Predictive tool for recombinant protein production in Escherichia coli shake-flask cultures using an on-line monitoring system

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

A Computer-aided noninterfering on-line technique for monitoring oxygen-transfer characteristics during fermentation processes

The role of computers in the monitoring and control of fermentation processes has increased steadfastly. The ultimate utility of the machines will not depend on the availability of online sensors but also on the availability of techniques that combine direct measurements, leading towards estimates of variable closely related to the microbial process or its control. In this article, a methodology for on-line and noninterfering evaluation of the volumetric mass-transfer coefficient K(l)a is developed. A detailed presentation of the procedure, called "the static method," is given. Its feasibility is proved through implementation of the method on an antibiotic fermentation process. These experiments indicate that operator actions meant to modify the oxygen-transfer conditions can be checked on-line. The quantitative value of the static method is ascertained by comparing the experimental results with K(l)a estimates obtained with the "gassing-out" method. A sensitivity analysis was carried out, revealing the need for temperature and pressure corrections and showing that the precision of the oxygen analyzer determines the precision of the static method.     

Lactobacilli prevent hydroxy radical production and inhibit Escherichia coli and Enterococcus growth in system mimicking colon fermentation

Aims: To explore the effect of Lactobacillus on redox state of colon chyme. Methods and Results: Nine Lactobacillus strains were studied for the inhibition of lipid peroxide formation in Fe²⁺/ascorbate system and for their ability to chelate ‘free’ ferrous ion. The result shows both properties were strain specific and no relationship between them was found. Both properties of Lactobacillus paracasei Fn032, Lactobacillus rhamnosus GG (LGG) and Lactobacillus sp. Fn001 were successively decreasing. LGG and Fn032 significantly decreased hydroxyl radicals (P < 0·01) in colonic fermentation model, in which considerable hydroxyl radicals occurred spontaneously. Addition of ferrous ion induced the production of hydroxyl radicals, which could be significantly inhibited by LGG, Fn032 (P < 0·01) and Fn001 (P < 0·05). Ferrous ion significantly induced the growth of Enterococcus and Escherichia coli, which could be inhibited by all three Lactobacillus strains. Escherichia coli and Enterococcus show significantly positive correlation with hydroxyl radicals with R of 0·96 (P = 0·0002) and 0·91 (P = 0·0017), respectively. Conclusions: Antioxidative Lactobacillus could modulate redox state in colonic fermentation system, which is related to their free radical–scavenging ability or antibacterial effect. Significance and Impact of the Study: This study proves that Lactobacillus strain could influence the redox state of gut chyme. Evaluation of antioxidative ability might be a powerful method for screening probiotic Lactobacillus strains.     

A two-step fermentation process for efficient production of penta-N-acetyl-chitopentaose in recombinant Escherichia coli

The nodC gene from Mesorhizobium loti was cloned into E. coli, leading to production of chitin oligosaccharides (COs)—mainly penta-N-acetyl-chitopentaose. A two-step fermentation procedure was then developed which gave 930 mg CO/L with a productivity of 37 mg/l·h.     

A rapid procedure for production of human basic fibroblast growth factor in Escherichia coli cells.

Human basic fibroblast growth factor has been expressed in Escherichia coli cells at a level of 2-3 mg/l culture, using a rapid procedure which requires only simple DNA manipulative work. The recombinant material has the same potency as natural basic fibroblast growth factor from bovine pituitary glands.     

Online monitoring of Escherichia coli ghost production

Controlled expression of cloned phi X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex. Production of bacterial ghosts is routinely monitored by classical microbiological procedures. These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis. Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts. To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture. For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells. The total quantitative analysis procedure takes less than 2 min. The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.     

A decreasing feeding profile for the optimization of ethanol production in a recombinant Escherichia coli fed-batch fermentation

Summary A decreasing feed flowrate was used to optimize the ethanol volumetric productivity and was found to be optimal and equal to 1.8 g ethanol/h·L with a recombinant Escherichia coli with plasmid pLOI297. This productivity is more than double in previous reports. High substrate concentrations favor ethanol production when the microorganisms are in the exponential phase and when they are not inhibited.     

Systematic high-yield production of human secreted proteins in Escherichia coli

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.     

