Enhanced beauvericin production with in situ adsorption in mycelial liquid culture of Fusarium redolens Dzf2

Fusarium redolens Dzf2, an endophytic fungal species, is a high producer of the antibiotic compound beauvericin (BEA). However, the BEA produced by the F. redolens Dzf2 fungus was retained mainly as an intracellular product. This study was to evaluate an integrated fermentation-in situ product recovery process for enhancement of BEA production in F. redolens Dzf2 myelical culture. A macroporous polystyrene resin (X-5) was selected as the sorbent and added to the mycelial culture flasks (enclosed in a nylon bag). With 2 g resin added to 40 ml medium in each flask in the early stationary growth phase (day 5), the volumetric BEA yield (on day 7) was increased from 194 to 265 mg l^?1, with 65% being adsorbed to the resin phase. With resin renewal plus glucose feeding (on day 7), the BEA production was increased even more dramatically to 400 mg l^?1 (on day 9), double of the yield in the batch control culture. The results show that in situ adsorption was an effective strategy for enhancing the BEA production and also facilitating its recovery in the mycelial liquid culture.     

Immobilized Candida antarctica lipase B on ZnO nanowires/macroporous silica composites for catalyzing chiral resolution of (R,S)-2-octanol

ZnO nanowires were successfully introduced into a macroporous SiO₂ by in situ hydrothermal growth in 3D pores. The obtained composites were characterized by SEM and XRD, and used as supports to immobilize Candida antarctica lipase B (CALB) through adsorption. The high specific surface area (233 m²/g) and strong electrostatic interaction resulted that the average loading amount of the composite supports (196.8 mg/g) was 3–4 times of that of macroporous SiO₂ and approximate to that of a silica-based mesoporous material. Both adsorption capacity and the activity of the CALB immobilized on the composite supports almost kept unchanged as the samples were soaked in buffer solution for 48 h. The chiral resolution of 2-octanol was catalyzed by immobilized CALB. A maximum molar conversion of 49.1% was achieved with 99% enantiomeric excess of (R)-2-octanol acetate under the optimal condition: a reaction using 1.0 mol/L (R,S)-2-octanol, 2.0 mol/L vinyl acetate and 4.0 wt.% water content at 60 °C for 8 h. After fifteen recycles the immobilized lipase could retain 96.9% of relative activity and 93.8% of relative enantioselectivity.     

Production of l-phenylalanine from glucose by metabolic engineering of wild type Escherichia coli W3110

The market of l-phenylalanine has been stimulated by the great demand for the low-calorie sweetener aspartame. In this paper, the effects of pivotal genes on l-phenylalanine production were evaluated by metabolic engineering of wild type Escherichia coli. The bifunctional PheA protein contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by l-phenylalanine). The catalytic domain of PheA was overexpressed to increase l-phenylalanine production. It was firstly indicated that this domain could enhance the metabolic influx to overproduce l-phenylalanine and improve the survival ability under m-Fluoro-dl-phenylalanine stress. Furthermore, the fermentation performance of aroG feedback inhibition resistant mutants was firstly compared, aroG29 and aroG15 increased the l-phenylalanine concentration by 5-fold. After that the expression of aroK and ydiB was also elevated, and the l-phenylalanine yield on cell (0.79 g/g) and maximum l-phenylalanine productivity (0.073 g/L/h) were subsequently doubled. Meanwhile, the l-phenylalanine yield on glucose increased from 0.124 g/g to 0.153 g/g. It was found that genes ydiB and aroK could elevate the l-phenylalanine yield and productivity and shorten the lag phase.     

HPLC-DAD analysis of arbutin produced from hydroquinone in a biotransformation process in Origanum majorana L. shoot culture

