Construction and optimization of ergothioneine-producing Escherichia coli / 生物工程学报

Ergothioneine (ERG) is a natural antioxidant that has been widely used in the fields of food, medicine and cosmetics. Compared with traditional plant extraction and chemical synthesis approaches, microbial synthesis of ergothioneine has many advantages, such as the short production cycle and low cost, and thus has attracted intensive attention. In order to engineer an ergothioneine high-yielding Escherichia coli strain, the ergothioneine synthesis gene cluster egtABCDE from Mycobacterium smegmatis and egt1 from Schizosaccharomyces pombe were introduced into E. coli BL21(DE3) to generate a strain E1-A1 harboring the ergothioneine biosynthesis pathway. As a result, (95.58±3.2) mg/L ergothioneine was produced in flask cultures. To further increase ergothioneine yield, the relevant enzymes for biosynthesis of histidine, methionine, and cysteine, the three precursor amino acids of ergothioneine, were overexpressed. Individual overexpression of serAT410STOP and thrA resulted in an ergothioneine titer of (134.83±4.22) mg/L and (130.26±3.34) mg/L, respectively, while co-overexpression of serAT410STOP and thrA increased the production of ergothioneine to (144.97±5.40) mg/L. Eventually, by adopting a fed-batch fermentation strategy in 3 L fermenter, the optimized strain E1-A1-thrA-serA* produced 548.75 mg/L and 710.53 mg/L ergothioneine in glucose inorganic salt medium and rich medium, respectively.

Construction and optimization of cordycepin-producing Saccharomyces cerevisiae / 生物工程学报

Cordycepin is the key active component of medicinal fungus Cordyceps militaris, and it shows multiple functional activities such as anti-tumor and anti-virus. Cordycepin was conventionally produced by liquid fermentation of C. militaris, but the long production cycle and the low productivity constrained its development and application. In this study, two key genes for cordycepin biosynthesis (ScCNS1 and ScCNS2) were introduced into Saccharomyces cerevisiae S288C, producing 67.32 mg/L cordycepin at 240 h. Analysis of gene expression profiles indicated that ZWF1, PRS4, ADE4, ScCNS1 and ScCNS2 which encode enzymes involved in pentose phosphate pathway, purine metabolism and cordycepin biosynthesis pathway, were significantly up-regulated in the late phage of fermentation. Optimization of fermentation medium determined that 50 g/L initial glucose followed by feeding, supplemented with 5 mmol/L Cu²⁺ and 1.0 g/L adenine were the best condition. Fed-batch fermentation using the engineered yeast in a 5 L stirred fermenter produced 137.27 mg/L cordycepin at 144 h, with a productivity up to 0.95 mg/(L·h) reached, which was 240% higher than that of the control.

Improvement of ethanol production from sweet sorghum juice under batch and fed-batch fermentations: effects of sugar levels, nitrogen supplementation, and feeding regimes

Background: Fermentation process development has been very important for efficient ethanol production. Improvement of ethanol production efficiency from sweet sorghum juice (SSJ) under normal gravity (NG, 160 g/L of sugar), high gravity (HG, 200 and 240 g/L of sugar) and very high gravity (VHG, 280 and 320 g/L of sugar) conditions by nutrient supplementation and alternative feeding regimes (batch and fed-batch systems) was investigated using a highly ethanol-tolerant strain, Saccharomyces cerevisiae NP01. Results: In the batch fermentations without yeast extract, HG fermentation at 200 g/L of sugar showed the highest ethanol concentration (PE, 90.0 g/L) and ethanol productivity (QE, 1.25 g/L·h). With yeast extract supplementation (9 g/L), the ethanol production efficiency increased at all sugar concentrations. The highest PE (112.5 g/L) and QE (1.56 g/L·h) were observed with the VHG fermentation at 280 g/L of sugar. In the fed-batch fermentations, two feeding regimes, i.e., stepwise and continuous feedings, were studied at sugar concentrations of 280 g/L. Continuous feeding gave better results with the highest PE and QE of 112.9 g/L and 2.35 g/L·h, respectively, at a feeding time of 9 h and feeding rate of 40 g sugar/h. Conclusions: In the batch fermentation, nitrogen supplementation resulted in 4 to 32 g/L increases in ethanol production, depending on the initial sugar level in the SSJ. Under the VHG condition, with sufficient nitrogen, the fed-batch fermentation with continuous feeding resulted in a similar PE and increased QP by 51% compared to those in the batch fermentation.

