Hierarchical amino acid utilization and its influence on fermentation dynamics: rifamycin B fermentation using Amycolatopsis mediterranei S699, a case study

Background  

Industrial fermentation typically uses complex nitrogen substrates which consist of mixture of amino acids. The uptake of amino acids is known to be mediated by several amino acid transporters with certain preferences. However, models to predict this preferential uptake are not available. We present the stoichiometry for the utilization of amino acids as a sole carbon and nitrogen substrate or along with glucose as an additional carbon source. In the former case, the excess nitrogen provided by the amino acids is excreted by the organism in the form of ammonia. We have developed a cybernetic model to predict the sequence and kinetics of uptake of amino acids. The model is based on the assumption that the growth on a specific substrate is dependent on key enzyme(s) responsible for the uptake and assimilation of the substrates. These enzymes may be regulated by mechanisms of nitrogen catabolite repression. The model hypothesizes that the organism is an optimal strategist and invests resources for the uptake of a substrate that are proportional to the returns.     

Design of methanol Feed control in Pichia pastoris fermentations based upon a growth model

The methylotrophic yeast Pichia pastoris is an effective system for recombinant protein productions that utilizes methanol as an inducer, and also as carbon and energy source for a Mut(+) (methanol utilization plus) strain. Pichia fermentation is conducted in a fed-batch mode to obtain a high cell density for a high productivity. An accurate methanol control is required in the methanol fed-batch phase (induction phase) in the fermentation. A simple "on-off" control strategy is inadequate for precise control of methanol concentrations in the fermentor. In this paper we employed a PID (proportional, integral and derivative) control system for the methanol concentration control and designed the PID controller settings on the basis of a Pichia growth model. The closed-loop system was built with four components: PID controller, methanol feed pump, fermentation process, and methanol sensor. First, modeling and transfer functions for all components were derived, followed by frequency response analysis, a powerful method for calculating the optimal PID parameters K(c) (controller gain), tau(I) (controller integral time constant), and tau(D) (controller derivative time constant). Bode stability criteria were used to develop the stability diagram for evaluating the designed settings during the entire methanol fed-batch phase. Fermentations were conducted using four Pichia strains, each expressing a different protein, to verify the control performance with optimal PID settings. The results showed that the methanol concentration matched the set point very well with only small overshoot when the set point was switched, which indicated that a very good control performance was achieved. The method developed in this paper is robust and can serve as a framework for the design of other PID feedback control systems in biological processes.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers

AIMS: The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. METHODS AND RESULTS: In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l(-1) sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) - P(HB-co-HV)--of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l(-1)) in a 3.0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2.7 g l(-1), respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2.5 g l(-1). The polymer was characterized by proton NMR. CONCLUSIONS: Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.     

Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.

Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.     

Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae

We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C₄-C₆ and C₃-C₈ chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.This revised version was published online in February 2005 with corrections to Table 1.     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Evaluation of native potential probiotic bacteria using an in vitro ruminal fermentation system

Rumen microbiota provides an important source of protein to grazing animals and produces volatile fatty acids (VFA), the main energy source for ruminants generated by fibre fermentation. Probiotics can be used to modulate rumen fermentation, and native microbiota is a source of potentially useful microorganisms. In this work, ruminal bacterial strains were isolated and subsequently identified, and their potential to modify fermentation patterns with wheat straw, microcrystalline cellulose and oat xylan as substrates was assessed by in vitro gas production and VFA fermentation patterns. Four of the isolates were identified as Pseudobutyrivibrio ruminis and two corresponded to new members of the Lachnospiraceae family. The addition of one P. ruminis (strain 50C) and one Lachnospiraceae (strain 21C) to the fermentation system which used wheat straw as the substrate significantly increased total VFA concentration without altering the total gas produced in one case and showed a decrease in total gas production in the other. All bacterial strains induced higher butyric acid concentrations with the three substrates (up to 31 mM in the case of Lachnospiraceae 21C incubated with oat xylan and 25 mM in microcrystalline cellulose fermenters to which P. ruminis 50C had been added) compared to the control, which had concentrations of <1 mM. Analysis of the fermentation products suggested that the addition of probiotics to the fermentation system had the potential to induce metabolic shifts that would result in better energy yields. These results show that native bacteria have promising features as fermentation modulators, thereby justifying further research to assess their use as probiotics for ruminants.     

Characterization of a prototype industrial on-line analyzer for bicarbonate/carbonate monitoring

In many biological reactors bicarbonate is the major species determining pH buffering capacity, or alkalinity. In anaerobic digesters bicarbonate levels should be within 10 to 50 mM for stable operation. Bicarbonate alkalinity in wastewater treatment processes in routinely measured off-line titrimetrically. Recently we have described the principle of a novel on-line method of measuring bicarbonate alkalinity. In the prototype device described here, a continuous stream (15 cm(3) min(-1)) of the substrate to be monitored was saturated with gaseous CO2, acidified by the addition of excess acid, and the rate of carbon dioxide evolution, proportional to the concentration of bicarbonate/carbonate in the liquid flow, continuously measured by a sensitive gas meter. The instrument was robust and its response was satisfactory for wastewater treatment process control applications, with linearity in the range 5 to 50 mM HCO3(-), a response time in the order of 30 min, and accuracy of the order of 7% in the concentration range 5 to 50 mM sodium bicarbonate. The device was not affected by interference from volatile fatty acids, does not make use of pH probes which in many wastes are subject to fouling, and may form the basis of a digester control strategy. (c) 1994 John Wiley & Sons, Inc.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

