Comparative metabolic profiling-based improvement of rapamycin production by Streptomyces hygroscopicus

Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds.     

Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography–ion mobility–mass spectrometry

A method has been developed for metabolite profiling of the salivary metabolome based on protein precipitation and ultra-high performance liquid chromatography coupled with ion mobility-mass spectrometry (UHPLC–IM–MS). The developed method requires 0.5 mL of human saliva, which is easily obtainable by passive drool. Standard protocols have been established for the collection, storage and pre-treatment of saliva. The use of UHPLC allows rapid global metabolic profiling for biomarker discovery with a cycle time of 15 min. Mass spectrometry imparts the ability to analyse a diverse number of species reproducibly over a wide dynamic range, which is essential for profiling of biofluids. The combination of UHPLC with IM–MS provides an added dimension enabling complex metabolic samples to be separated on the basis of retention time, ion mobility and mass-to-charge ratio in a single chromatographic run. The developed method has been applied to targeted metabolite identification and untargeted metabolite profiling of saliva samples collected before and after exercise-induced physiological stress. δ-Valerolactam has been identified as a potential biomarker on the basis of retention time, MS/MS spectrum and ion mobility drift time.     

A metabolomics-based method for studying the effect of yfcC gene in Escherichia coli on metabolism

Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography–mass spectrometry for Escherichia coli. It was found that physiological saline at −80 °C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference.     

Optimization of γ-amino butyric acid production in a newly isolated Lactobacillus brevis

An isolate from kimchi, identified as Lactobacillus brevis, accumulated γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter, in the culture medium. Optimal culture conditions for growth of L. brevis and production of GABA were 6 % (w/v) l-glutamic acid, 4 % (w/v) maltose, 2 % (w/v) yeast extract, 1 % (w/v) NaCl, 1 % (w/v) CaCl₂, 2 g Tween 80/l, and 0.02 mM pyridoxal 5′-phosphate at initial pH 5.25 and 37 °C. GABA reached 44.4 g/l after 72 h cultivation with a conversion rate 99.7 %, based on the amount (6 %) of l-glutamic acid added. GABA was purified using ion exchange column chromatography with 70 % recovery and 97 % purity.     

Optimization of Cultivating Conditions for Triterpenoids Production from Antrodia cinnmomea

The submerged cultivating conditions for triterpenoids production from Antrodia cinnamomea were optimized using uniform design method and the one-factor-at-a-time method was adopted to investigate the effect of plants oils and glucose supply on triterpenoids production and mycelia growth. Corn starch and culturing time were identified as more significant variables for triterpenoids production. The optimal conditions for triterpenoids production was 20.0 g/L corn starch, 20.0 g/L wheat bran, 1.85 g/L MgSO4, initial pH 3 and 16 days of cultivation. In addition, investigation of plant oils and glucose supply showed that 0.3 % (v/v) olive oil supply at the beginning of fermentation stimulated mycelia growth and significantly increased triterpenoids production; 0.2 % (w/v) glucose supplement at 10th day enhanced production of triterpenoids with slight effect on biomass, which is reported for the first time. The triterpenoids production experimentally obtained under the optimal conditions was 7.23 % (w/w). The uniform design method may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoids production from A. cinnamomea.     

Influence of Selenium on the Production of T-2 Toxin by <Emphasis Type="Italic">Fusarium poae</Emphasis>

The objective of this study was to investigate the effects of selenium on the production of T-2 toxin by a Fusarium poae strain cultured in a synthetic medium containing different concentrations of selenium. The T-2 toxin contents in fermentative products were evaluated by a high performance liquid chromatography (HPLC). The results showed that the production of T-2 toxin was correlated with the concentration of selenium added to the medium. In all three treatments, the addition of 1 mg/L selenium to the medium resulted in a lower toxin yield than the control (0 mg/L); the yield of the toxin began to increase when selenium concentration was 10 mg/L, while it decreased again at 20 mg/L. In summary, T-2 toxin yield in the fermentative product was affected by the addition of selenium to the medium, and a selenium concentration of 20 mg/L produced the maximum inhibitory effect of T-2 toxin yield in the fermentative product of F. poae.     

