Construction of spore mutants of Bacillus subtilis for the development as a host for foreign protein production

For the development of Bacillus subtilis as a host for foreign protein synthesis, three types of sigma factor deleted mutants (spoIIAC, spoIIIG and spoIIIC) were constructed by antibiotic marker insertion using plasmid vector-mediated method or LFH (Long Flanking Homology)-PCR. Mother cell specific sigma factor mutants of B. subtilis (^K), B. subtilis DB104 spoIIIC (km ^r)::pMK101, had two to three times higher subtilisin activity than the wild type DB104::pMK101. Subtilisin expression by the other two mutants, B. subtilis DB104 spoIIAC (km ^r)::pMK101 and DB104 spoIIIG (km ^r)::pMK101, which are pre-spore specific sigma factor (^F and ^G) deleted strains, was similar to, or less than that of the wild type.     

用遗传工程的方法构建一个分泌型高表达的枯草杆菌碱性蛋白酶E(Subtilisin E)的枯草杆菌质粒-宿主系统

将克隆了枯草杆菌蛋白酶E基因aprE的枯草杆菌质粒pPZW101和一个组成型強启动子Psk连接构建成质粒pPZW102,转入碱性和中性蛋白酶缺失枯草杆菌DB104并进行表达。此质粒-宿主系统具有Subtilisin E高表达和外分泌的特点。     

Physiological and genetic strategies for enhanced subtilisin production by Bacillus subtilis.

Defined minimal media conditions were used to assess and subsequently enhance the production of subtilisin by genetically characterized Bacillus subtilis strains. Subtilisin production was initiated by the exhaustion or limitation of ammonium in batch and fed-batch cultures. Expression of the subtilisin gene (aprE) was monitored with a chromosomal aprE::lacZ gene fusion. The beta-galactosidase production driven by this fusion reflected subtilisin accumulation in the culture medium. Subtilisin gene expression was temporally extended in sporulation-deficient strains (spoIIG), relative to co-genic sporogenous strains, resulting in enhanced subtilisin production. Ammonium exhaustion not only triggered subtilisin production in asporogenous spoIIG mutants but also shifted carbon metabolism from acetate production to acetate uptake and resulted in the formation of multiple septa in a significant fraction of the cell population. Fed-batch culture techniques, employing the spoIIG strain, were investigated as a means to further extend subtilisin production. The constant provision of ammonium resulted in linear growth, with doubling times of 11 and 36 h in each of two independent experiments. At the lower growth rate, the responses elicited (subtilisin production, glucose metabolism, and morphological changes) during the feeding regime closely approximated the ammonium starvation response, while at the higher growth rate a partial starvation response was observed.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

Multi-stage high cell continuous fermentation for high productivity and titer

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.     

Production of excreted human epidermal growth factor (hEGF) by an efficient recombinant Escherichia coli system

Recombinant Escherichia coli JM101 strains harbouring plasmids pWKW2 or lacUV5par8EGF, both encoding human epidermal growth factor (hEGF), were used in fermentations to optimize levels of excreted hEGF. Medium composition, inducer level, growth stage at induction and culture conditions, were optimized with respect to volumetric production of the recombinant protein. MMBL medium, with glucose at 5 g/l and tryptone as nitrogen source, was chosen. Isopropyl-β- -thiogalactopyranoside(IPTG) concentrations of 0.1 mM for E. coli JM101[pWKW2] and 0.2 mM for E. coli K-12 JM101[lacUV5par8EGF], were found to give the best hEGF production levels. The volumetric yields of hEGF were maximal when the cultures were induced in the mid-logarithmic phase. Growth temperature had a significant effect on hEGF yield. A simple continuous fed-batch process for cultivation of E. coli JM101[pWKW2] was developed. The maximum concentration of excreted hEGF attained in continuous fed-batch cultivation was 325 mg/l, as compared to 175 mg/l, in batch cultivation. The hEGF produced from the continuous fed-batch cultivation was substantiated by SDS-PAGE and immunoblotting.     

High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium

A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli.     

