Expression of Vitreoscilla hemoglobin in Escherichia coli enhances ribosome and tRNA levels: a flow field-flow fractionation study

Asymmetrical flow field-flow fractionation (FFF) was used to separate and quantitate 70S ribosomes, the 30S and 50S subunits, and tRNA in one single analytical procedure. The method was applied to an investigation of the effect of Vitreoscilla hemoglobin (VHb) on the translational machinery of the recombinant Escherichia coli cells. The number of active 70S ribosomes per cell increased dramatically, more than 2-fold, as did also the tRNA levels for the VHb-expressing strain relative to VHb-negative control at the end of a 30-h fed-batch cultivation. This was accompanied by a corresponding 61% increase of a cloned marker enzyme activity. The results clearly indicate that VHb promotes the level of translational components. There should be many other cases in bioengineering where it is important to relate the protein production level in a bioreactor to the ribosome and tRNA levels.     

Enhanced ribosome and tRNA contents in Escherichia coli expressing a truncated Vitreoscilla hemoglobin mutant analyzed by flow field-flow fractionation

The ribosome and tRNA levels of Escherichia coli cells, transformed with a native or mutated Vitreoscilla hemoglobin genes (vhb), were investigated using asymmetrical flow field-flow fractionation (AFFFF). Mutagenesis of vhb by error-prone PCR was carried out to alter the growth behavior of microaerobically cultivated native VHb-expressing E. coli. A VHb mutant, pVMT1, was identified, which was able to reach a remarkably high final A₆₀₀ of 15, the value of which being 160% higher than that of a VHb control carrying pVHb8 (A₆₀₀ 5.8). AFFFF revealed that cells expressing mutant vhbs showed up to a doubling in the number of active 70S ribosomes cell^–1, an almost 3-fold increase in the number of tRNAs cell^–1, and up to a 26% increase in the mass fraction of active 70S ribosomes.     

Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K. pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli control carrying the parental plasmid without any hemoglobin gene.     

Error-prone PCR of Vitreoscilla hemoglobin (VHb) to support the growth of microaerobic Escherichia coli

Expression of the gene encoding bacterial hemoglobin (VHb) from Vitreoscilla has been previously used to improve recombinant cell growth and enhance product formation under microaerobic conditions. It is very likely that the properties of VHb are not optimized for foreign hosts; therefore, we used error-prone PCR to generate a number of randomly mutated vhb genes to be expressed and studied in Escherichia coli. In addition, the mutated VHb proteins also contained an extension of eight residues (MTMITPSF) at the amino terminus. VHb mutants were screened for improved growth properties under microaerobic conditions and 15 clones expressing mutated hemoglobin protein were selected for further characterization and cultivated in a microaerobic bioreactor to analyze the physiological effects of novel VHb proteins on cell growth. The expression of four VHb mutants, carried by pVM20, pVM50, pVM104, and pVM134, were able to enhance microaerobic growth of E. coli by approximately 22%, 155%, 50%, and 90%, respectively, with a concomitant decrease of acetate excretion into the culture medium. The vhb gene in pVM20 contains two mutations substituting residues Glu19(A17) and Glu137(H23) to Gly. pVM50 expresses a VHb protein carrying two mutations: His36(C1) to Arg36 and Gln66(E20) to Arg66. pVM104 and pVM134 express VHb proteins carrying the mutations Ala56(E10) to Gly and Ile24(B5) to Thr, respectively. Our experiments also indicate that the positive effects elicited by mutant VHb-expression from pVM20 and pVM50 are linked to the peptide tail. Removal of the N-terminal sequence reduced cell growth approximately 23% and 53%, respectively, relative to wild-type controls. These results clearly demonstrate that it is possible to obtain mutated VHb proteins with improved characteristics for improving microaerobic growth of E. coli by using combined mutation techniques, addition of a peptide tail, and random error-prone PCR.     

