Enhanced production of avermectin B1a by medium optimization and glucose feeding with Streptomyces avermilitis

Avermectin B1a is an important macrolide antibiotic with potent anthlemintic and insecticidal activity. The objective of this study was to enhance productivity of avermectin B1a by optimizing culture medium and glucose feeding fermentation. Results showed that B1a concentration was increased by 48.6% through optimization of nitrogen, carbon sources as well as supplement of 0.2 mM Co²⁺. It was found that a maximum B1a concentration of 826 mg/l was attained by glucose-feeding in a laboratory scale fermentor, which was two-fold higher than that in the control fermentation. Meanwhile, the proportion of B1a in the total products was increased by 6% with respect to the control process. These results would be very useful for maximizing productivity of avermectin B1a in an up-scaled fermentation.     

Improvement of tautomycin production in <Emphasis Type="Italic">Streptomyces spiroverticillatus</Emphasis> by feeding glucose and maleic anhydride

Optimization of the feeding process for tautomycin production by Streptomyces spiroverticillatus was performed using glucose and/or maleic anhydride. The feeding of glucose was based on the reducing sugar content (lower than 8 g/L) at a cultivation time of 40 h. After addition of 2% (w/v) glucose, the biomass increased from 21 to 28 g/L, and that of tautomycin from 572.06 to 837.6 mg/L. Moreover, 1723.1 mg/L of tautomycin (increased by 201.21%) was obtained by feeding 0.2% (w/v) maleic anhydride solution at a pH between 4 and 7 in the broth. For the experiments in the 15 L fermentor, tautomycin content reached its highest level (1714.7 mg/L), which was 199.7% higher than that of control by feeding both glucose and maleic anhydride.     

Antioxidants enhanced production of destruxin E from cultivation of Metarhizium anisopliae

The effect of antioxidants on the production of an important cyclohexadepsipeptide congener destruxin E (dtx E) was investigated using the entomopathogenic fungus Metarhizium anisopliae F061. In shaker flask cultivations, 0.015% of menadione-enhanced dtx E production of 220.4 mg/l compared to the control cultivation 90.2 mg/l, which was illustrated by stimulation of dtx E biosynthesis through two electron reduction DT-diaphorase processes in cultivation of M. anisopliae. In 5-l stirred-tank bioreactor cultivation with menadione addition and of control pH 4.0, a yield of 454.6 mg/l of dtx E was obtained after 7 days, and was nearly 30 and 15-fold higher than that from no pH control, and controlled pH 2.0 cultivations, respectively. Further cultivation in a 20-l airlift bioreactor, at pH 4.0, dtx E obtained on the 9th day was 406.0 mg/l, which was much higher than the standard cultivation of no pH control yield 203.3 mg/l on the 11th day. Thus, the present study provides useful information for enhancing dtx E production in cultivation.     

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.     

Oxygen uptake rate regulation during cell growth phase for improving avermectin B<Subscript>1a</Subscript> batch fermentation on a pilot scale (2 m<Superscript>3</Superscript>)

Avermectin B_1a batch fermentation of Streptomyces avermitilis in a 2 m³ fermentor was investigated by oxygen uptake rate (OUR) regulation during cell growth phase. OUR was controlled by adjusting of aeration and agitation. Result showed that OUR strongly affected cell growth and antibiotics production. Avermectin B_1a biosynthesis could be effectively enhanced when OUR was stably regulated at an appropriate level in batch fermentation of S. avermitilis. Avermectin B_1a yield reached 5568 ± 111 mg/l by controlling maximal OUR between 15 and 20 mmol/l/h during cell growth phase, which was increased by 21.8% compared with the control (maximal OUR above 20 mmol/l/h). The stimulation effect on avermectin B_1a production could be attributed to the improved supply of propionic acid and acetic acid, the precursors of avermectin B_1a, in the cells. Hence, this OUR control method during cell growth phase may be a simple and applicable way to improve industrial production of avermectin.     

Transformation of cholesterol to testosterone byMycobacterium sp.

Summary The production of testosterone from cholesterol via a single-step microbial transformation process was investigated. A supplement of44% glucose and 1% peptone into a synthetic medium, pH control at 6.0 and continuous feeding of cholesterol were the most important parameters. Under optimal conditions, the maximum molar conversion rate of testosterone from cholesterol was up to 42.68% in 2.5-L surface-aerated fermentor after 100 h cultivation. The reduction of androst-4-ene-3.17-dione, an intermediate product, to testosterone was catalyzed by 17-hydroxysteroid dehydrogenase inMycobacterium sp. Testosterone was isolated from the fermentation broth by the addition of Amberlite XAD-7 resin and was further purified by flash chromatography on a silica gel column. After crystallization in acetone, testosterone could be obtained as needle crystals.     