The hydroquinone glucoside arbutin is a plant derived compound medically applied due to its uroantiseptic activity. It also has skin whitening properties and thus is widely used in dermatology and cosmetology. Origanum majorana L. (Lamiaceae) is known to produce arbutin, however the content of the compound in cultivated plants is very variable and low. Since plant cell and tissue cultures are capable to perform specific biotransformation reactions including glucosylation, this investigation targeted the formation of arbutin from hydroquinone in agitated O. majorana shoot cultures. For this purpose different doses of hydroquinone (96, 144, 192, 288 and 384 mg/L of medium) were added to the culture flasks in one, two or three portions. Arbutin was qualitatively and quantitatively determined in methanol extracts from dry biomass and lyophilized media using HPLC-DAD. Cells of O. majorana shoot cultures efficiently converted hydroquinone into arbutin. The product was accumulated in the biomass and was not observed (or in trace amounts) in the medium samples. Different doses as well as portioning of the precursor had a significant impact on the biotransformation process. Arbutin accumulation increased from 0.23 ± 0.03 mg/g DW up to 52.6 ± 4.8 mg/g DW in the biomass. The highest product content was observed after the addition of 192 mg/L hydroquinone in three portions. The highest efficiency of the biotransformation process, i.e. 67.5 ± 5.2% was calculated for a dose of 96 mg/L precursor divided into three portions. After further optimization of the biotransformation process, O. majorana shoot cultures could serve as a rich source of arbutin.     

Bioprocess engineering to produce 10-hydroxystearic acid from oleic acid by recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Microbial hydroxylation of long chain fatty acids has been extensively investigated. However, biotransformation productivity remains below ca. 1.0 g/g cell dry weight (CDW)/h under process conditions. In the present study, a highly efficient microbial hydroxylation process to convert oleic acid into 10-hydroxystearic acid was developed. A recombinant Escherichia coli expressing ohyA, the gene encoding oleate hydratase of Stenotrophomonas maltophilia, was used as the biocatalyst. Investigation of the ohyA expression and biotransformation conditions (e.g., inducer concentration, gene expression period before initiating biotransformation, mixing condition of reaction medium) enabled 10-hydroxystearic acid to accumulate to a final concentration of approximately 46 g/L in the culture medium. The specific product formation rate and product yield reached approximately 2.0 g/g CDW/h (i.e., 110 U/g CDW) and 91%, respectively. The specific product formation rate was more than 3-fold higher than those of a bioprocess using wild type Stenotrophomonas sp. cells. Additionally, the product of the whole-cell biotransformation was recovered at a yield of 70.9% and a purity of 99.7% via solvent fraction crystallization at low temperature. These results will contribute to developing a biological process for hydroxylation of oleic acid.     

Biotransformation of 2-phenylethanol to phenylacetaldehyde in a two-phase fed-batch system

Phenylacetaldehyde (PA) can be produced by the oxidation of 2-phenylethanol (PE) through biotransformation. In order to prevent substrate and product inhibitions and the transformation of the PA to phenylacetic acid (PAA), utilization of a two-phase system is very attractive. Gluconobacter oxydans B-72 was used as the microorganism and iso-octane as the solvent. The effect of initial substrate concentration on the PA production was investigated in single- and two-phase systems. In the single-phase system, substrate inhibition occurred above 5 g/l, and in the two-phase system, above 7.5 g/l. Substrate inhibition kinetics were also studied in the two-phase system and kinetic constants were determined as r_max=0.64 g/l min, K_M=8.15 g/l, K_PA=2.5 g/l. Because it was observed that two-phase system is insufficient to remove the substrate inhibition effect, fed-batch operation was utilised in this study. For 7.5 g/l of PE, 1.65, 3.85, and 7.35 g/l of PA were obtained in the single-phase, two-phase, and two-phase three fed-batch systems, respectively. Effect of biotransformation time, initial substrate concentration, agitation speed, and fed-batch number on the PA production was investigated in a two-phase fed-batch system by the response surface methodology (RSM). The optimum values were found as 3 fed-batch number, 2.75 g/l initial substrate concentration, 150 rpm agitation speed, and 65 min of one batch biotransformation time. In order to verify these results, an experiment was performed at these optimum conditions and 7.10 g/l of PA concentration was obtained.     

Structural stability of acetyl saponins in different solvents and separation materials

Two acetyl saponins, 2″-O-acetylplatycodin D and 3″-O-acetylplatycodin D from Platycodon Radix were selected for their structure stability study. Different solvents, stationary phases and temperatures were employed to study the structural inter-conversion of acetyl group in two acetyl saponins. The results showed that the reaction of acetyl transfer was faster in water than other solvents, and comparing to the normal/reverse silica gels, the reaction of acetyl migration almost did not happen during the process of purification by macroporous resin. The activation energy and enthalpy of 2″-APD converted into 3″-APD reaction were 63.01 kJ mol⁻¹, and 7.48 kJ mol⁻¹, respectively. Low polar solvent, macroporous resin and low temperature may be more suitable for the separation and purification of acetyl saponins.     

