-day moromi process to 30 days. Meanwhile, BC-moromi cultured in 20% molasses generated significantly lower GABA (118 mg/L) production compared to other percentages, however the concentration boosted for TH-moromi in 20% molasses (159 mg/L). The combination of BC-TH percentages (5%–20%) gave significantly lower GABA (83–137 mg/L) than the singular 20% TH treatment. The results promote the use of TH in producing high-GABA soy sauce.

Fructooligosaccharides can be produced by direct enzymatic conversion from sucrose-rich sugarcane syrup (SS) consisting of 58.93% sucrose yielding 21.28 g FOS/100 g sucrose. This study evaluated the prebiotic effect of unpurified/purified SS containing FOS for the modulation of the human intestinal microbial composition and short-chain fatty acid production. The unpurified and purified FOS substrates, which were a mixture of 1-kestose, nystose and 1F-fructosylnystose, were supplemented into human faecal culture using a pH-controlled batch fermentation system and significantly increased the Bifidobacterium counts after 5 h fermentation, while Bacteroides/Prevotella counts were highest throughout 24 h fermentation. Meanwhile, Lactobacillus/Enterococcus exhibited a slight increase after 5 h fermentation before reaching a plateau afterwards. The steady Bacteroides/Prevotella growth and increased Bifidobacterium population promoted an increase in the production of short-chain fatty acids acetate (58 ± 2.70 mM), propionate (9.19 ± 5.94 mM) and butyrate (7.15 ± 2.28 mM). These results provide evidence that representative gut microbiota could utilise the enzymatically synthesised FOS to generate short-chain fatty acids as metabolites in pH-controlled conditions, thus FOS from SS are a potential prebiotic ingredient for foods and health drinks.

For centuries, Azadirachta indica or neem has been utilized as a primary source of medicine due to its antimicrobial, larvacidal, antimalarial and antifungal properties. Recently, its potential as an effective biopesticide has garnered attention, especially towards efficient and continuous production of its bioactive compounds. The present study investigated the effect of the plant growth regulators (PGRs) thiadiazuron (TDZ) and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of colored callus formation and subsequent accumulation of azadirachtin (AZA) in A. indica. An efficient protocol was established for micropropagation and colored callus production of this species, followed by quantification of AZA (a mixture of azadirachtin A and B) and its safety assessment. For induction of the callus, leaf and petiole explants obtained from a young growing neem plant were excised and cultured on Murashige and Skoog (MS) medium supplemented with TDZ (0.2&ndash, 0.6 mg L-1) and 2,4-D (0.2&ndash, 0.6 mg L-1), either applied singly or in combination. Callus was successfully induced from both explant types at different rates, where media with 0.6 mg L-1 of TDZ resulted in the highest fresh weight (3.38 &plusmn, 0.08 g). In general, media with a single hormone (particularly TDZ) was more effective in producing a high mass of callus compared to combined PGRs. A culture duration of six weeks resulted in the production of green, brown and cream colored callus. The highest callus weight and accumulation of AZA was recorded in green callus [214.53 &plusmn, 33.63 mg g-1 dry weight (DW)] induced using TDZ. On the other hand, small amounts of AZA were detected in both brown and cream callus. Further experimentation indicated that the green callus with the highest AZA was found to be non-toxic (LC50 at 4606 &micro, g mL-1) to the zebrafish animal model. These results suggested that the addition of different PGRs during in vitro culture could prominently affect callus and secondary metabolite production and can further be manipulated as a sustainable method for the production of a natural and environmentally friendly pesticide.

This study investigates the effects of viscosity, friction, and sonication on the morphology and the production of lovastatin, (+)-geodin, and sulochrin by Aspergillus terreus ATCC 20542. Sodium alginate and gelatine were used to protect the fungal pellet from mechanical force by increasing the media viscosity. Sodium alginate stimulated the production of lovastatin by up to 329.0% and sulochrin by 128.7%, with inhibitory effect on (+)-geodin production at all concentrations used. However, the use of gelatine to increase viscosity significantly suppressed lovastatin, (+)-geodin, and sulochrin's production (maximum reduction at day 9 of 42.7, 60.8, and 68.3%, respectively), which indicated that the types of chemical play a major role in metabolite production. Higher viscosity increased both pellet biomass and size in all conditions. Friction significantly increased (+)-geodin's titre by 1527.5%, lovastatin by 511.1%, and sulochrin by 784.4% while reducing pellet biomass and size. Conversely, sonication produced disperse filamentous morphology with significantly lower metabolites. Sodium alginate-induced lovastatin and sulochrin production suggest that these metabolites are not affected by viscosity; rather, their production is affected by the specific action of certain chemicals. In contrast, low viscosity adversely affected (+)-geodin's production, while pellet disintegration can cause a significant production of (+)-geodin.