Effect of PTS modifications on L-tryptophan production in Escherichia coli / 生物工程学报

L-tryptophan, one of the aromatic amino acids, is widely used in the fields of medicine, food and feed additives. The phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) plays an important role in glucose transport and phosphorylation in Escherichia coli. PTS-mediated regulation dominates the carbohydrates' uptake and metabolism in E. coli. We constructed L-tryptophan-producing bacteria containing two typical PTS mutations (ptsHIcrrglf⁻ glk⁺ and ptsG⁻) by Red homologous recombination system, and studied in 50 L jar fermenter using fed-batch fermentation. Both PTS system mutants had a great impact on the biomass (increasing 47.0% and 17.6%, respectively), L-tryptophan production (increasing 25.9% and 9.4%, respectively), glucose conversion rate (increasing 26.5% and 17.4%, respectively) and byproduct acetic acid generation (slightly increased and decreased，respectively).

Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation. The present work demonstrated the production, purification and characterization of recombinant human antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process. Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration (312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency (99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95 and 94%, respectively. The concentrated solution was further purified by the single step ion exchange chromatography with the recovery yield and purity of 55 and >98%, respectively. The purified rhAT was characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and Liquid chromatography mass spectrometry (LC-MS) analysis. The biological activity of rhAT was analyzed as heparin cofactor to meet the therapeutic grade applications. Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.

User-friendly optimization approach of fed-batch fermentation conditions for the production of iturin A using artificial neural networks and support vector machine

Background In the field of microbial fermentation technology, how to optimize the fermentation conditions is of great crucial for practical applications. Here, we use artificial neural networks (ANNs) and support vector machine (SVM) to offer a series of effective optimization methods for the production of iturin A. The concentration levels of asparagine (Asn), glutamic acid (Glu) and proline (Pro) (mg/L) were set as independent variables, while the iturin A titer (U/mL) was set as dependent variable. General regression neural network (GRNN), multilayer feed-forward neural networks (MLFNs) and the SVM were developed. Comparisons were made among different ANNs and the SVM. Results The GRNN has the lowest RMS error (457.88) and the shortest training time (1 s), with a steady fluctuation during repeated experiments, whereas the MLFNs have comparatively higher RMS errors and longer training times, which have a significant fluctuation with the change of nodes. In terms of the SVM, it also has a relatively low RMS error (466.13), with a short training time (1 s). Conclusion According to the modeling results, the GRNN is considered as the most suitable ANN model for the design of the fed-batch fermentation conditions for the production of iturin A because of its high robustness and precision, and the SVM is also considered as a very suitable alternative model. Under the tolerance of 30%, the prediction accuracies of the GRNN and SVM are both 100% respectively in repeated experiments.

Effect of kLa and Fed-batch Strategies for Enhanced Production of Xylitol by Debaryomyces nepalensis NCYC 3413.

Aims: To evaluate the effect of volumetric oxygen transfer coefficient on the production of xylitol by Debaryomyces nepalensis and to enhance the yield and productivity of xylitol by fed-batch fermentation using xylose as substrate. Place and Duration of Study: All experiments were performed at the Applied and Industrial Microbiology Laboratory, Indian Institute of Technology Madras, from March 2013 to May 2014. Methodology: Batch cultivations were carried out in a 7.5 L fermentor under various oxygen transfer coefficients in the range 12 to 39.6 h-1 in order to understand the effect of oxygen on xylitol production. Fed-batch studies were performed in 2.5 L bioreactor with a working volume of 1 L. The cultures were initially grown as batch cultures. Feed containing xylose and nitrogen source was added to the medium intermittently. Samples were periodically collected at regular intervals of time and the concentrations of xylose, xylitol and glycerol were determined by HPLC. Results: Maximal xylitol yield (0.64 g/g) and productivity (0.43 g/L·h) were obtained at kLa 13.68 h-1. The effect of pH was also studied at this kLa. A pH value of 6.0 was found to be favorable for xylitol accumulation. Fed-batch fermentation involving feeding of xylose and nitrogen source was used for xylitol production by D. nepalensis. Within the fed-batch phase, the yield of xylitol was 0.83 g/g and the productivity was increased to 0.83 g/L.h with a final product concentration of 90 g/L. Conclusion: Higher kLa favors biomass production whereas product formation was favored at lower kLa. Fed-batch process resulted in enhancement of final product concentration by 73%.

Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product

Background: Recombinant proteins, including antibodies and antibody fragments, often contain disulfide bond bridges that are necessary for their folding, stability and function. Production of disulfide-bond-containing proteins in the periplasm of Escherichia coli has been very useful, due to unique characteristics of the periplasm, for obtaining fully active and correctly folded products and for alleviating downstream processing. Results: In this study, fed-batch cultivation of Escherichia coli (E. coli) for production of Fab D1.3, which is an anti-hen egg white lysozyme (HEWL) antibody fragment was carried out at 37ºC, and the bacterial cells were induced by adding 0.1 mM IPTG to the culture medium. Fermentor was sampled over the course of fermentation; the bacterial cells were centrifugally separated from the culture broth and subjected to osmotic shock (with excluding HEWL) and sonication procedures. The resulting fractions were analysed for Fab using a combination of ELISA, SDS-PAGE and Western blotting and changes in product titre, location, and form was assessed throughout growth. It was shown that osmotic shock released the Fab from the periplasm very efficiently and its efficacy was 20-45% more than sonication. This study demonstrates that, at high cell density cultivation in fermentor, target product can appear inside and outside the cells, depending on the time of induction. The maximum amount of Fab (47 mg/l) in the periplasm was reached at 14 hrs cultivation (4 hrs post induction), being suitable time for cell harvest, selective periplasmic extraction and downstream capture. The Fab increasingly leaked into the culture medium, and reached its maximum culture medium titre of ~78 mg/l after 6 hrs post induction. After 16 hrs cultivation (6 hrs post induction) the amount of Fab remained constant in different locations within and outside the cells. Western blot analysis of cell fractions showed that certain amount of the Fab was also produced in the cells as insoluble form. Conclusions: In this work we showed that the production of Fab in the periplasm during high cell density cultivation of E. coli in fermentor can be challenging as the product may appear in various locations within and outside the cells. To exploit the advantages of the periplasmic expression systems for purification in downstream processing, bacterial cells should be harvested when they maintain the majority of the target protein in their periplasmic space (i.e. 4 hrs post induction).

Production of pullulan by a thermotolerant Aureobasidium pullulans strain in non-stirred fed batch fermentation process

Total 95 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 11 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RG-5, produced highest pullulan (37.1±1.0 g/l) at 42ºC, pH 5.5 in 48h of incubation with 3% sucrose and 0.5% ammonium sulphate in a non-stirred fed batch fermentor of 6 liters capacity. The two liters of initial volume of fermentation medium was further fed with the 2 liters in two successive batches at 5 h interval into the fermentor. The sterile air was supplied only for 10h at the rate of 0.5 vvm.

Enhance 1, 3-propanediol production from crude glycerol in batch and fed-batch fermentation with two-phase pH-controlled strategy

The batch fermentation of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae SU6 at different crude glycerol concentration (40-100 g l-1), pH (6.5-7.5) and temperature (31-40ºC) combined with two-phase pH-controlled strategy was investigated. Effect of feeding rate (0.10-0.15 L h-1) was studied in fed-batch fermentation. In batch fermentation, the optimal condition was 60 g l-1 crude glycerol, pH control at 6.5 and cultivation temperature at 37ºC. The maximum 1,3-PD of 20 g l-1, the yield of 0.34 g 1,3-PD g-1 glycerol consumed and the productivity of 1.25 g l-1 h-1 were achieved at 16 hrs cultivation. The by-products were acetic acid and succinic acid at 2.7 and 1.1 g l-1, respectively. Two-phase pH-controlled strategy gave better results (24.95 g l-1 1,3-PD and 1.78 g l-1 h-1 productivity) than constant pH-controlled strategy (20 g l-1 and 1.25 g l-1 h-1, respectively) at 16 hrs incubation. In fed-batch fermentation, the maximum 1,3-PD of 45.35 g l-1 was achieved at constant feeding rate of 0.1 L h-1. The yield and productivity were 0.44 g g-1 and 1.94 g l-1 h-1, respectively. The fed-batch fermentation with constant feeding at 0.1 L h-1 with two-phase pH-controlled strategy gave 2.2 folds higher 1,3 PD concentration than the batch fermentation with two-phase pH-controlled strategy. This demonstrated the great impact of combination of pH control and feeding strategies in fed-batch fermentation on enhancing 1,3-propanediol production.

Calculation of fermentation parameters from the results of a fed-batch test taking account of the volume of biomass in the fermenting medium

The volume of biomass in the fermenting medium may significantly affect the values of fermentation parameters calculated from the measured concentrations of the substrates and/or products. The corrections proposed in this paper should be evaluated and, depending on their magnitude, considered in order to obtain more representative results. A numerical example is presented.

O volume da biomassa no meio em fermentação pode afetar significativamente os parâmetros do processo calculados a partir das medidas das concentrações de substratos e/ou produtos. Correções propostas neste trabalho deveriam ser avaliadas e, dependendo de seus valores, consideradas a fim de obter resultados mais representativos. Apresenta-se um exemplo numérico.

High Cell Density Culture of Lactobacillus plantarum Lp-2 / 中国生物工程杂志

High cell density culture of Lactobacillus plantaru is a significant step of the preparation of lactic acid bacteria starter.Firstly,this experiment is designed to analyze the effects of different dissolved oxygen and pH on the batch fermentation of Lactobacillus plantarum.And then,on the base of the batch fermentation,fed-batch fermentation was studied,companied by chemic neutralization in the experiment to achieve the aim of high cell density cultivation.Finally,in the comparison of two kinds of feeding mode for fed-batch operation in 10L fermentor,it was demonstrated that two kinds of feeding mode made cell concentration increase differently,in which pH feedback feeding can control sucrose concentration within a certain lower range,therefore obtaining the maximum cell concentration.With the pH feedback feeding,the dry cell weight was up to 13.56g/L,which increased 90.05% over that of batch culture.