地衣芽孢杆菌2709碱性蛋白酶基因的克隆、序列测定及其表达

以克隆的地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增片段为探针。通过原位杂交从2709基因文库中筛选出两个含有完整的2709碱性蛋白酶基因的阳性克隆：Psci和Psc7。对Psc7中的插入片段构建若干亚克隆后测定了其全部DNA序列,结果显示该插入片段含2709碱性蛋白酶及其信号肽与导肽(Pro—peptide)在内的全部编码序列(1140碱基对)及长度分别为299和832碱基对的上、下游序列,该序列同M．Jacobs等克隆的地衣芽孢杆菌NcIB 6816的subtlisin Carlsberg基因序列显示了极高的同源性。通过枯草杆菌-大肠杆菌穿梭质粒Pbe2将克隆的2709碱性蛋白酶基因转入到蛋白酶缺陷型的枯草芽孢杆菌DB104中,结果表明2709碱性蛋白酶基因在枯草芽孢杆菌中得到了明显的表达。     

Fed-batch culture for L-lysine production via on-line state estimation and control

Computer application for fed-batch culture of Brevibacterium flavum for L-lysine production has been developed. The organisms are auxotrophic mutants for L-homoserine and are resistant to S-(2-aminoethyl)-L-cysteine. Adaptive control is applied for substrate addition. The sugar concentration is estimated using online respiratory measurement. During the period of fed-batch culture, the total sugar concentration is maintained at a given value. The cultivation strategy results in high productivity and high conversion yield.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Increase in efficiency of media utilization for recombinant protein production in Chinese hamster ovary culture through dilution

Animal cells are extensively used for the large-scale production of recombinant proteins. Processes and genetically engineered cell lines have been developed to enhance longevity of the culture and increase protein productivity. In this study, we tested the effect of diluting a culture of Chinese hamster ovary (CHO) cells with phosphate-buffered saline (PBS) on cell growth and efficiency of media utilization. An immunoglobulin G-expressing CHO cell line was cultured in CD CHO media followed by dilution of the culture with PBS after the end of the exponential phase. A 28% and 61% increase in protein yield per milliliter of media was observed in the diluted culture in the batch and fed-batch mode with glucose and protein hydrolysate feeding, respectively. To aid in analyzing the potential causes of this observed increase, an unstructured mathematical model was constructed using previously reported kinetics to simulate cell growth, nutrient utilization, and protein production. The model predicts an increase in recombinant protein yield per milliliter of media in PBS diluted cultures under both batch and fed-batch conditions, and suggests that this observed increase could at least partly be due to a decrease in inhibitor concentration in the diluted culture.     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture

Production of toluene cis-glycol (TCG) was investigated using recombinant Escherichia coli strains that express toluene dioxygenase under the tac promoter. E. coli TG2 was selected as the host for the recombinant plasmid, pTAC365, because the TCG yield was 64% higher than with strain JM105 as the host. By using fed-batch culture, TCG production could be improved by 4.4-fold compared with batch cultures, with a toluene vapor feed. A further improvement of 1.2-fold was obtained by using a two-liquid phase culture system, but the improvement was 1.6-fold when the feed rate of toluene vapor was reduced by 75%. When the period of growth in fed-batch culture was extended and the reduced vapor feed was used, the maximum TCG concentration increased to 4 g/l, an overall improvement of 10-fold compared with the batch culture system used initially. However, product consumption was observed during the late stages of fed-batch growth and in stationary phase, resulting in the formation of o-cresol, 3-methylcatechol and benzyl alcohol. We conclude that the key to optimizing TCG production by recombinants is to prolong growth of the cells to ensure sustained growth-linked product formation, and to optimize the supply of the toxic substrate, toluene, but further work is needed to eliminate by-product formation.     

Over-expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli

Human interferon-gamma (hIFN-gamma) was expressed in Escherichia coli BL21(DE3) under the control of the T7 promoter. Glucose was used as the sole source of carbon and energy with simple exponential feeding rate in fed-batch process. Cell density of recombinant E. coli was reached to 100 g dry wt l(-1) under both constant (0.12 h(-1)) and variable (0.12-0.52 h(-1)) specific growth rates. In the variable specific growth rate fed-batch process, plasmid stability and specific yield of rhIFN-gamma were greater than constant specific growth rate fed-batch process. The final specific yield and overall productivity of rhIFN-gamma were 0.35 +/- 0.02 g rhIFN-gamma g(-1) dry cell wt and 0.9 +/- 0.05 g rhIFN-gamma l(-1) h(-1) in the variable specific growth rate fed-batch process, respectively.     

