Identification and fermentation optimization of protopectinase-overproducing strain Aspergillus niger CD-01 for pectin production

In order to solve the citrus peel resource waste problem protopectinase-overproducing strain CD-01 for pectin production and minimize the drawbacks of chemical extraction of pectin, a was isolated from a pit soil dumped with perished orange in Changde City, Hunan Province of China. The strain CD-01 had the same morphology and 28S rRNA gene sequence （FJ184995） as that of Aspergillus niger （ATCC 64028）. It was thus identified and named as Aspergillus niger CD-01. The fermentation condition was optimized based on L9（34） orthogonal experimental design and the variances analyses. The results show that the optimal condition for producing pectin is as follows： time 36 h, temperature 35 ℃, pH 5, and urea as the nitrogen source. Under this condition, the pectin yield can reach up to 24.5%. This shows a great potential of Aspergillus niger CD-01 in pectin extraction from citrus.

Optimization of Fermentation Medium for Epothilones Production with Sequential Statistical Approach

A sequential statistical approach was applied to optimizing the fermentation medium of epothilones（Epos） production by means of a mutant which was obtained by treating polyangium cellulosum ATCC 15384 with nitrite and ultraviolet. The effects of different carbon sources and nitrogen sources on the fermentation medium were tested, and the suitable ones were selected. Then a uniform design was employed to design the experiments. A linear model was developed for identifying the significant components in fermentation medium, while a third degree polynomial model was used for studying the relationship between the concentration of the components in fermentation medium and the yield of Epos（YEPs）. A pattern search method was used for searching the optimum fermentation medium in the test space, which was as follows（g/L）： potassium nitrate 8.00, soybean peptone 17.60, potassium hydrogen phos- phate 1.00, beef extraction 6.46, yeast extraction 1.00, calcium chloride 0.25, sodium chloride 1.00 and ferric chloride 0.02. The optimum fermentation medium was expected to result in a yield of Epos（YEPs） of 2.48 mg/L. The validation experiments with the optimum medium were performed in triplicate and the average yield of Epos was 2.45 mg/L which was 7.78 times higher than that of Epos prepared without optimization.

Isolation of Cordyceps ophioglossoides L2 from Fruit Body and Optimization of Fermentation Conditions for Its Mycelial Growth

A strain isolated from the fruiting body of a fungus parasitized on Elaphomyces was identified as Cordyceps ophioglossoides based on the morphological characteristics and the analysis of ITS-5.8s rDNA sequence. The optimal medium, composition （g·L^-1）, containing sucrose 66.0, yeast powder 10.0, silkworm chrysalises digest 30.0, MgSO4· 7H2O 0.4, and KH2PO4 0.4, Was found using fractional factorial design ancl a central composite design, and the optimization of cultural conditions obtained a result of seed age 6 days, inoculum size 6% （by volume）, initial pH 5.6, temperature 24℃, shaking speed 160 ·＇min^-1 by one-factor-at-a-time method. The maximum biomass reached about 20.2 g·L^-1 after 90 hours culture under the optimal conditions. Elementary nharmaeclogical actlwtties showed that mycelia of C. ophioglossoides L2 from submerged culture promoted Uterus growth in estrogen- depleted mice. In the 15-litre scale-up fermentation, the mycelial biomass was around 19.1 g·L^-1, indicating a promising prospect for this biotechnoloagy and the potency to develoo its medical value.

Optimization of rice wine fermentation process based on the simultaneous saccharification and fermentation kinetic model

Chinese rice wine making is a typical simultaneous saccharification and fermentation （SSF） process. During the fermentation process, temperature is one of the key parameters which decide the quality of Chinese rice wine. To optimize the SSF process for Chinese rice wine brewing, the effects of temperature on the kinetic parameters of yeast growth and ethanol production at various temperatures were determined in batch cultures using a mathematical model. The kinetic parameters as a function of temperature were evaluated using the software Origin8.0. Combing these functions with the mathematical model, an appropriate form of the model equations for the SSF considering the effects of temperature were developed. The kinetic parameters were found to fit the experimental data satisfactorily with the developed temperature-dependent model. The temperature profile for maximizing the ethanol production for rice wine fermentation was determined by genetic algorithm. The optimum temperature profile began at a low temperature of 26℃ up to 30 h. The operating temperature increased rapidly to 31.9 ℃, and then decreased slowly to 18℃ at 65 h. Thereafter, the temperature was maintained at 18 ℃ until the end of fermentation. A maximum ethanol production of 89.3 g.L 1 was attained. Conceivably, our model would facilitate the improvement of Chinese rice wine production at the industrial scale.