A novel anticancer and antifungus phenazine derivative from a marine actinomycete BM-17

A marine actinomycete, designated strain BM-17, was isolated from a sediment sample collected in the Arctic Ocean. The strain was identified as Nocardia dassonvillei based on morphological, cultural, physiological, biochemical characteristics, along with the cell wall analysis and 16S rDNA gene sequence analysis. A new secondary metabolite (1), N-(2-hydroxyphenyl)-2-phenazinamine (NHP), and six known antibiotics (2–7) have been isolated from the saline culture broth of the stain by sequentially purification over macroporous resin D101, silica gel, Sephadex LH-20 column chromatography and preparative HPLC after the stain was incubated in soy bean media at 28 °C for 7 days. The chemical structures of the compounds were elucidated on the basis of spectroscopic analysis, including two-dimensional (2D) NMR and HR-ESI-MS data. The new compound showed significant antifungal activity against Candida albicans, with a MIC of 64 μg/ml and high cancer cell cytotoxicity against HepG2, A549, HCT-116 and COC1 cells.     

An integrated bioreactor-expanded bed adsorption system for the removal of acetate to enhance the production of alpha-interferon-2b by Escherichia coli

A stirred tank bioreactor (STB) integrated with an expanded bed adsorption (EBA) system containing anion-exchange resin (Diaion WA30) was developed for in situ removal of acetate to increase the production of α-interferon-2b (α-PrIFN-2b) by Escherichia coli (E. coli). Although the total acetate (9.79 g/L) secreted by E. coli in the integrated STB/EBA system was higher than that in a bioreactor with dispersed resin or a conventional batch bioreactor, cell growth (14.97 g/L) and α-PrIFN-2b production (867.4 μg/L) were significantly improved owing to the high efficiency of acetate removal from the culture. The production of α-PrIFN-2b in the integrated STB/EBA system was improved by 3-fold and 1.4-fold over that obtained in a conventional batch bioreactor and a bioreactor containing dispersed resins, respectively.     

Kinetics of lipase recovery from the aqueous phase of biodiesel production by macroporous resin adsorption and reuse of the adsorbed lipase for biodiesel preparation

A commercial macroporous resin (D3520) was screened for lipase recovery by adsorption from the aqueous phase of biodiesel production. The influences of several factors on the adsorption kinetics were investigated. It was found that the kinetic behavior of lipase adsorption by macroporous resin could be well described by pseudo-first-order model. Temperature had no significant effects on lipase adsorption, while resin-to-protein ratio (R) significantly affected both rate constant (k₁) and equilibrium adsorption capacity (Q_e). No lipase was adsorbed when mixing (shaking) was not performed; however, protein recovery reached 98% after the adsorption was conducted at 200 rpm for 5 h in a shaker. The presence of methanol and glycerol showed significant negative influence on lipase adsorption kinetics. Particularly, increasing glycerol concentration could dramatically decrease k₁ but not impact Q_e. Biodiesel was found to dramatically decrease Q_e even present at a concentration as low as 0.02%, while k₁ was found to increase with biodiesel concentration. The adsorbed lipase showed a relatively stable catalytic activity in tert-butanol system, but poor stability in solvent-free system when used for biodiesel preparation. Oil and biodiesel were also found to adsorb onto resin during transesterification in solvent-free system. Therefore, the resin had to be washed by anhydrous methanol before re-used for lipase recovery.     

Efficient production of α-arbutin by whole-cell biocatalysis using immobilized hydroquinone as a glucosyl acceptor

The aim of the present study is to develop an efficient and cost-effective method for α-arbutin production by using whole-cell of Xanthomonas maltophilia BT-112 as a biocatalyst. Hydroquinone (HQ), substrate for the bioconversion as glucosyl acceptor, was immobilized on H107 macroporous resin to reduce its toxic effect on the cells, and the optimal reaction conditions for α-arbutin synthesis were investigated. When 350 g/L H107 resin (254.5 mM HQ) and 20 g/L (4.2 U/g) of cells were shaken in 10 mL Na₂HPO₄–KH₂PO₄ buffer (50 mM, pH 6.5) containing 509 mM sucrose at 35 °C with 150 rpm for 48 h, the final yield of α-arbutin reached 65.9 g/L with a conversion yield of 95.2% based on the amount of HQ supplied. The α-arbutin production was 202% higher than that of the control (free HQ) and the cells maintained its full activity for almost six consecutive batch reactions, indicating a potential for reducing production costs. Additionally, the product was one-step isolated and identified as α-arbutin by ¹³C NMR and ¹H NMR analysis. In conclusion, the combination of whole cells and immobilized hydroquinone (IMHQ) is a promising approach for economical and industrial-scale production of α-arbutin.     