Carbon source is a key component of metabolites synthesis in microorganisms. This work examined the effects of selected carbon sources in the form of carbohydrates, on the growth of Aspergillus terreus ATCC 20542 and the production of lovastatin. Slowly metabolised carbohydrates, such as D-galactose (consumption rate, r=3.11), produced a high microbial biomass, XFINAL (9.44 g/L) compared to other carbohydrates, but with a low biomass yield coefficient (YLOV/X=1.68). In contrast, D-ribose (YLOV/X=) which showed moderate biomass growth (XFINAL=8.78 g/L) and consumption rate (r=5.44 g/day), produced the highest lovastatin amount (51.81 mg/L, day 6). These indicate little correlation between biomass growth and lovastatin production. Notably, culture consisting of pellets with short hairy surface feature is associated with enhanced lovastatin production. Our findings suggest that the production of lovastatin by Aspergillus terreus is highly influenced by the choice of carbohydrates that will shape the pellet morphology rather than the rate of carbohydrates metabolism.

Purpose This study aimed to assess the effect of nitrogen, salt and pre-culture conditions on the production of lovastatin in A. terreus ATCC 20542. Methods Different combinations of nitrogen sources, salts and pre-culture combinations were applied in the fermentation media and lovastatin yield was analysed chromatographically. Result The exclusion of MnSO4 ·5H2O, CuSO4·5H2O and FeCl3·6H2O were shown to significantly improve lovastatin production (282%), while KH2PO4, MgSO4·7H2O, and NaCl and ZnSO4·7H2 O were indispensable for good lovastatin production. Simple nitrogen source (ammonia) was unfavourable for morphology, growth and lovastatin production. In contrast, yeast extract (complex nitrogen source) produced the highest lovastatin yield (25.52 mg/L), while powdered soybean favoured the production of co-metabolites ((+)-geodin and sulochrin). Intermediate lactose: yeast extract (5:4) ratio produced the optimal lovastatin yield (12.33 mg/L) during pre-culture, while high (5:2) or low (5:6) lactose to yeast extract ratio produced significantly lower lovastatin yield (7.98 mg/L and 9.12 mg/L, respectively). High spore concentration, up to 10 7 spores/L was shown to be beneficial for lovastatin, but not for co-metabolite production, while higher spore age was shown to be beneficial for all of its metabolites. Conclusion The findings from these investigations could be used for future cultivation of A. terreus in the production of desired metabolites.

Crude glycerol (CG) can be used as a substrate for microbial bioconversion. However, due to presence of many impurities, many microorganisms are unable to utilise this substrate efficiently. The present study is trying to improve CG using as the feedstock of Aspergillus terreus for the production of lovastatin, (+)-geodin, and sulochrin. The CG was pretreated chemically (solvents) and physically (activated carbon (AC) and water softener (WS)) to separate most of the impurities from the CG. For solvent pretreatments, petroleum ether (PE) produced the largest increase of lovastatin (92.8%) when compared to positive control and pure glycerol (PG) and up to 820% when compared to negative control (CG). In contrast, diethyl ether (DE) produced the largest increase in (+)-geodin at 80.81% (versus CG) and 176.23% (versus PG). The largest increase in toluene (Tol) was observed in sulochrin production, at 67.22% (versus CG) and 183.85% (versus PG). For physical pretreatments, the pattern of metabolite production in AC (lovastatin: 20.65 mg/L, (+)-geodin: 7.42 mg/L, sulochrin: 11.74 mg/L) resembled PG (lovastatin: 21.8 mg/L, (+)-geodin: 8.60 mg/L, sulochrin: 8.18 mg/L), while WS (lovastatin: 11.25 mg/L, (+)-geodin: 15.38 mg/L, sulochrin: 16.85 mg/L) resembled CG (lovastatin: 7.1 mg/L, (+)-geodin: 17.10 mg/L, sulochrin: 14.78 mg/L) at day 6 of fermentation. These results indicate that solvent pretreatments on CG are excellent for metabolites production in A. terreus, depending on the solvents used. In contrast, physical pretreatments are only feasible for (+)-geodin and sulochrin production. Therefore, different strategies can be employed to manipulate the A. terreus bioconversion using improved CG by using a few simple pretreatment strategies.

