Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Effects of agitation and aeration on the production of extracellular glucose oxidase from a recombinant Saccharomycescerevisiae

A recombinant strain of Saccharomyces cerevisiae, GAL-GO2, was developed to facilitate the production of extracellular glucose oxidase (GOD). The recombinant strain secreted 85% (8.7?U/ml) of the total GOD (10.3?U/ml) produced in shake flask culture. For further enhancement of GOD production, optimization of the speed of agitation and the rate of aeration in a stirred tank fermentor was carried out. Response surface methodology with appropriate statistical experimental design was employed for this purpose. The maximal level of extracellular GOD was achieved when the speed of agitation and the rate of aeration were 420?rpm and 0.25?vvm, respectively. The enzyme production was increased by 74% compared to the level obtained under unoptimized conditions.     

Kojic acid production byAspergillus flavus using gelatinized and hydrolyzed sago starch as carbon sources

Direct conversion of gelatinized sago starch into kojic acid byAspergillus flavus strain having amylolytic enzymes was carried out at two different scales of submerged batch fermentation in a 250-mL shake flask and in a 50-L stirred-tank fermentor. For comparison, fermentations were also carried out using glucose and glucose hydrolyzate from enzymic hydrolysis of sago starch as carbon sources. During kojic acid fermentation of starch, starch was first hydrolyzed to glucose by the action of α-amylase and glucoamylase during active growth phase. The glucose remaining during the production phase (non-growing phase) was then converted to kojic acid. Kojic acid production (23.5g/L) using 100 g/L sago starch in a shake flask was comparable to fermentation of glucose (31.5 g/L) and glucose hydrolyzate (27.9 g/L) but in the 50-L fermentor was greatly reduced due to non-optimal aeration conditions. Kojic acid production using glucose was higher in the 50-L fermentor than in the shake flask.     

Effect of agitation and aeration on the production of nitrile hydratase by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor

Aims: To evaluate the effect of different physicochemical parameters such as agitation, aeration and pH on the growth and nitrile hydratase production by Rhodococcus erythropolis MTCC 1526 in a stirred tank reactor. Methods and Results: Rhodococcus erythropolis MTCC 1526 was grown in 7‐l reactor at different agitation, aeration and controlled pH. The optimum conditions for batch cultivation in the reactor were an agitation rate of 200 rev min^?1, aeration 0·5 v/v/m at controlled pH 8. In this condition, the increase in nitrile hydratase activity was almost threefold compared to that in the shake flask. Conclusion: Agitation and aeration rate affected the dissolved‐oxygen concentration in the reactor which in turn affected the growth and enzyme production. Significance and Impact of the Study: Cultivation of R. erythropolis MTCC 1526 in the reactor was found to have significant effect on the growth and nitrile hydratase production when compared to the shake flask.     

Implementation of a transhydrogenase-like shunt to counter redox imbalance during xylose fermentation in Saccharomyces cerevisiae

Three enzymes responsible for the transhydrogenase-like shunt, including malic enzyme (encoded by MAE1), malate dehydrogenase (MDH2), and pyruvate carboxylase (PYC2), were overexpressed to regulate the redox state in xylose-fermenting recombinant Saccharomyces cerevisiae. The YPH499XU/MAE1 strain was constructed by overexpressing native Mae1p in the YPH499XU strain expressing xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis, and native xylulokinase. Analysis of the xylose fermentation profile under semi-anaerobic conditions revealed that the ethanol yield in the YPH499XU/MAE1 strain (0.38?±?0.01 g g^?1 xylose consumed) was improved from that of the control strain (0.31?±?0.01 g g^?1 xylose consumed). Reduced xylitol production was also observed in YPH499XU/MAE1, suggesting that the redox balance was altered by Mae1p overexpression. Analysis of intracellular metabolites showed that the redox imbalance during xylose fermentation was partly relieved in the transformant. The specific ethanol production rate in the YPH499XU/MAE1–MDH2 strain was 1.25-fold higher than that of YPH499XU/MAE1 due to the additional overexpression of Mdh2p, whereas the ethanol yield was identical to that of YPH499XU/MAE1. The specific xylose consumption rate was drastically increased in the YPH499XU/MAE1–MDH2–PYC2 strain. However, poor ethanol yield as well as increased production of xylitol was observed. These results demonstrate that the transhydrogenase function implemented in S. cerevisiae can regulate the redox state of yeast cells.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

代谢工程改造大肠杆菌合成丙二酸

丙二酸是一种重要的有机二元羧酸,其应用价值遍及化工、医药、食品等领域。本文以大肠杆菌为底盘细胞,过表达了ppc、aspC、panD、pa0132、yneI和pyc基因,成功构建了丙二酸合成重组菌株大肠杆菌BL21(TPP)。该菌株在摇瓶发酵条件下,丙二酸产量达到0.61 g/L。在5 L发酵罐水平,采用间歇补料的方式丙二酸的积累量达3.32 g/L。本研究应用了融合蛋白技术,将ppc和aspC、pa0132和yneI分别进行融合表达,构建了工程菌BL21(SCR)。在摇瓶发酵水平,该菌株丙二酸的积累量达到了0.83 g/L,较出发菌株BL21(TPP)提高了36%。在5 L发酵罐中,工程菌BL21(SCR)的丙二酸产量最高达5.61 g/L,较出发菌株BL21(TPP)提高了69%。本研究实现了丙二酸在大肠杆菌中的生物合成,为构建丙二酸合成的细胞工厂提供了理论依据和技术基础,同时也对其他二元羧酸的生物合成具有启发和指导意义。     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 under submerged fermentation conditions

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran–3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4°C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70°C, which will be useful for its spray drying.     