Optimization of Fermentation Process for the Production of Bacteriocins from Lactic Acid Bacteria

[Objective]This study aimed to improve the yield of bacteriocins from lactic acid bacteria by optimizing the fermentation process for production of bacteriocins from lactic acid bacteria.[Method]By single-factor analysis,fermentation temperature,seed age,inoculation volume,fermentation duration and fermentation media p H were optimized to determine the best fermentation process.The inhibitory zone of bacteriocins from lactic acid bacteria was analyzed with oxford cup method,based on which the fermentation process was evaluated.[Result]The optimal fermentation process was optimized：fermentation temperature 37℃,seed age 14 h,inoculation volume 2%,fermentation duration 48 h,fermentation media p H 5.0.[Conclusion]Under the optimized fermentation conditions,the yield of bacteriocins from lactic acid bacteria was improved significantly.

Fermentation Performance and Characterization of Cold-Adapted Lipase Produced with Pseudomonas Lip35

Strain of Pseudomonas Lip35 producing lipase was isolated in a refrigerator. Lipase production and characterization of this strain were investigated under different conditions. The Pseudomonas was cultivated in shaking flasks in a fermentation medium in various nutritional and physical environments. Lipase production has been influenced by the presence of yeast-extract, soybean powder, NaCI, and Tween-80. Maximum lipase productivity was obtained when the physical environment of the fermentation medium was optimal for 67 h. The production of lipase reached 58.9 U·mL^-1. The lipase of Pseudomonas Lip35 can be considered to be inducible, but the inducer had little influence on the production of lipase. The lipase was characterized and showed high lipolytic activity from pH 7.5-8.0. The optimum temperature was observed at 20℃ and the thermal inactivation of lipase was obvious at 60℃. The lipase activity was inhibited by K＋, stimulated by Ca^2＋, and thermostability decreased in the presence of Ca^2＋, therefore the lipase was Ca^2＋ -dependent cold-adapted enzyme.

Reduction of foaming and enhancement of ascomycin production in rational Streptomyces hygroscopicus fermentation

Foaming reduces the working volume and limits the biosynthesis of macrolide immunosuppressant ascomycin(FK520) in the batch fermentation process of Streptomyces hygroscopicus FS-35 in a 7.5 L bioreactor. To find the relation between FK520 production and foaming, effects of 10 fermentation parameters including organic acids and membrane permeability were investigated. The results suggest that acetate accumulation caused by short period oxygen de ficiency and fast consumption of glucose is the reason for increased foaming and declined FK520 production. Therefore, a fed-batch fermentation strategy was developed to reduce the accumulation of acetate. After optimization, the maximum acetate concentration dropped from 320 mg·L-1to 157 mg·L-1, decreased by 50.8%, and the maximum foam height reduced from 5.32 cm to 3.74 cm, decreased by 29.7%, while the maximum FK520 production increased from 375 mg·L-1to 421 mg·L-1, improved by 12%.

Study on Optimal Fermentation and Storage Time of Soybean Food （Thua-Nao）in Thailand

The soybean cultivar Tadang Muangpai was used to improve the productive quality of Thua-Nao and reduce the concentration of aflatoxin to less than 20 ppb. It was conducted at CMFCRC, Chiangmai, Thailand between Dec. 2006 and Mar. 2007. Soybean was boiled for 5 hours and then fermented at different time to create natural bacterial species, mainly Bacillus spp. Thua-Nao could be stored up to 90 days after in storage. Nutritional value, food value, and microorganisms content were investigated during fermentation and storage. Also, aflatoxin content of Thua-Nao was recorded during storage. The results showed that 3 days of soybean fermentation gave the best performance of Thua-Nao in term of nutritional value （protein = 47.12%）, food value, and content of Bacillus spp. （2.78 × 10^9 CFU/g）. Without being harmed from aflatoxin, Thua-Nao could be stored not more than 23 days in normal room （Tmax. = 33.9 ℃, Tmin. = 15.8 ℃） and not more than 36 days in climate-controlled room （Tmax. = 20 ℃, Tmin. = 15 ℃）.