High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer

Expression of a recombinant pyranose oxidase (P2O) from the basidiomycete Trametes ochracea has been increased 10-fold in shaking flask cultures of Escherichia coli BL21(DE3) harboring plasmid pSE33 by optimizing the composition of the culture medium using an experimental design approach. Inexpensive lactose was used as a medium component and inducer of expression of the P2O gene, which is under the control of a trc promoter. The expression system was studied in detail in batch and fed-batch cultivations with the aim to improve the expression level of active recombinant protein and to minimize the formation of inclusion bodies. In batch cultivations, the highest specific P2O activity of 0.9 U (mg of soluble protein)(-1) was measured in oxygen-limited cultures grown at 25 degrees C. The highest overall volumetric productivity of 33 mg of active P2O per liter and hour (corresponding to 345U (L h)(-1)) has been determined in a high-density fed-batch process with a feed-forward exponential feeding strategy. During the fed-batch process, lactose was added intermittently to the culture. A final biomass concentration of 33 g L(-1) (based on cell dry weight) was obtained. Compared to shaking flask cultures in not optimized culture media, the overall volumetric P2O productivity has been improved by a factor of 110 using the fed-batch strategy and the optimized culture medium. Recombinant P2O was expressed in the cytoplasm with 9% of the total soluble protein being active P2O. In terms of physical and enzyme kinetic properties, the purified recombinant P2O was found to be similar to the previously published data of P2O isolated from its original host.     

Effect of branch frequency in Aspergillus oryzae on protein secretion and culture viscosity.

Highly branched mutants of two strains of Aspergillus oryzae (IFO4177, which produces alpha-amylase, and a transformant of IFO4177 [AMG#13], which produces heterologous glucoamylase in addition to alpha-amylase) were generated by UV or nitrous acid mutagenesis. Four mutants of the parental strain (IFO4177), which were 10 to 50% more branched than the parental strain, were studied in stirred batch culture and no differences were observed in either the amount or the rate of enzyme production. Five mutants of the transformed parental strain (AMG#13), which were 20 to 58% more branched than the parental strain, were studied in either batch, fed-batch or continuous culture. In batch culture, three of the mutants produced more glucoamylase than the transformed parental strain, although only two mutants produced more glucoamylase and alpha-amylase combined. No increase in enzyme production was observed in either chemostat or fed-batch culture. Cultures of highly branched mutants were less viscous than those of the parental and transformed parental strains. A linear relationship was found between the degree of branching (measured as hyphal growth unit length) and culture viscosity (measured as the torque exerted on the rheometer impeller) for these strains. DOT-controlled fed-batch cultures (in which the medium feed rate was determined by the DOT) were thus inoculated with either the transformed parent or highly branched mutants of the transformed parent to determine whether the reduced viscosity would improve aeration and give higher enzyme yields. The average rate of medium addition was higher for the two highly branched mutants (ca. 8.3 g medium h(-1)) than for the parental strain (5.7 g medium h(-1)). Specific enzyme production in the DOT controlled fed-batch cultures was similar for all three strains (approx. 0.24 g alpha-amylase and glucoamylase [g of biomass](-1)), but one of the highly branched mutants made more total enzyme (24.3 +/- 0.2 g alpha-amylase and glucoamylase) than the parental strain (21.7 +/- 0.4 g alpha-amylase and glucoamylase).     

地衣芽孢杆菌BF-002高产芽孢的氮源流加工艺研究*

目的: 提高地衣芽孢杆菌BF-002的芽孢产量,实现氮源流加过程的自动化控制,降低生产成本,为其他芽孢杆菌提高芽孢产率的研究提供一种思路。方法: 通过摇瓶做单因素实验,筛选最佳温度和碳氮源,在此基础上进行5 L发酵罐实验。初始添加不同浓度的氮源,探索芽孢形成与氮源的关系。提出相对氨基氮的概念,通过恒速补料、间歇补料和基于尾气CO₂浓度反馈流加三个策略控制相对氨基氮浓度水平。采用Python语言编写计算机控制程序,实现基于尾气CO₂浓度反馈流加策略的自动化控制。结果: 摇瓶筛选最佳温度及碳氮源分别为:37℃、葡萄糖、鱼粉蛋白胨、豆粕。上罐结果表明,相对氨基氮浓度越低芽孢率越高,采用基于尾气CO₂浓度反馈流加能将相对氨基氮控制在8.42 mg/OD₆₀₀水平,芽孢量可达4.25×10⁹ cfu/mL。利用计算机程序自动控制低价氮源氯化铵的流加,可以使芽孢量达到1.87×10¹⁰ cfu/mL,是前期最优批次的4.4倍,同时降低原料成本。结论: 将相对氨基氮浓度控制在适宜水平可以得到芽孢量较高的培养液,自动流加氯化铵策略能降低生产成本并实现自动化控制,为研究芽孢杆菌产孢提供一种思路。     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.