Biocatalytic synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate with Candida krusei SW2026: A practical process for high enantiopurity and product titer

Ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), a key intermediate in the production of angiotensin-converting enzyme (ACE) inhibitors, was prepared by the microbial reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Among 63 microorganisms tested, Candida krusei SW2026, for the first time, was proven to be a highly effective biocatalyst in this reduction process, leading to the (R)-enantiomer in 99.7% ee and 95.1% yield at 2.5 g/L of OPBE (under optimal conditions of 30 °C, pH 6.6, and in the presence of 5% glucose as co-substrate). In order to achieve higher product concentration with desired enantiopurity and yield for application in large-scale production, strategies such as substrate fed-batch and aqueous/organic biphasic system were successfully conducted in the biotransformation reaction. At 20 g/L of OPBE, the enantiomeric excess (ee), yield, and product concentration were enhanced to 97.4%, 82.0%, and 16.6 g/L, respectively, in water/dibutyl phthalate biphasic system, compared with 87.5%, 45.8%, and 9.2 g/L in aqueous medium. This study provides an attractive process of (R)-HPBE production for potential green chemistry applications.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Novel biotransformation process of podophyllotoxin to produce podophyllic acid and picropodophyllotoxin by Pseudomonas aeruginosa CCTCC AB93066, Part II: Process optimization

This work optimized the novel biotransformation process of podophyllotoxin to produce podophyllic acid by Pseudomonas aeruginosa CCTCC AB93066. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/l of yeast extract and 5 g/l of peptone were favorable for podophyllic acid production (i.e. 25.3 ± 3.7 mg/l), while not beneficial for the cell growth of P. aeruginosa. This indicated that the accumulation of podophyllic acid was not corresponded well to the cell growth of P. aeruginosa. 0 g/l of sucrose was beneficial for podophyllic acid production (i.e. 34.3 ± 3.9 mg/l), which led to high podophyllotoxin conversion (i.e. 98.2 ± 0.1%). 1 g/l of NaCl was the best for podophyllic acid production (i.e. 47.6 ± 4.0 mg/l). Secondly, the production of podophyllic acid was significantly enhanced by fed-batch biotransformation. When each 100 mg/l of podophyllotoxin was added to the biotransformation system after 4, 10 and 25 h of culture, respectively, podophyllic acid concentration reached 99.9 ± 12.3 mg/l, enhanced by 284% comparing to one-time addition (i.e. 26.0 ± 2.1 mg/l). The fundamental information obtained in this study provides a simple and efficient way to produce podophyllic acid.     

Bioconversion of phenylpyruvic acid to l-phenylalanine by mixed-gel immobilization of Escherichia coli EP8-10

A mixed-gel of κ-carrageenan and gelatin was used in l-phenylalanine production. The mixed-gel, containing 87.5% κ-carrageenan and 12.5% gelatin [the total gel concentration was 4 wt%], showed the best performance and was selected for further study with Escherichia coli EP8-10. The optimum pH and temperature were 8.5 and 37 °C, respectively. The effects of trehalose and Mg²⁺ were studied in the mixed-gel immobilization. Their optimum concentrations were 5 × 10^?2 and 2 × 10^?3 mol/L, respectively. Under the optimal conditions, 98.3% of the phenylpyruvic acid (PPA) was converted to l-phenylalanine. The activity recovery of the transaminase enzyme in the mixed-gel immobilization was higher than that in single κ-carrageenan immobilization, which was 93.6%. The total PPA conversion rate was over 80% in all 15 batches, suggesting great sustainability in the mixed-gel immobilization. The maximum reaction rate (r_max) was calculated to be 4.75 × 10^?2 mol/(L g h).     