Biostimulation of anaerobic BTEX biodegradation under fermentative methanogenic conditions at source-zone groundwater contaminated with a biodiesel blend (B20)

Field experiments were conducted to assess the potential for anaerobic biostimulation to enhance BTEX biodegradation under fermentative methanogenic conditions in groundwater impacted by a biodiesel blend (B20, consisting of 20 % v/v biodiesel and 80 % v/v diesel). B20 (100 L) was released at each of two plots through an area of 1 m² that was excavated down to the water table, 1.6 m below ground surface. One release was biostimulated with ammonium acetate, which was added weekly through injection wells near the source zone over 15 months. The other release was not biostimulated and served as a baseline control simulating natural attenuation. Ammonium acetate addition stimulated the development of strongly anaerobic conditions, as indicated by near-saturation methane concentrations. BTEX removal began within 8 months in the biostimulated source zone, but not in the natural attenuation control, where BTEX concentrations were still increasing (due to source dissolution) 2 years after the release. Phylogenetic analysis using quantitative PCR indicated an increase in concentration and relative abundance of Archaea (Crenarchaeota and Euryarchaeota), Geobacteraceae (Geobacter and Pelobacter spp.) and sulfate-reducing bacteria (Desulfovibrio, Desulfomicrobium, Desulfuromusa, and Desulfuromonas) in the biostimulated plot relative to the control. Apparently, biostimulation fortuitously enhanced the growth of putative anaerobic BTEX degraders and associated commensal microorganisms that consume acetate and H₂, and enhance the thermodynamic feasibility of BTEX fermentation. This is the first field study to suggest that anaerobic-methanogenic biostimulation could enhance source zone bioremediation of groundwater aquifers impacted by biodiesel blends.     

The production of poly(3-hydroxybutyrate) [P(3HB)] by a newly isolated Bacillus sp. ST1C using liquid waste from biodiesel production

A newly isolated poly(3-hydroxybutyrate) [P(3HB)] producing strain, ST1C, was identified as Bacillus aryabhattai based on its morphological, biochemical and molecular characteristics. It synthesized and accumulated relatively high amounts of P(3HB). The aim of this work was to establish if it could convert an inexpensive liquid waste product from the production of biodiesel, biodiesel liquid waste (BLW), to P(3HB). Using a mineral salt medium (MSM) containing 2.0 % (v/v) glycerol present in the BLW and both normal batch and a draw and fill culture method, B. aryabhattai ST1C produced a maximum P(3HB) content and biomass concentration of 72.31 % dry cell weight (DCW) and 7.24 g/L, respectively, over a 24 h cultivation period in the draw and fill cultivation method. From 24 h to the end of cultivation at 72 h both the P(3HB) content and the biomass concentrations continuously reduced. Concentrations of glycerol in the BLW in this MSM above 3.0 % (v/v) or from pure glycerol (PG) or with an added NaCl concentration of greater than 3.0 % significantly reduced both the maximum P(3HB) content and the biomass concentrations.     

Isolation of biosurfactant-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production

An efficient biosurfactant-producing native Pseudomonas aeruginosa RS29 has been isolated from crude oil contaminated soil. Isolation was followed by optimization of different factors to achieve maximum production of biosurfactant in terms of surface tension reduction (STR) and emulsification index (E24). The isolated strain produced highest biosurfactant in the presence of glycerol after 48 h of incubation at 37.5°C, with pH range of 7–8 and at salinity <0.8% (w/v). The extent of STR and the E24 of medium with different nitrogen sources were investigated and found to be maximal for sodium nitrate (26.3 mN/m, E24?=?80%) and potassium nitrate (26.4 mN/m, E24?=?79%). The production of biomass by the designated strain was found to be maximal in ammonium-nitrate-containing medium as compared to the other nitrogen sources. A kinetic study revealed that biosurfactant production is positively correlated with growth of P. aeruginosa, and highest STR was achieved (27.0 mN/m) after 44 h of growth. The biosurfactant was produced as a primary metabolite and 6 g/L crude biosurfactant was extracted by chloroform:methanol (2:1). The critical micelle concentration of the biosurfactant was 90 mg/L. The absorption bands of the FTIR spectra confirmed the rhamnolipid nature of the biosurfactant. The biosurfactant was thermostable (up to 121°C for 15 min) and could withstand a wide range of pH (2–10) and NaCl concentration (2%–10% w/v). The extracted biosurfactant had good foaming and emulsifying activities and was of satisfactory quality in terms of stability (temperature, pH and salinity) and foaming activity.     

Influence of triacontanol and jasmonic acid on metabolomics during early stages of root induction in cultured tissue of tomato (<Emphasis Type="Italic">Lycopersicon esculentum)</Emphasis>