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

The existence of a conditional lethal temperature-sensitive mutant affecting peptidyl-tRNA hydrolase in Escherichia coli suggests that this enzyme is essential to cell survival. We report here the isolation of both chromosomal and multicopy suppressors of this mutant in pth, the gene encoding the hydrolase. In one case, the cloned gene responsible for suppression is shown to be lysV, one of three genes encoding the unique lysine acceptor tRNA; 10 other cloned tRNA genes are without effect. Overexpression of lysV leading to a 2- to 3-fold increase in tRNA(Lys) concentration overcomes the shortage of peptidyl-tRNA hydrolase activity in the cell at non-permissive temperature. Conversely, in pth, supN double mutants, where the tRNA(Lys) concentration is reduced due to the conversion of lysV to an ochre suppressor (supN), the thermosensitivity of the initial pth mutant becomes accentuated. Thus, cells carrying both mutations show practically no growth at 39 degrees C, a temperature at which the pth mutant grows almost normally. Growth of the double mutant is restored by the expression of lysV from a plasmid. These results indicate that the limitation of growth in mutants of E.coli deficient in Pth is due to the sequestration of tRNA(Lys) as peptidyl-tRNA. This is consistent with previous observations that this tRNA is particularly prone to premature dissociation from the ribosome.     

Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli

When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and 'rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of 'stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity.     

Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: A proteome-level study

Vitreoscilla haemoglobin (VHb) expression in heterologous host was shown to enhance growth and oxygen utilization capabilities under oxygen-limited conditions. The exact mechanism by which VHb enhances the oxygen utilization under oxygen-limiting conditions is still unknown. In order to understand the role of VHb in promoting oxygen utilization, changes in the total protein profile of E. coli expressing the vgb gene under its native promoter was analysed. Two-dimensional difference gel electrophoresis (2D DIGE) was employed to quantify the differentially expressed proteins under oxygen-limiting conditions. Overexpression of proteins involved in aerobic metabolic pathways and suppression of proteins involved in non-oxidative metabolic pathways shown in this study indicates that the cells expressing VHb prefer aerobic metabolic pathways even under oxygen limitation. Under these conditions, the expression levels of proteins involved in central metabolic pathways, cellular adaptation and cell division were also found to be altered. These results imply that Vitreoscilla haemoglobin expression alters aerobic metabolism specifically, in addition to altering proteins involved in other pathways, the significance of which is not clear as of now.     

Cloning and Expression of the Vitreoscilla Hemoglobin Gene in Enterobacter Aerogenes: Effect on Cell Growth and Oxygen Uptake

The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxi-dative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5–2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2–5-fold higher activity for these enzymes than cells at the exponential phase.     

Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-beta-D-thiogalactopyranoside- (IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (Vhb(-)) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb(-) control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 mumol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 60%, respectively. Increasing VHb concentration to 3.8 mumol/g DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb(+) cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADH/NADPH transhydrogenase while the VHb(-) cells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb(+) cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb(+) cells. (c) 1996 John Wiley & Sons, Inc.     

Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic regulation by proteomic analysis

Heterologous expression of Vitreoscilla hemoglobin (VHb) has been reported to improve cell growth, protein synthesis, metabolite productivity and nitric oxide detoxification. Although it has been proposed that such phenomenon is attributed to the enhancement of respiration and energy metabolism by facilitating oxygen delivery, the mechanism of VHb action remains to be elucidated. In the present study, changes of protein expression profile in Escherichia coli as a consequence of VHb production was investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting. Total protein extracts derived from cells expressing native green fluorescent protein (GFPuv) and chimeric VHbGFPuv grown in Luria-Bertani broth were prepared by sonic disintegration. One hundred microgram of proteins was individually electrophoresed in IEF-agarose rod gels followed by gradient SDS-PAGE gels. Protein spots were excised from the gels, digested to peptide fragments by trypsin, and analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Results revealed that expression of VHbGFPuv caused an entire disappearance of tryptophanase as well as down-regulated proteins involved in various metabolic pathways, e.g. glycerol kinase, isocitrate dehydrogenase, aldehyde dehydrogenase, and D-glucose-D-galactose binding protein. Phenotypic assay of cellular indole production confirmed the differentially expressed tryptophanase enzymes in which cells expressing chimeric VHbGFP demonstrated a complete indole-negative reaction. Supplementation of delta-aminolevulinic acid (ALA) to the culture medium enhanced expression of glyceraldehyde-3-phosphate dehydrogenase and glycerol kinase. Our findings herein shed light on the functional roles of VHb on cellular carbon and nitrogen consumptions as well as regulation of other metabolic pathway intermediates, possibly by autoregulation of the catabolite repressor regulons.     