Natural mycelial biomass (MB) and extracted exopolysaccharide (EPS) from the pre-grown Malaysian Ganoderma lucidum mushroom are both considered as high-end materials due to their high commercial value in the aquaculture industry. To evaluate their potential toxicity as a fish-feed supplement for the fungivore red hybrid Tilapia (Oreochromis sp.), both MB (250−5000 μg/mL) and EPS (62.5−3000 μg/mL) were subjected to zebrafish embryo toxicity (ZFET) assay, and the effects on zebrafish embryos (ZE) early development were analyzed between 24−120 hours of post-exposure (HPE). MB and EPS showed no toxic effect towards the ZE with LC50 of 1650 μg/mL and 2648.38 μg/mL, respectively. MB at concentrations between 250−5000 μg/mL and EPS at 3000 μg/mL showed no significant changes in ZE hatching. No significant changes in the ZE heart rate were detected following treatment with both tested compounds (MB: 250−2000 μg/mL and EPS: 62.5−3000 μg/mL) as compared to untreated embryos (135.5 beats/min). Furthermore, teratogenic effects of both MB and EPS (

Lovastatin is widely prescribed to reduce elevated levels of cholesterol and prevent heart-related diseases. Cultivation of Aspergillus terreus (ATCC 20542) with carbohydrates or low-value feedstocks such as glycerol produces lovastatin as a secondary metabolite and (+)-geodin as a by-product. An A. terreus mutant strain was developed (gedCΔ) with a disrupted (+)-geodin biosynthesis pathway. The gedCΔ mutant was created by inserting the antibiotic marker hygromycin B (hyg) within the gedC gene that encodes emodin anthrone polyketide synthase (PKS), a primary gene responsible for initiating (+)-geodin biosynthesis. The effects of emodin anthrone PKS gene disruption on (+)-geodin and lovastatin biosynthesis and the production of the precursors acetyl-CoA and malonyl-CoA were investigated with cultures based on glycerol alone and in combination with lactose. The gedCΔ strain showed improved lovastatin production, particularly when cultivated on the glycerol-lactose mixture, increasing lovastatin production by 80% (113 mg/L) while simultaneously inhibiting (+)-geodin biosynthesis compared to the wild-type strain. This study thus shows that suppression of the (+)-geodin pathway increases lovastatin yield and demonstrates a practical approach of manipulating carbon flux by modulating enzyme activity.

In this study, a selected γ-aminobutyric acid (GABA)-rich Malaysian strain Aspergillus oryzae NSK was collected from soy sauce koji. The strain was used to explore the effect of using renewable native sugar syrup, sugarcane, nipa, and molasses as fermentable substrates for developing a novel functional GABA soy sauce. We evaluated the strain using the chosen native sugars for 7 days using shake flask fermentation at 30 °C. The results showed optimum GABA concentration was achieved using cane molasses as the fermentable substrate (354.08 mg/L), followed by sugarcane syrup (320.7 mg/L) and nipa syrup (232.07 mg/L). Cane molasses was subsequently utilized as a substrate to determine the most suitable concentration for A. oryzae NSK to enhance GABA production and was determined as 50% g/L of glucose standard cane molasses. Our findings indicate that cane molasses can be used as a GABA-rich ingredient to develop a new starter culture for A. oryzae NSK soy sauce production.

The use of pure substrate represents a significant proportion of the cost of manufacturing a drug such as lovastatin. This study explores the production of lovastatin and (+)-geodin by Aspergillus terreus ATCC 20542 using biodiesel-derived crude glycerol (CG) as a feedstock. Shake flask experiments showed reduced lovastatin production and glycerol consumption in the presence of 10–50 g/L CG with respect to pure glycerol controls. At 50 g/L, lovastatin and (+)-geodin production was significantly reduced by 82 and 73%, respectively. The lowest lovastatin inhibition was detected in 30 g/L of CG (48%), which was accompanied by a significant rise in (+)-geodin production (338%). Further investigation was performed on three major impurities found in CG, namely methanol (MeOH), sodium chloride (NaCl), and fatty acids (oleic acid and palmitic acid (PA), soap). None was particularly inhibitory for lovastatin, except soap and PAs, which reduced its production by more than 50% at all concentrations tested. In contrast, (+)-geodin was inhibited in the presence of MeOH and PA by up to 46 and 91%, respectively. These observations indicate that partial purification of CG would be potentially useful in improving production of lovastatin and (+)-geodin by A. terreus.