Coupled strategy based on regulator manipulation and medium optimization empowers the biosynthetic overproduction of lincomycin

The biosynthesis of bioactive secondary metabolites,specifically antibiotics,is of great scientific and economic importance.The control of antibiotic production typically involves different processes and molecular mechanism.Despite numerous efforts to improve antibiotic yields,joint engineering strategies for combining genetic manipulation with fermentation optimization remain finite.Lincomycin A(Lin-A),a lincosamide antibiotic,is industrially fermented by Streptomyces lincolnensis.Herein,the leucine-responsive regulatory protein(Lrp)-type regulator SLCG_4846 was confirmed to directly inhibit the lincomycin biosynthesis,whereas indirectly controlled the transcription of SLCG_2919,the first reported repressor in S.lincolnensis.Inactivation of SLCG_4846 in the high-yield S.lincolnensis LA219X(LA219XΔ4846)increases the Lin-A production and deletion of SLCG_2919 in LA219XΔ4846 exhibits superimposed yield increment.Given the effect of the double deletion on cellular primary metabolism of S.lincolnensis,Plackett-Burman design,steepest ascent and response surface methodologies were utilized and employed to optimize the seed medium of this double mutant in shake flask,and Lin-A yield using optimal seed medium was significantly increased over the control.Above strategies were performed in a 15-L fermenter.The maximal yield of Lin-A in LA219XΔ4846-2919 reached 6.56 g/L at 216 h,55.1%higher than that in LA219X at the parental cultivation(4.23 g/L).This study not only showcases the potential of this strategy to boost lincomycin production,but also could empower the development of high-performance actinomycetes for other antibiotics.

Optimizing production ofasperolide A,a potential anti-tumor tetranorditerpenoid originally produced by the algal-derived endophytic fungus Aspergillus wentii EN-48

The marine algal-derived endophytic fungus Aspergillus wentii EN-48 produces the potential anti-tumor agent asperolide A, a tetranorlabdane diterpenoid active against lung cancer. However, the fermentation yield of asperolide A was very low and only produced in static cultures. Static fermentation conditions of A. wentii EN-48 were optimized employing response surface methodology to enhance the production of asperolide A. The optimized conditions resulted in a 13.9-fold yield enhancement, which matched the predicted value, and the optimized conditions were successfully used in scale-up fermentation for the production of asperolide A. Exogenous addition of plant hormones （especially 10 pmol/L methyl jasmonate） stimulated asperolide A production. To our knowledge, this is first optimized production of an asperolide by a marine-derived fungus. The optimization is effective and valuable to supply material for further anti-tumor mechanism studies and preclinical evaluation of asperolide A and other norditerpenoids.

Statistical culture-based strategies to enhance chlamydospore production by Trichoderma harzianum SH2303 in liquid fermentation

Trichoderma-based formulations are applied as commercial biocontrol agents for soil-borne plant path- ogens. Chlamydospores are active propagules in Trichoderma spp., but their production is currently limited due to a lack of optimal liquid fermentation technology. In this study, we explored response surface methodologies for opti- mizing fermentation technology in Trichoderma SH2303. Our initial studies, using the Plackett-Burman design, iden- tified cornmeal, glycerol, and initial pH levels as the most significant factors （P〈0.05） for enhancing the production of chlamydospores. Subsequently, we applied the Box-Behnken design to study the interactions between, and optimal levels of, a number of factors in chlamydospore production. These statistically predicted results indicated that the highest number of chlamydospores （3.6×108 spores/ml） would be obtained under the following condition： corn flour 62.86 g/L, glycerol 7.54 ml/L, pH 4.17, and 6-d incubation in liquid fermentation. We validated these predicted values via three repeated experiments using the optimal culture and achieved maximum chlamydospores of 4.5×108 spores/ml, which approximately a 8-fold increase in the number of chlamydospores produced by T. harzianurn SH2303 compared with that before optimization. These optimized values could help make chlamydospore production cost-efficient in the future development of novel biocontrol agents.