Vitreoscilla hemoglobin. Intracellular localization and binding to membranes

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.     

Intracellular expression of Vitreoscilla hemoglobin modifies microaerobic Escherichia coli metabolism through elevated concentration and specific activity of cytochrome o

The function of the reversible oxygen-binding hemoprotein from Vitreoscilla (VHb), which enhances oxygen-limited cell growth and recombinant protein production when functionally expressed in Escherichia coli, was investigated in wild-type E. coli and in E. coli mutants lacking one of the two terminal oxidases, cytochrome o complex (aerobic terminal oxidase, Cyo) or cytochrome d complex (microaerobic terminal oxidase, Cyd). Deconvolution of VHb, cytochrome o, and cytochrome d bands from in vivo absorption spectra revealed a 5-fold enhancement in cytochrome o content and a 1.5-fold increment in cytochrome d by VHb under microaerobic environments (dissolved oxygen less than 2% air saturation). Based upon oxygen uptake kinetics measurements of these mutants, the apparent oxygen affinity of the Cyo(+), Cyd(-) E. coli was increased in the presence of VHb, but no difference in the apparent K(m) was observed for the Cyo(-), Cyd(+) strain. Results suggest that the expression of VHb in E. coli increases the level and activity of terminal oxidases and thereby improves the efficiency of microaerobic respiration and growth.     

Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant chinese hamster ovary cells

Previous studies suggest that secretion of cloned proteins synthesized by recombinant Chinese hamster ovary (CHO) cells can be adenosine triphosphate (ATP) limited. Other research indicates that the presence of cloned Vitreoscilla hemoglobin (VHb) enhances ATP production in oxygen-limited Escherichia coli. To evaluate the influence of VHb expression on recombinant CHO cell productivity, the vhb gene has been fused to the mouse mammary tumor virus (MMTV) promoter and cloned in a CHO cell line previously engineered to express human tissue plasminogen activator (tPA). Western blot analysis confirms dexamethasone-inducible VHb expression in all of the clones tested. Batch cultivation experiments with one VHb-expressing clone and the parental CHO-tPA expressing cells. The VHb-expressing clone exhibits specific tPA production 40 to 100% greater than the parental CHO-tPA culture. (c) 1994 John Wiley & Sons, Inc.     

The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.     

在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。     

Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.     

Vitreoscilla hemoglobin expression in engineered Escherichia coli: improved performance in high cell-density batch cultivations

High cell-density cultivations are the preferred system for biomolecules production by Escherichia coli. It has been previously demonstrated that a strain of E. coli with a modified substrate transport system is able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. The use of elevated amounts of glucose from the beginning of the cultivation, eliminates the existence of substrate gradients due to deficient mixing at large-scale. However, the large amounts of oxygen demanded resulted in microaerobic conditions after some hours of cultivation, even at small-scale. In this work, the effect of expressing the Vitreoscilla hemoglobin (VHb) in the engineered strain during batch cultures using high-glucose concentrations was tested. Together, the expression of VHb and the modified substrate transport system resulted in a 33% increase of biomass production compared to the parental strain (W3110) lacking the VHb in batch cultivations using 25 g/L of glucose. When 50 g/L of glucose were used, expression of VHb in the modified strain led to 11% higher biomass production compared to W3110. The VHb also increased the growth rates of the strains by about 30% in the aerobic phase and more than 200% in the microaerobic phase of batch cultivation.