基于辅因子调控对大肠杆菌两阶段发酵产丁二酸的影响

考察了E.coli NZN111及其重组菌株E.coli NZN111/pTrc99a-pncB发酵生产丁二酸的性能。E.coli NZN111两阶段发酵丁二酸的同时,会造成丙酮酸的大量积累。研究发现:通过过量表达烟酸转磷酸核糖激酶,两阶段发酵重组菌株E.coli NZN111/pTrc99a-pncB,减少丙酮酸的积累且无副产物乙酸生成,提高丁二酸的产量,丁二酸得率和耗糖速率分别提高了139%和20%。     

Expression of secreted insulin-like growth factor-1 in Escherichia coli

The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1 or somatomedin-C) fused to LamB and OmpF secretion leader sequences in Escherichia coli have been investigated. Expression and secretion of IGF-1 was achieved. The major portion of this secreted IGF-1 accumulated in the periplasmic space as insoluble aggregates. A small amount of IGF-1 was found folded in its native conformation in the medium. The lamB and ompF signal sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20 micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted into the periplasm and exported into the medium confirmed the precise removal of the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated to be active in a cell proliferation bioassay.     

High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.     

产琥珀酸工程菌株的发酵工艺条件优化

对实验室构建的产琥珀酸大肠杆菌工程菌株(E.coliQZ1111)进行发酵工艺条件研究。以AM1低盐培养基为基础,研究不同C、N源及其质量浓度,培养基初始pH和发酵温度等因素对琥珀酸的影响,并在5L发酵罐中进行了补料-分批发酵实验。优化后的发酵条件为葡萄糖20g/L,玉米浆10g/L,pH6.4,发酵温度37℃。在5L发酵罐中培养,琥珀酸产量达到47.9g/L。     

Flux through citrate synthase limits the growth of ethanologenic Escherichia coli KO11 during xylose fermentation

Previous studies have shown that high levels of complex nutrients (Luria broth or 5% corn steep liquor) were necessary for rapid ethanol production by the ethanologenic strain Escherichia coli KO11. Although this strain is prototrophic, cell density and ethanol production remained low in mineral salts media (10% xylose) unless complex nutrients were added. The basis for this nutrient requirement was identified as a regulatory problem created by metabolic engineering of an ethanol pathway. Cells must partition pyruvate between competing needs for biosynthesis and regeneration of NAD(+). Expression of low-K(m) Zymomonas mobilis pdc (pyruvate decarboxylase) in KO11 reduced the flow of pyruvate carbon into native fermentation pathways as desired, but it also restricted the flow of carbon skeletons into the 2-ketoglutarate arm of the tricarboxylic acid pathway (biosynthesis). In mineral salts medium containing 1% corn steep liquor and 10% xylose, the detrimental effect of metabolic engineering was substantially reduced by addition of pyruvate. A similar benefit was also observed when acetaldehyde, 2-ketoglutarate, or glutamate was added. In E. coli, citrate synthase links the cellular abundance of NADH to the supply of 2-ketoglutarate for glutamate biosynthesis. This enzyme is allosterically regulated and inhibited by high NADH concentrations. In addition, citrate synthase catalyzes the first committed step in 2-ketoglutarate synthesis. Oxidation of NADH by added acetaldehyde (or pyruvate) would be expected to increase the activity of E. coli citrate synthase and direct more carbon into 2-ketoglutarate, and this may explain the stimulation of growth. This hypothesis was tested, in part, by cloning the Bacillus subtilis citZ gene encoding an NADH-insensitive citrate synthase. Expression of recombinant citZ in KO11 was accompanied by increases in cell growth and ethanol production, which substantially reduced the need for complex nutrients.     

Effect of cell growth rate on the performance of a two-stage continuous culture system in a recombinant Escherichia coli fermentation

As part of the process optimization of a two-stage continuous culture system, the effect of growth rate mu(2) (app) on the performance of the second stage (production stage) was studied in a recombinant Escherichia coli K12 (DeltaH1Deltatrp/pPLc23trpA1). Important parameters considered were specific gene expression rate, plasmid content, and plasmid stability, all of which were closely related to the cell growth rate and the production rate of the cloned gene product (trpalpha). When operating conditions were maintained constant (T(1) = 35 degrees C, D(1) = 0.9 h(-1), T(2) = 40 degrees C, and D(2) = 0.7 h(-1)) and mu(2) (app) was varied, plasmid content in the second stage showed its maximum at mu(2) (app) = 0.4 h(-1) and decreased thereafter. Specific gene expression rate linearly increased with increasing mu(2) (app), while plasmid stability decreased. Optimum cell growth rate giving the maximum value in overall productivity was observed at around mu(2) (app) = 0.4 h(-1). The contribution or role of the three parameters, specific gene expression rate, plasmid content, and plasmid stability in exhibiting the maximum productivity at the optimal mu(2) (app) is discussed.