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of theβ-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose.However,extracellular lactase production was still restricted to the process of cell lysis.In this study,lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside.The purified enzyme was identified as a neutral lactase with an optimum pH of 9.To facilitate extracellular production of lactase,a putative mannoprotein KLLA0_E01057g of K.lactis was knocked out.It could effectively promote cell wall degradation and lactase production after lyticase treatment,which showed potential on other extracellular enzyme preparation.After optimizing the fermentation conditions,the lactase yield from mannoprotein-deficient K.lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Engineering Komagataella phaffii to biosynthesize cordycepin from methanol which drives global metabolic alterations at the transcription level

Cordycepin has the potential to be an alternative to the disputed herbicide glyphosate.However,current laborious and time-consuming production strategies at low yields based on Cordyceps militaris lead to extremely high cost and restrict its application in the field of agriculture.In this study,Komagataella phaffii(syn.Pichia pastoris)was engineered to biosynthesize cordycepin from methanol,which could be converted from CO_(2).Combined with fermentation optimization,cordycepin content in broth reached as high as 2.68±0.04 g/L within 168 h,around 15.95 mg/(L⋅h)in productivity.Additionally,a deaminated product of cordycepin was identified at neutral or weakly alkaline starting pH during fermentation.Transcriptome analysis found the yeast producing cordycepin was experiencing severe inhibition in methanol assimilation and peroxisome biogenesis,responsible for delayed growth and decreased carbon flux to pentose phosphate pathway(PPP)which led to lack of precursor supply.Amino acid interconversion and disruption in RNA metabolism were also due to accumu-lation of cordycepin.The study provided a unique platform for the manufacture of cordycepin based on the emerging non-conventional yeast and gave practical strategies for further optimization of the microbial cell factory.

Combinatorial mutagenesis of Bacillus amyloliquefaciens for efficient production of protease

As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γ irradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.

Genomics-guided discovery of a new and significantly better source of anticancer natural drug FK228

FK228 is an FDA-approved anticancer drug naturally produced by Chromobacterium violaceum No.968 up to 19 mg/L in a pilot industry-scale batch fermentation.Here we report a genomics-guided discovery of Burkholderia thailandensis MSMB43 as a new and significantly better source of FK228.The genome of B.thailandensis MSMB43 was found to contain a functional biosynthetic gene cluster highly homologous to that of FK228 in C.violaceum No.968,and the bacterium indeed produces authentic FK228.By simple fermentation in shaking flasks in a preferred M8 medium,B.thailandensis MSMB43 produced FK228 up to 67.7 mg/L;by fedbatch fermentation in a 20-L fermentor in M8 medium,B.thailandensis MSMB43 produced FK228 up to 115.9 mg/L,which is 95 fold higher than that of C.violaceum No.968 under the same laboratory fermentation conditions.RT-PCR analysis indicated that the high FK228 yield of B.thailandensis MSMB43 was due to high expression of biosynthetic genes,represented by Bth_depA,during the fermentation process.Further genetic manipulation resulted in a recombinant strain,B.thailandensis MSMB43/pBMTL3-tdpR,which harbors a broad host-range vector expressing the thailandepsin biosynthetic pathway regulatory gene tdpR.This engineered strain produced up to 168.5 mg/L of FK228 in fed-batch fermentation in a 20-L fermentor in M8 medium.Therefore,the wild-type B.thailandensis MSMB43 or its engineered derivative could potentially be a good starting point for an industrial process to improve FK228 production for its expanding use in therapy.

Constitutive expression of codon optimized Trichoderma reesei TrCel5A in Pichia pastoris using GAP promoter

To address the deficient activity of TrCel5A in naturally secreted cellulase preparation,this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris.A recombinant TrCel5A was screened out after gene optimization,synthesis,and expression.The biochemical and enzymatic properties of the new recombinant were characterized.As a result,optimization of shake-flask fermentation of the recombinant was obtained at 28℃,2%inoculum volume,an initial pH of 6.0,as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L,respectively.Under the above-optimized conditions,the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation.To further enhance enzyme production,pilot-scale cultivation was evaluated using 5-L bioreactors.Using high-cell-density fermentation,the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L.In addition,the kinetic factors,including K_(m) and V_(max) values for TrCel5A,were detected to be 5.1 mg/mL and 265.9μmol/(min.mg),respectively.Thus,TrCel5A was effectively expressed in P.pastoris under the GAP promoter,and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.