E. coli filament formation induced by heterologous S-layer expression

Escherichia coli is a rod-shaped intestinal bacterium which has a size of 1.1-1.5 μm x 2.0-6.0?μm. The fast cell division process and the uncomplicated living conditions have turned E. coli into a widely used host in genetic engineering and into one of the best studied microorganisms of all. We used E. coli BL21(DE3) as host for heterologous expression of S-layer proteins of Lysinibacillus sphaericus JG-A12 in order to enable a fast and high efficient protein production. The S-layer expression induced in E. coli an unusual elongation of the cells, thus producing filaments of > 100 μm in length. In the stationary growth phase, E. coli filaments develop tube-like structures that contain E. coli single cells. Fluorescence microscopic analyses of S-layer expressing E. coli cells that were stained with membrane stain FM (?) 5-95 verify the membrane origin of the tubes. Analyses of DAPI stained GFP-S-layer expressing E. coli support the assumption of a disordered cell division that is induced by the huge amount of recombinant S-layer proteins. However, the underlying mechanism is still not characterized in detail. These results describe the occurrence of a novel stable cell form of E. coli as a result of a disordered cell division process.     

Mycobacterial mammalian cell entry protein 1A (Mce1A)-mediated adherence enhances the chemokine production by A549 alveolar epithelial cells

Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non-phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP-1 and IL-8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP-1 and IL-8 production. However, Mce1A-coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells.     

In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli

Lycopene is produced by recombinant Escherichia coli expressing genes to encode for the lycopene biosynthesis. However, the productivity of lycopene seemed to be limited by many factors including product toxicity. In the present study, we have investigated physiology of recombinant E. coli during biosynthesis and in situ recovery of lycopene based on an organic/aqueous two-phase system. Lycopene, the 40-carbon molecule product, was little extracted from recombinant E. coli cells to octane or decane phase. However, partial digestion of cell walls with lysozyme promoted extraction of lycopene into the organic phases. Engineering of an organic/aqueous two-phase system allowed recombinant E. coli cells to produce ca. 40% larger amount of lycopene compared to that in a conventional aqueous single-phase system. Optimization of the in situ product recovery process will lead to further increase of product concentration and productivity.     

A study of the intracellular fate of recombinant human interleukin 2 in Escherichia coli

The expression of the human IL-2 recombinant gene in E. coli cells was studied. The processes which take place during thermo-induced expression and effect the state of the product were investigated. Experimental data on the membrane localisation of IL-2, the formation of aggregates (inclusion bodies) and polymers were obtained. It was determined that temperature significantly influence the kinetics of the processes of intracellular IL-2 production and IL-2 stability. It is supposed that the cell membrane state plays a determining role in these processes via temperature mediation. Thus, the formation of inclusion bodies described for a number of E. coli recombinant strains is probably stipulated not only by recombinant polypeptides properties, but also by cellular interactions.     

Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase

N-acetyl-d-neuraminic acid (NeuAc; sialic acid) is a precursor for the manufacture of many pharmaceutical drugs, such as anti-influenza virus agents. To develop a whole cell process for NeuAc production, genes of Anabaena sp. CH1 N-acetyl-d-glucosamine 2-epimerase (bage) and Escherichia coli N-acetyl-d-neuraminic acid lyase (nanA) were cloned and expressed in E. coli BL21 (DE3). The expressed bGlcNAc 2-epimerase was purified from the crude cell extract of IPTG-induced E. coli BL21 (DE3) (pET-bage) to homogeneity by nickel-chelate chromatography. The molecular mass of the purified bGlcNAc 2-epimerase was determined to be 42kDa by SDS-PAGE. The pH and temperature optima of the recombinant bGlcNAc 2-epimerase were pH 7.0 and 50 degrees C, respectively, and only needs 20mum ATP for maximal activity. The specific activity of bGlcNAc 2-epimerase (124Umg(-1) protein) for the conversion of N-acetyl-d-glucosamine to N-acetyl-d-manosamine was about four-fold higher than that of porcine N-acetyl-d-glucosamine 2-epimerase. A stirred glass vessel containing transformed E. coli cells expressing age gene from Anabaena sp. CH1 and NeuAc lyase gene from E. coli NovaBlue separately was used for the conversion of GlcNAc and pyruvate to NeuAc. A maximal productivity of 10.2gNeuAcl(-1)h(-1) with 33.3% conversion yield from GlcNAc could be obtained in a 12-h reaction. The recombinant E. coli cells can be reused for more than eight cycles with a productivity of >8.0gNeuAcL(-1)h(-1). In this process, the expensive activator, ATP, necessary for maximal activity of GlcNAc 2-epimerase in free enzyme system can be omitted.     

Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase

Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.     

Differential adhesion of major surface proteins 1a and 1b of the ehrlichial cattle pathogen Anaplasma marginale to bovine erythrocytes and tick cells