Influence of n-triacontanol (TRIA) and jasmonic acid (JA) on metabolic profiling during root morphogenesis was studied in Lycopersicon esculentum (cv. PKM-1). Proton nuclear magnetic resonance (¹H NMR) based metabolomics was employed to investigate the variations in metabolic profile. Chenomx NMR suite v.8.1 was used to identify and quantify metabolites based on their respective signature spectra. The levels of 47 metabolites were monitored for 72 h at specific time intervals (0, 3, 6, 9, 12, 24, 36, 48 and 72 h). Principal component analysis was performed to determine the variations in the metabolic profile between control and treatments during in vitro rhizogenesis. TRIA was observed to promote early root emergence (24 h) and also influence the metabolic variation during rhizogenesis between 9 and 24 h post exposure. Compounds such as IAA, ATP, NADPH, UDP-N-acetylglucosamine and gallate predominated at 9 h. Unlike TRIA, JA was unable to promote an early root induction. However, it influenced the synthesis of a relatively higher concentration of IAA at 6 h when compared to ATP, NADPH and trigonelline at 9 h. In the presence of both TRIA and JA (TRIA?+?JA), significant changes in the metabolic profiles were observed 24 h post exposure and the rooting was observed only after 72 h. The study suggests that TRIA may accelerate in vitro rhizogenesis of cultured tomato tissues by mainly increasing the synthesis of other growth promoting metabolites. But in the presence of JA, TRIA’s effect appears to be reduced.     

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils

A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12–14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production—glycerol—is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.     

Enhanced production of triterpenoid in submerged cultures of Antrodia cinnamomea with the addition of citrus peel extract

In recent years, Antrodia cinnamomea has become a well-known medicinal mushroom in Taiwan. Triterpenoids are considered one of the most biologically active components found in A. cinnamomea. The aim of this research is to investigate the feasibility of enhancing triterpenoid production in shake flask cultures of A. cinnamomea by adding citrus peel extract. As a result of its containing essential oils, citrus peel extract is inhibitory to mycelial growth. In the experiments, the appropriate adding time is determined to be on day 7. Of the various citrus peel extracts tested, tangerine proves to be the most effective in enhancing polyphenol and triterpenoid production. With an addition of 2 % (v/v), the content and production of total polyphenols rises from 5.95 mg/g DW of the control and 56.73 mg/L to 23.52 mg/g DW and 224.39 mg/L, respectively, on day 28. The production of triterpenoids also increases from 99.93 to 1,028.02 mg/L, for more than a tenfold increase. An optimal level of tangerine peel additive is determined to be around 4 %. Furthermore, when compared with the mycelia of the control culture, the profiles of the HPLC analysis show that the mycelia cultured with the tangerine-peel addition contain more kinds of triterpenoids. This study demonstrates that the addition of citrus peel extract effectively enhances the production of bioactive metabolites in the submerged cultures of A. cinnamomea.     

Bioconversion of waste office paper to hydrogen using pretreated rumen fluid inoculum

In this study, a microbial consortium from an acid-treated rumen fluid was used to improve the yields of H₂ production from paper residues in batch reactors. The anaerobic batch reactors, which contained paper and cellulose, were operated under three conditions: (1) 0.5 g paper/L, (2) 2 g paper/L, and (3) 4 g paper/L. Cellulase was added to promote the hydrolysis of paper to soluble sugars. The H₂ yields were 5.51, 4.65, and 3.96 mmol H₂/g COD, respectively, with substrate degradation ranging from 56 to 65.4 %. Butyric acid was the primary soluble metabolite in the three reactors, but pronounced solventogenesis was detected in the reactors incubated with increased paper concentrations (2.0 and 4.0 g/L). A substantial prevalence of Clostridium acetobutylicum (99 % similarity) was observed in the acid-treated rumen fluid, which has been recognized as an efficient H₂-producing strain in addition to ethanol and n-butanol which were also detected in the reactors.     

Rational design of a synthetic Entner–Doudoroff pathway for enhancing glucose transformation to isobutanol in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Isobutanol as a more desirable biofuel has attracted much attention. In our previous work, an isobutanol-producing strain Escherichia coli LA09 had been obtained by rational redox status improvement under guidance of the genome-scale metabolic model. However, the low transformation from sugar to isobutanol is a limiting factor for isobutanol production by E. coli LA09. In this study, the intracellular metabolic profiles of the isobutanol-producing E. coli LA09 with different initial glucose concentrations were investigated and the metabolic reaction of fructose 6-phosphate to 1, 6-diphosphate fructose in glycolytic pathway was identified as the rate-limiting step of glucose transformation. Thus, redesigned carbon catabolism was implemented by altering flux of sugar metabolism. Here, the heterologous Entner–Doudoroff (ED) pathway from Zymomonas mobilis was constructed, and the adaptation of upper and lower parts of ED pathway was further improved with artificial promoters to alleviate the accumulation of toxic intermediate metabolite 2-keto-3-deoxy-6-phospho-gluconate (KDPG). Finally, the best isobutanol-producing E. coli ED02 with higher glucose transformation and isobutanol production was obtained. In the fermentation of strain E. coli ED02 with 45 g/L initial glucose, the isobutanol titer, yield and average producing rate were, respectively, increased by 56.8, 47.4 and 88.1% to 13.67 g/L, 0.50 C-mol/C-mol and 0.456 g/(L × h) in a shorter time of 30 h, compared with that of the starting strain E. coli LA09.     

Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)⁺, but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO₃ (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.