Optimization of a biotransformation process to produce 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin

The developed tandem biotransformation process for the directional biosynthesis of a designed compound 4-(2,3,5,6-tetramethylpyrazine-1)-4′-demethylepipodophyllotoxin (4-TMP-DMEP) by Alternaria alternata S-f6 was systematically optimized. 28 °C of culture temperature and 120 rpm of rotary shaker speed were suitable for the accumulation of 4-TMP-DMEP. The production (i.e., 11.1 ± 1.4 mg/L) of 4-TMP-DMEP was remarkably improved by using an initial yeast extract concentration of 2.5 g/L. 2.0 g/L of Span 80 was beneficial for the 4-TMP-DMEP production (i.e., 25.0 ± 1.5 mg/L). Furthermore, the 4-TMP-DMEP production was remarkably improved by one pulse feeding of 50 mg/L of DMEP on day 6 and two pulse feedings of 40 mg/L of TMP on days 8 and 14 when its residual level was below 50 mg/L and 10 mg/L, respectively. The 4-TMP-DMEP production of 45.1 ± 1.6 mg/L was obtained in the fed-batch biotransformation process, which was enhanced by 726% and 256%, comparing to that (i.e., 5.4 ± 0.4 mg/L and 0.9 mg/L/day) obtained in the batch biotransformation before optimization.     

l-Phenylalanine catabolism and l-phenyllactic acid production by a phototrophic bacterium,Rubrivivax benzoatilyticus JA2

A phototrophic bacterium (Rubrivivax benzoatilyticus JA2) grows at the expense of l-phenylalanine as sole source of nitrogen but not as carbon source. Near stoichiometric yields of l-phenylpyruvic acid (0.4 mM) and l-phenyllactate (0.4 mM) were observed from l-phenylalanine (0.9 mM consumed). Aminotransfarase and dehydrogenase activities involved in the formation of l-phenylpyruvic acid and l-phenyllactate were demonstrated unequivocally in Rubrivivax benzoatilyticus JA2. Growth conditions and carbon sources had an influence on l-phenyllactate production. The process yielded a maximum of 0.92 mM l-phenyllactate from l-phenylalanine (1 mM) when fructose served as carbon source for R. benzoatilyticus JA2.     

Metabolic engineering of Escherichia coli for the production of 5-aminovalerate and glutarate as C5 platform chemicals

5-Aminovalerate (5AVA) is the precursor of valerolactam, a potential building block for producing nylon 5, and is a C5 platform chemical for synthesizing 5-hydroxyvalerate, glutarate, and 1,5-pentanediol. Escherichia coli was metabolically engineered for the production of 5-aminovalerate (5AVA) and glutarate. When the recombinant E. coli WL3110 strain expressing the Pseudomonas putida davAB genes encoding delta-aminovaleramidase and lysine 2-monooxygenase, respectively, were cultured in a medium containing 20 g/L of glucose and 10 g/L of l-lysine, 3.6 g/L of 5AVA was produced by converting 7 g/L of l-lysine. When the davAB genes were introduced into recombinant E. coli strainXQ56allowing enhanced l-lysine synthesis, 0.27 and 0.5 g/L of 5AVA were produced directly from glucose by batch and fed-batch cultures, respectively. Further conversion of 5AVA into glutarate could be demonstrated by expression of the P. putida gabTD genes encoding 5AVA aminotransferase and glutarate semialdehyde dehydrogenase. When recombinant E. coli WL3110 strain expressing the davAB and gabTD genes was cultured in a medium containing 20 g/L glucose, 10 g/L l-lysine and 10 g/L α-ketoglutarate, 1.7 g/L of glutarate was produced.     

Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4–5 days at 30 °C. The optimum condition for biotransformation of EAP involved cultivation at 37 °C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.     

Biotransformation of betulin to betulone by growing and resting cells of the actinobacterium Rhodococcus rhodochrous IEGM 66

The ability of Rhodococcus actinobacteria to transform betulin to betulone was proved and reported for the first time. Betulone, the product of regioselective oxidation of a 3β-hydroxyl group of betulin, is a useful intermediate in the synthesis of novel biologically active compounds. Of 56 strains of Rhodococcus tested, Rhodococcus rhodochrous IEGM 66 was selected because it had the highest betulin-transforming ability. It was shown that R. rhodochrous IEGM 66 growing cells transformed 0.5 g/L betulin to betulone with 45% conversion rate within 240 h. A substantial reduction in the time of betulin (0.5 g/L) biotransformation was achieved by using resting cells, which catalyzed the production of 75% betulone after 96 h. At higher initial betulin concentrations (1.0–3.0 g/L), resting cells catalyzed 40–60% betulone production within 24 h.