Anaplasma marginale is a tick-borne ehrlichial pathogen of cattle for which six major surface proteins (MSPs) have been described. The MSP1 complex, a heterodimer composed of MSP1a and MSP1b, was shown to induce a protective immune response in cattle and both proteins have been identified as putative adhesins for bovine erythrocytes. In this study the role of MSP1a and MSP1b as adhesins for bovine erythrocytes and tick cells was defined. msp1alpha and msp1beta1 genes from the Oklahoma isolate of A. marginale were cloned and expressed in Escherichia coli K-12 under the control of endogenous and tac promoters for both low and high level protein expression. Expression of the recombinant polypeptides was confirmed and localised on the surface of transformed E. coli. The adhesion properties of MSP1a and MSP1b were determined by allowing recombinant E. coli expressing these surface polypetides to react with bovine erythrocytes, Dermacentor variabilis gut cells and cultured tick cells derived from embryonic Ixodes scapularis. Adhesion of the recombinant E. coli to the three cell types was determined using recovery adhesion and microtiter haemagglutination assays, and by light and electron microscopy. MSP1a was shown by all methods tested to be an adhesin for bovine erythrocytes and both native and cultured tick cells. In contrast, recombinant E. coli expressing MSP1b adhered only to bovine erythrocytes and not to tick cells. When low expression vectors were used, single E. coli expressing MSP1a was seen adhered to individual tick cells while reaction of tick cells with the E. coli/MSP1a/high expression vector resulted in adhesion of multiple bacteria per cell. With electron microscopy, fusion of E. coli cell membranes expressing MSP1a or MSP1b with erythrocyte membranes was observed, as well as fusion of tick cell membranes with E. coli membranes expressing MSP1a. These studies demonstrated differential adhesion for MSP1a and MSP1b for which MSP1a is an A. marginale adhesin for both bovine erythrocytes and tick cells while MSP1b is an adhesin only for bovine erythrocytes. The role of the MSP1 complex, therefore, appears to vary among vertebrate and invertebrate hosts.     

重组大肠杆菌高密度发酵研究进展

重组大肠杆菌的高密度发酵是提高基因工程产品产量的一个非常有效的手段,是现代发酵工程研究的一个热点。本文就高密度发酵中影响重组大肠杆菌发酵产率的几个因素,包括宿主菌、培养基、培养条件、补料方法以及高密度发酵过程中存在的问题和对策加以讨论,着重探讨了高密度下大肠杆菌产生的有害代谢副产物———乙酸的产生机制、抑制作用机理,以及控制乙酸积累的技术方法 。     

Proteome analysis of recombinant Escherichia coli producing human glucagon-like peptide-1

The proteomic response of recombinant Escherichia coli producing human glucagon-like peptide-1 was analyzed by two-dimensional gel electrophoresis. Protein spots in two-dimensional gel could be identified by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their expression profiles were compared with those of nonproducing cells. Thirty-five intracellular proteins exhibited differential expression levels between the production and control strains. These changes reflected physiological responses to heterologous peptide production in recombinant E. coli. Specifically, physiological changes included the down-regulation of proteins involved in the central carbon metabolism, biosynthesis of cellular building blocks and peptides, and up-regulation of cell protection proteins and some sugar transport proteins. This comprehensive analysis would provide useful information for understanding physiological alterations to heterologous peptide production and for designing efficient metabolic engineering strategies for the production of recombinant peptides in E. coli.     

Expression of the Escherichia coli pntA and pntB genes, encoding nicotinamide nucleotide transhydrogenase, in Saccharomyces cerevisiae and its effect on product formation during anaerobic glucose fermentation.

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD+ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD+ by NADPH and by NADH in the presence of NADP+, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD+, NADPH, and NADP+ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD+.     

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.     

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.     

Assessment of physiological conditions in E. coli fermentations by epifluorescent microscopy and image analysis

The development of monitoring methods for assessing the physiological state of microorganisms during recombinant fermentation processes has been encouraged by the need to evaluate the influence of processing conditions in recombinant protein production. In this work, a technique based on microscopy and image analysis was developed that allows the simultaneous quantification of parameters associated with viability and fluorescent protein production in recombinant Escherichia coli fermentations. Images obtained from light microscopy with phase contrast are used to assess the total number of cells in a given sample and, from epifluorescence microscopy, both protein producing and injured cells are evaluated using two different fluorochromes: propidium iodide and enhanced yellow fluorescent protein. This technique revealed the existence of different cell populations in the recombinant E. coli fermentation broth that were evaluated along four batch fermentations, complementing information obtained with standard techniques to study the effects of the temperature and induction time in recombinant protein production processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.     

Comparison of the Escherichia coli proteomes for recombinant human growth hormone producing and nonproducing fermentations

Two-dimensional electrophoretic analyses of Escherichia coli cells producing recombinant human growth hormone (Nutropin) in fermentations were conducted. The resulting two-dimensional protein profiles were compared with those of nonproducing (blank) cells. A qualitative comparison was performed to address regulatory issues in the biopharmaceutical industry, and a semiquantitative comparison was performed to reveal information about the physiological state of the cells. The protein spots unique to production fermentation profiles were all related to recombinant human growth hormone (hGH); these included intact hGH, charge variants of hGH, and a proteolytically cleaved form of hGH, as expected. There were no E. coli host cell proteins unique to either the production or blank fermentation profiles. Rather, all detectable differences in E. coli proteins were quantitative in nature. Specifically, the levels of IbpA (inclusion body binding protein A), Ivy (inhibitor of vertebrate lysozyme), and a cleaved form of GroEL (Hsp60 homolog) were higher in hGH production profiles, whereas the levels of GlmU protein and PspA (phage shock protein A) were higher in blank profiles. In general, the high degree of similarity between proteomes for hGH-producing and nonproducing cells suggests that E. coli proteins from a nonproducing (blank) fermentation are appropriate for eliciting antibodies that are then used in immunoassays to measure host cell proteins in samples from production fermentations.     

Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827

L-Homophenylalanine (l-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the l-HPA production, N-acylamino acid racemase (NAAAR) and l-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l-enantioselection to hydrolyze the NAc-l-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-d-HPA to l-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-l-HPA, in the early stage of conversion and a low productivity of 0.83 mmol l-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l-HPA could be reached in 1 h, with a productivity of 10 mmol l-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l-HPA to be reported.     

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.     

Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia coli

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l(-1) (OD600 approximately 60) and the production of 700 mg l(-1) of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l(-1) of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l(-1) with E. coli as the production host.