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d ‐xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro‐organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499‐SsXR‐AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a β‐d ‐glucosidase (Aspergillus aculeatus β‐glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5‐fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499‐XR‐ScMAL11‐BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.     

Improving the production of S-adenosyl-L-methionine in Escherichia coli by overexpressing metk

S-adenosyl-L-methionine (SAM) has important applications in many fields including chemical therapy and pharmaceutical industry. In this study, the recombinant Escherichia coli strain was constructed for effective production of SAM by introducing the SAM synthase gene (metK). This strain produced 34.5?mg/L of SAM in basic medium in shake flask. Yeast extract, pH, and loaded volume had a significant positive effect on the yield of SAM. Their optimal values were 35?g/L, 7.5, and 30?mL, respectively. The final conditions optimized were as follows: glucose 20, g/L; peptone, 40?g/L; yeast extract, 35?g/L; NaCl, 10?g/L; MgSO₄, 1.2?g/L; L-methionine, 1?g/L; rotate speed, 220?rpm; loaded volume, 30?mL; inoculation, 1%; temperature, 37°C; and initial medium, pH 7.5. The recombinant strain produced 128.2?mg/L of SAM under the above conditions in shake flask. The production of SAM in a 5?L fermentor was also investigated. The maximal biomass of the recombinant strain was 60.4?g/L after the cells were cultured for 20?hr, and the highest yield of SAM was 300.9?mg/L after induction for 8?hr in a 5?L fermentor. This study provides a good foundation for the future production and use of SAM.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol‐3‐phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4‐fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC‐TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4‐fold compared with 3.6% for the wild‐type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3‐fold improvement, compared with 3.6% for the wild‐type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343–347. © 2012 Wiley Periodicals, Inc.     

Effect of aeration and agitation on synthesis of poly (γ-glutamic acid) in batch cultures of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NCIM 2324

The effects of aeration and agitation on the production and molecular weight of poly (γ-glutamic acid) (PGA) were systematically investigated in batch fermentor cultures of Bacillus licheniformis NCIM 2324. A high aeration rate and agitation speed enhanced the growth of B. licheniformis NCIM 2324, but did not always lead to high PGA production. Additionally, PGA production actually decreased at very high aeration rates and agitation speeds. The maximum PGA concentration was obtained at 750 rpm and 1 vvm. Rheological studies revealed that fermentation broth during production of PGA exhibited pseudoplastic behavior. The effects of aeration and agitation on the molecular weight of PGA were also studied, and the rate and extent of the decrease in the molecular weight of PGA as a function of time were found to be much greater at high aeration than low aeration. The PGA production of 46.34 g/L with a specific productivity of 0.17 g-PGA/g-biomass/ h and a PGA yield of 0.48 with respect to total substrate observed in the present study are much higher than the values reported in previously conducted studies.     

酿酒酵母中双链RNA介导基因沉默技术研究GRE3基因表达抑制

RNA沉默技术作为探索基因功能的实验手段应用于多种生物. 以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象，检测酿酒酵母双链RNA介导的基因沉默效应. 以pESC-LEU为骨架，构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499. 用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调. 结果表明，双链RNA介导的基因沉默技术，能够用作降低酿酒酵母某一特定基因表达水平的工具. 并有助于理解芽殖酵母的RNA干扰现象.     

Oxygen transfer and mycelial growth in a tubular loop fermentor

A 22 m long. 20 liter tubular loop fermentor (TLF) has been tested for oxygen transfer characteristics and as a reactor for mycelial growth. Model calculations show that the flow pressure drop has an important influence on the axial oxygen profiles. A design model that accounts for this influence is presented. Using the model, K_L a values are calculated from the results of sulfite oxidation experiments. These are correlated with power consumption and aeration rates. The K_L a dependence on aeration rate was found to be less than found with tank reactors. The growth kinetics of three metabolite-producing mycelial organisms in the TLF are presented: a Streptomyces, a Fusarium, and a Acrophialophora. In order to determine the influence of reactor type on the growth and product formation, these cultures have been grown in tanks and shake flasks. The antibiotic, product spectrum of Streptomyces is compared on the basis of inhibition tests and it is shown that the distribution of products is reactor dependent. The Fusarium culture produced a previously unknown metabolite, whose concentration in the loop fermentor was four times higher than in a shake flask. The Acrophialophora culture grew twice as fast in the loop fermentor, but produced essentially none of the specific product. Power Consumptions of up to 8 kW/m³ in the tubular fermentor did not appear to harm the mycelia.     

STATISTICAL OPTIMIZATION OF GREEN FLUORESCENT PROTEIN PRODUCTION FROM Escherichia coli BL21(DE3)

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box–Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD_600nm) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.     

Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase

An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.