Modeling and observer design for recombinant Escherichia coli strain

A mathematical model for recombinant bacteria which includes foreign protein production is developed. The experimental system consists of an Escherichia Coli strain and plasmid pIT34 containing genes for bioluminescence and production of a protein, β-galactosidase. This recombinant strain is constructed to facilitate on-line estimation and control in a complex bioprocess. Several batch experiments are designed and performed to validate the developed model. The design of a model structure, the identification of the model parameters and the estimation problem are three parts of a joint design problem. A nonlinear observer is designed and an experimental evaluation is performed on a batch fermentation process to estimate the substrate consumption.     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.     

Persistence of Escherichia coli recombinant strains in experimental animals

Recombinant E. coli strains, obtained by gene engineering techniques and capable of producing human alpha-interferon and HIV proteins, were studied. The cultures under study were completely eliminated from the body of experimental animals (mice) in 48 hours, and generalization of the infectious process took place. The study revealed that these recombinant strains had low virulence and were weakly adhesive, nontoxigenic and weakly toxic. Thus, the recombinant strains under study could be classified with class 3-4 of danger according to the "Classification of Strains of Industrial Microorganisms by the Degree of Their Danger".     

Process control for enhanced L-phenylalanine production using different recombinant Escherichia coli strains

A novel fed-batch approach for the production of L-phenylalanine (L-Phe) with recombinant E. coli is presented concerning the on-line control of the key fermentation parameters glucose and tyrosine. Two different production strains possessing either the tyrosine feedback resistant aroF(fbr) (encoding tyrosine feedback resistant DAHP-synthase (3-desoxy-D-arabino-heptusonate-7-phosphate)) or the wild-type aroF(wt) were used as model systems to elucidate the necessity of finding an individual process optimum for each genotype. With the aid of tyrosine control, wild-type aroF(wt) could be used for L-Phe production achieving higher final L-Phe titers (34 g/L) than the aroF(fbr) strain (28 g/L) and providing higher DAHP-synthase activities. With on-line glucose control, an optimum glucose concentration of 5 g/L could be identified that allowed a sufficient carbon supply for L-Phe production while at the same time an overflow metabolism leading to acetate by-product formation was avoided. The process approach is suitable for other production strains not only in lab-scale but also in pilot-scale bioreactors.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Electrophysical monitoring of culture process of recombinant Escherichia coli strains

A novel method for monitoring the cell culture process has been developed. The method is based on the measurements of electro-optical characteristics of cell suspension, calculation of cell structure parameters, and the relationship between accumulation of proteins and change of these parameters' employment. Application of the method for the monitoring of a culture process of a recombinant strain is considered. The process of growth of recombinant strains cannot be sufficiently predicted and the direct measurement of cell culture parameters is unlikely to be the most efficient way of solving the problem.Escherichia coli plasmid-free and recombinant strains synthesizing the fusion protein consisting of tumor necrosis factor-alpha (TNF) and thymosin-alpha(1) (T) were studied. It was found that cytoplasmic electroconductivity of the strains investigated increased during the culture process. The accumulation of insoluble recombinant pThy-315-encoded hybrid protein TNF(SINGLEBOND)T in cells resulted in a decrease of the membrane dielectric permeability. To determine variations of membrane dielectric permeability the amount of insoluble recombinant protein TNF(SINGLEBOND)T in the bacterial cells should be calculated.     

Rational design of an improved induction scheme for recombinant Escherichia coli

In Escherichia coli, strong overexpression of a recombinant protein has been shown to be deleterious due to a heavy metabolic burden on the host cell, which may completely cease cell growth before maximum product accumulation has occurred. Aiming at a reduction of very high product formation rates, we engineered E. coli strains by mutating the Leloir pathway for galactose metabolization, so that galactose can be utilized to induce lac derived promoters. The induction with galactose was effective in every strain and expression construct tested, and it reduced the metabolic burden on a highly overproducing clone so that cell growth and product accumulation could be maintained for several generations.     

FT-IR study of heterologous protein expression in recombinant Escherichia coli strains

Two recombinant Escherichia coli strains expressing different levels of an interferon fusion protein as inclusion bodies have been studied by Fourier transform infrared (FT-IR) microspectroscopy. A marker band at 1628 cm(-1) allowed monitoring of the protein expression by direct analysis of cell pellets in a rapid, non-invasive and quantitative way. The results demonstrate that FT-IR microspectroscopy is a technique of potential biotechnological interest for studying inclusion body formation.     

Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations

Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e.g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes.     

Improvement of Escherichia coli strains overproducing lysine using recombinant DNA techniques

Summary Several genes of the lysine biosynthetic pathway were cloned separately on the high copy number plasmid pBR322 (Richaud et al. 1981). These hybrid plasmids were used to transform an Escherichia coli strain TOC R 21 that overproduces lysine due to mutations altering the aspartokinase reaction. The synthesis of lysine was studied in these different strains. It appears that only plasmids containing the dapA gene (encoding dihydrodipicolinate synthetase) lead to an increase in lysine production. This result allows us to identify this reaction as the limiting biosynthetic step in strain TOC R 21 and indicates that such a method of gene amplification can be used to improve strains overproducing metabolites.     

Directed modification of Escherichia coli metabolism for the design of threonine-producing strains

The modification of Escherichia coli K-12 metabolism leading to threonine overproduction is the most studied system in synthetic biology that has been used to elaborate the majority of the currently known approaches to constructing microbial producers. They include optimization of biosynthesis through search for rate-limiting stages, modification of substrate and product transport, elimination of side metabolic pathways and degradation systems, reinforcement of the regeneration of coenzymes that are required for product biosynthesis, and exclusion of futile cycles and metabolic pathways with low energy efficiency. Extensive research in functional genomics made it possible to selectively remove the “unnecessary genes,” the functions of which are useless for producing a strain or adversely affect its properties. In total, using various approaches to designing threonine-producing strains, over 150 genome loci that affect more than 30% genes in E. coli were directly modified, thus providing interesting data for researchers in the field of microbial synthesis, as well as in related biological sciences. This review is dedicated to the assessment of genetic engineering modifications in E. coli metabolism (primarily, on the basis of modern patent literature) that ensure threonine overproduction.     

Escherichia coli K12 relA strains as safe hosts for expression of recombinant DNA

Most Escherichia coli K12 strains survive for a relatively long time outside the laboratory. Under the same conditions the isoallelic E. coli K12 relA mutants die faster because they lack the stringent response. The killing rate is increased by using a plasmid-encoded suicide system consisting of the phage T7 lysozyme gene driven by the E. coli alkaline phosphatase gene promoter (phoA). Cells containing this system were rapidly and effectively killed as soon as phosphate was made limiting. The combination of the chromosomal relA mutation and a conditional suicide system of this type provides an effective means of biological containment for recombinant E. coli strains.     

N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains

Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.     

Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.     

Specific selection for virulent urinary tract infectious Escherichia coli strains during catheter-associated biofilm formation

Biofilm-associated bacterial infections have a major impact on artificial implants such as urinary catheters, often with devastating consequences. The capacity of a microorganism to form a biofilm on a surface depends on the nature of the surface and its conditioning. When a urinary catheter is exposed to urine, various components adsorb onto the surface and form a conditioning film, which becomes the real interface where microbial interaction takes place. It follows that the material constituting the catheter determines the composition of the conditioning film, which in turn influences which microorganisms can attach. Urinary tract infectious (UTI) Escherichia coli range in pathogenicity and the damage they cause--from benign asymptomatic bacteriuria (ABU) strains, which inflict no or few problems to the host, to uropathogenic E. coli (UPEC) strains, which are virulent and often cause severe symptoms and complications. We have found that whereas ABU strains produce better biofilms on polystyrene and glass, UPEC strains have a clear competitive advantage during biofilm growth on catheter surfaces. Our results indicate that some silicone and silicone-latex catheters actually select for and promote biofilm formation of the most virulent group of UTI E. coli strains, hardly a desirable situation for the catheterized patient.     

Purification of recombinant Bacillus cereus ResD-ResE proteins expressed in Escherichia coli strains

Recombinant E. coli strains expressing the Bacillus cereus ATCC 14579^T resD and resE genes fused with the ubiquitin gene were constructed, and purification of the ResD and ResE proteins was performed. The approach used in the study allowed us to increase the protein yield of the electrophoretic homogeneous ResD and ResE proteins without denaturation steps up to 150 mg per gram of wet cell weight.