Improved production of heterologous protein from Streptomyces lividans

Summary Protein-secreting procaryotic host organisms are currently being sought as alternatives to Escherichia coli for recombinant processing. In this study we examined how manipulation of the cultivation conditions can enhance heterologous protein production by Streptomyces lividans. The recombinant S. lividans used in this study expressed and excreted a Flavobacterium enzyme capable of hydrolyzing organophosphates. Initial shake-flask studies demonstrated that supplementing Luria-Bertani medium with moderate amounts of glucose (30 g/l), led to improved enzyme production. In fermentor studies with controlled pH, a further twofold increase in production was observed when glucose was fed continuously as compared to batch cultivation. This improved production in the glucose-fed culture may be related to a reduced accumulation of acids. Continuous feeding of both glucose and tryptone led to a further sixfold increase in production. In addition to enhancing production 25-fold, the efficiency of enzyme production and the specific activity of the excreted enzyme were also improved by glucose and tryptone feeding. These results demonstrate that in addition to genetic manipulations, optimization of cultivation conditions can lead to significant improvements in the production of heterologous proteins from Streptomyces. Offprint requests to: G. F. Payne     

Ethanol evolution rate: a new parameter to determine the feeding rate for the production of avermectins by Streptomyces avermitilis

A new parameter ethanol evolution rate (EER) was developed to aid in the determination of glucose feeding rate in avermectin production. The EER characterized the level of primary metabolism and its value was affected mainly by the supply of O2 and glucose. In an abnormal batch, over-feeding of glucose led to 2.5-fold increase of the maximum EER value compared to the normal one, and the production was thus decreased by nearly 80%. Together with other criteria, the EER helped to control utilization of substrate, so it has been successfully used to control glucose feeding in an industrial process.     

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Characterization of D-ribose biosynthesis in Bacillus subtilis JY200 deficient in transketolase gene

D-Ribose is a functional five-carbon sugar, which has been used for the commercial production of riboflavin. Mechanisms of d-ribose biosynthesis from xylose were investigated in the genetically engineered Bacillus subtilis JY200 with a deficiency in transketolase. A transketolase gene (tkt) disruption cassette in plasmid pUNKC was introduced into the chromosomal tkt gene in the wild type B. subtilis 168. Analysis of culture broth by thin layer chromatography confirmed that the disruption of tkt allowed B. subtilis JY200 to produce d-ribose. In a batch culture of B. subtilis JY200, a loss of cell viability was observed after glucose depletion. Fed-batch cultivation by feeding 400 gl(-1) glucose solution as a co-substrate was carried out to supply energy to xylose metabolism and to maintain cell viability throughout cultivation. Fed-batch cultivation of B. subtilis JY200 in a complex medium containing 11 gl(-1) xylose and 5 gl(-1) glucose initially gave the best result of 10.1 gl(-1)D-ribose concentration, 0.24 gg(-1)D-ribose yield and 0.29 gl(-1)h(-1) productivity, corresponding to 40-, 5- and 12-fold increases compared with those in the batch culture. A kinetic study of D-ribose production in fed-batch cultivations of B. subtilis JY200 suggested that xylose uptake might be critical to maximize D-ribose biosynthesis from xylose.     

Mathematical modeling of citric acid production by repeated batch culture

A mathematical model has been created for the process of citric acid biosynthesis by yeast (mutant strain Yarrowia lipolytica) cultivated by the repeated batch (RB) method on ethanol under conditions of nitrogen limitation. The model accounts for cell growth as a function of nitrogen concentration in the culture liquid; nitrogen uptake by growing cells; citric acid production; pH control in the fermentor by means of NaOH addition; and changes in system volume. The model represents a system of five nonlinear differential equations. Experimental measurements of cell concentration, citric acid concentration, and cultivation broth volume were used with the least squares method to determine the values of eight model parameters. The parameter values obtained were consistent with literature data and general concepts of cell growth and citric acid biosynthesis. The model has been used to predict optimum RB culture conditions.     

An effective and simplified scale-up strategy for acarbose fermentation based on the carbon source control

The scale-up strategy for acarbose fermentation by Actinoplanes sp. A56 was explored in this paper. The results obtained in shake-flask cultivation demonstrated that the ratio of maltose and glucose had significant effects on the biosynthesis of acarbose, and the feeding medium containing 3:1 (mass ratio) of maltose and glucose was favorable for acarbose production. Then the correlation of the carbon source concentration with acarbose production was further investigated in 100-l fermenter, and the results showed that 7.5–8.0 g of total sugar/100 ml and 4.0–4.5 g of reducing sugar/100 ml were optimal for acarbose production. Based on the results in 100-l fermenter, an effective and simplified scale-up strategy was successfully established for acarbose fermentation in a 30-m³ fermenter, by using total sugar and reducing sugar as the scale-up parameter. As a result, 4,327 mg of acarbose/l was obtained at 168 h of fermentation.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

Comparative metabolomics reveals the mechanism of avermectin production enhancement by <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine

It was found that S-adenosylmethionine (SAM) could effectively improve avermectin titer with 30–60 μg/mL addition to FH medium. To clearly elucidate the mechanism of SAM on intracellular metabolites of Streptomyces avermitilis, a GC–MS-based comparative metabolomics approach was carried out. First, 230 intracellular metabolites were identified and 14 of them remarkably influenced avermectin biosynthesis were discriminative biomarkers between non-SAM groups and SAM-treated groups by principal components analysis (PCA) and partial least squares (PLS). Based on further key metabolic pathway analyses, these biomarkers, such as glucose, oxaloacetic acid, fatty acids (in soybean oil), threonine, valine, and leucine, were identified as potentially beneficial precursors and added in medium. Compared with single-precursor feeding, the combined feeding of the precursors and SAM markedly increased the avermectin titer. The co-feeding approach not only directly verified our hypothesis on the mechanism of SAM by comparative metabolomics, but also provided a novel strategy to increase avermectin production.     

Batch and Fed-Batch Cultivation for Excretive Production of Human Epidermal Growth Factor (hEGF) with Recombinant E. Coli K12 System

Abstract

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32°C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Regulation of Amylase Synthesis in Bacillus circulans F-2

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.     

Strategy for the Improvement of Prodigiosin Production by a Serratia marcescens Mutant through Fed-Batch Fermentation

A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’, it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode.     

A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli

AIM: Development and optimization of an efficient and inexpensive medium for succinic acid production by Escherichia coli under anaerobic conditions. METHODS AND RESULTS: Initially, 0.8 gl(-1) of succinic acid was produced in 60 h in 300-ml medium. On optimization, glucose and peptone were replaced by cane molasses and corn steep liquor. Three hundred ml of this medium was inoculated with 4% (v/v) of seed inoculum, incubated at 39 degrees C for 72 h, resulted in 7.1 gl(-1) of succinic acid in 36 h. Scale up in a 10-l fermentor under conditions of controlled pH and continuous CO2 supply in this medium resulted in 17 gl(-1) of succinic acid in 30 h. CONCLUSIONS: A ninefold increase in succinic acid production was obtained in 500-ml anaerobic bottles with optimized medium having cane molasses and corn steep liquor as against initial medium containing glucose and peptone. However, a subsequent scale up in a 10-l fermentor resulted in a 2.5-fold increase in succinic acid production as against optimized medium used in 500-ml anaerobic bottles. SIGNIFICANCE AND IMPACT OF THE STUDY: Succinic acid production was enhanced in medium consisting of inexpensive carbon and nitrogen sources in a shorter span of time.     

Production of cellulase enzymes and hydrolysis of steam-exploded wood

Steam-exploded aspen has been examined as a candidate feedstock for both cellulose production and enzymatic hydrolysis of wood. Batch and fed-batch cultivation methods were evaluated and compared with previous experiments using ball-milled, crystalline cellulose (Solka Floe). Batch cultivation of Trichoderma reesei Rut C-30 on 9 wt% water-washed aspen yielded enzyme productivities and activities comparable to those obtained on Solka Floe (40 FP IU/L-h; 7. 5 FP IU/mL). Fed-batch cultivation of Rut C-30 resulted in higher enzyme productivities and tilers than batch cultivation (50 FP IU/L-h; 15 FP IU/mL). However, the overall enzyme production performance was lower than on Solka Floe at comparable cellulose feeding rates and concentrations. This may be due to the accumulation of steam explosion by-products and lignin in the fermentor.The hydroiysis of water-washed steam-exploded aspen was performed at different enzyme loadings and wood concentrations. Glucose production, using 10 and 15wt% suspension, showed that while glucose concentration increased with wood load, the yield of glucose derived from cellulose decreased. With 10wt% suspensions, it was possible to obtain a cellous conversion to glucose above 95%. Low cellulose levels in the hydrolyzates indicated that the filter paper activity ratios (approximately 1.5), a significant result since the fungus was grown exclusively on wood. mIt also suggested that the observed yield decrease is more likely to be caused by glucose than cellobiose inhibition of the enzymes.     

氮源对阿维菌素合成的影响及基于二氧化碳释放速率的阿维菌素发酵调控

中国是世界上最大的也是唯一的阿维菌素原料生产国,但在工业规模生产中与同类型大环内酯类抗生素相比其产量相对偏低。文中通过研究不同氮源对阿维链霉菌生长、代谢的影响,发现氮源在发酵中后期对菌丝活性、菌丝浓度以及阿维菌素B1a的合成都有较为显著的影响。在100 L生物反应器中,于发酵中后期基于二氧化碳释放速率(CER)控制补入酵母粉,效价达到8697mg/L,与原工艺相比,提高了26.9%。这一结论若在实际工业生产中应用,有望带来实际的经济效益。