L-丙氨酸厌氧发酵关键技术及产业化

传统氨基酸制造主要是通过化学合成或好氧发酵实现。相对于化学合成,微生物发酵可以实现以可再生资源为原料直接生产氨基酸,减少了对石油基原料的依赖,解决了化学合成高污染、高能耗等问题。好氧发酵具有生长快、产量高等特点,但好氧发酵中大量碳源用于细胞生长容易造成糖酸转化率低、能耗高等问题。厌氧发酵是近年来新出现的氨基酸生产模式,具有操作简单、无需通氧、糖酸转化率高容易接近理论最大值等优势。L-丙氨酸是国际上首个实现厌氧发酵产业化生产的氨基酸。本文以L-丙氨酸为例,综述了氨基酸厌氧发酵过程中的关键问题及其在产业化实施中的应用。未来,随着厌氧发酵关键技术在更多化合物生物制造技术中的突破,这种低成本、高效、低碳环保型发酵方式将会带来更大的经济价值和社会效益。     

Chlamydospore Production,Inoculation Methods and Pathogenicity of Fusarium oxysporum M12-4A,a Biocontrol for Striga hermonthica

Fusarium oxysporum isolate M12-4A is currently being evaluated for the biological control of Striga hermonthica . Inoculum production, inoculum delivery to the target, chlamydospore germination, and weed growth suppression of this weed-pathogen system were investigated. Liquid fermentation systems using organic material were evaluated for the production of large numbers of chlamydospores. A 1% sorghum straw powder (< 1 mm) substrate, exposed to black light at 21°C for 21 days, yielded 3.23 X 10 8 colony forming units (CFU) l -1 medium. A two-stage fermentation system using 5% w/v straw substrate under black light at 30°C for 14 days yielded 3.5 X 10 8 CFU l -1 medium. In vitro variations in chlamydospore germination were governed by the presence of exogenous carbon, nitrogen, and sorghum root exudates. Ammonium-nitrogen compounds and urea, in combination with glucose had a stronger stimulatory effect on chlamydospore germ tube growth than did potassium nitrate. Maximal germ tube elongation occurred when chlamydospores were exposed to urea at a C/N ratio of 10. Some mineral solutions and sorghum root exudates inhibited chlamydospore germ tube elongation; however, arabic gum, a complex polysaccharide, stimulated chlamydospore germ tube elongation and the production of secondary chlamydospores. In field trials, chlamydospore powder harvested from small-scale fermenters reduced S. hermonthica emergence by 92%. Complete inhibition of S. hermonthica emergence occurred when the chlamydospore powder was added to the soil at sowing and when sorghum seeds coated with chlamydospores were sown. Effective biological control of S. hermonthica was achieved using a simple fermentation system with sorghum straw as the inoculum growth substrate. For inoculum delivery to the farmers' fields, sorghum seeds were coated with the inoculum using arabic gum as the adhesive. This simple delivery system permits a uniform inoculation of the field as well as the proper positioning of the inoculum in the immediate environment of sorghum roots, where S. hermonthica attaches to its host. To facilitate a broad usage of F. oxysporum M12-4A for the biocontrol of S. hermonthica , we propose an inoculum production strategy based on a cottage industry model that utilizes a liquid fermentation process and inexpensive locally-available substrates including sorghum straw and arabic gum.     

Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405)

Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, >92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products - ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively.     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

Differential effect of valproate and its Delta2- and Delta4-unsaturated metabolites, on the beta-oxidation rate of long-chain and medium-chain fatty acids

Overall fatty acid oxidation rates were investigated in rat hepatocytes using [9,10-3H]-palmitic, [9,10-3H]-oleic, [9,10-3H]-myristic and [2,3-3H]-phenylpropionic acids. The effect of both valproate (VPA) (0-10 mM) and two of its unsaturated metabolites, Delta2(E)-VPA and Delta4-VPA (0-10 mM), on the overall 3H2O production rate was studied. The results give evidence of a general inhibitory effect of VPA on the beta-oxidation rate of all the tested substrates. Similar effects were observed with both VPA metabolites but these effects appeared to be dependent on the chain length of the substrate. When the effect on the oxidation of the medium-chain fatty acid 3-phenylpropionate (PPA) was studied, Delta2(E)-VPA at 0.5 mM caused a 94% inhibition of the overall beta-oxidation rate. However, with long-chain substrates, 0.5 mM Delta(4)-VPA was a more potent inhibitor (20-30% of control activity) than 0.5 mM Delta(2E)-VPA (60-80% of control activity). Our results suggest that VPA and/or its metabolites inhibit fatty acyl-CoA metabolism within the mitochondrion by two different mechanisms. The first mechanism involves CoASH sequestration, which affects the oxidation rate of all fatty acids with different chain length. The second mechanism is more specific in nature and involves selective inhibition of particular enzymes implicated in fatty acid beta-oxidation.     

Influence of the pH on (open) mixed culture fermentation of glucose: a chemostat study

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO(2)/H(2) production at lower pH values to formate production at higher pH values was observed. The ratio of CO(2)/H(2) versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO(2)/H(2). This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations.