Genetic engineering of Serratia marcescens with bacterial hemoglobin gene: effects on growth, oxygen utilization, and cell size

The bacterial hemoglobin from Vitreoscilla has been shown to increase growth yield and yield of genetically engineered product in Escherichia coli. To test the generality of this phenomenon, the approximately 560-bp bacterial (Vitreoscilla) hemoglobin gene (vgb) (including the native promoter), cloned into the vector pUC8 in two constructs containing about 1650 and 850 bp, respectively, of Vitreoscilla DNA downstream of vgb, was transformed into Serratia marcescens. After several transfers of the transformants on selective media, both plasmids became stable in this host and the resulting strains produced hemoglobin. Both transformants were compared, regarding growth in liquid Luria-Bertani (LB) medium, with untransformed S. marcescens and S. marcescens transformed with pUC8. The vgb-bearing strains had about 5 times lower maximum viable cell numbers than the strains without hemoglobin, but the former also had late log or early stationary phase cells that were 5-10 times larger than those of the latter. Further, on a dry cell mass basis the presence of vgb inhibited cell growth in liquid media. In contrast, growth of the vgb-bearing strains on LB plates based on cell mass (determined from colony size) was markedly enhanced compared with that of the pUC8 transformant. Respiration of the vgb-bearing strains was lower than that of the strains without vgb on a cell mass basis. These results show that the presence of vgb can have idiosyncratic effects and is not always an aid to cell growth so that its use for genetic engineering must be tested on a case by case basis.     

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.     

The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases.

Two Escherichia coli mutants that lack both cytochrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GV101, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GK100, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin but lacking the native promoter did not express the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respiration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide.     

透明颤菌血红蛋白的表达及对基因工程菌的影响

利用已克隆的透明颤菌(Vitreoscilla)血红蛋白基因(vgb),构建了一批复制类型和抗生标记不同的vgb表达载体,并就vgb基因表达及其对几种基因工程大肠杆菌的影响进行了初步研究。实验证明vgb基因的表达具有氧调控特性,在溶氧水平下跌至20％饱和度时迅速合成。Vgb基因的表达产物(Vitreoscilla Hemoglogin,VHb)可促进青霉素酰化酶和TNF、IL-2等基因工程菌在低氧条件下细胞生长和产物表达的状况,由于vgb基因的表达降低了细胞对氧的敏感程度,可望运用它来改善发酵过程中溶氧控制裕度。这些实验结果预示着vgb基因在耗氧生物过程中,如抗生素工业和基因工程菌高密度发酵,有着良好的应用前景。     

Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases

The bacterium, Vitreoscilla, can induce the synthesis of a homodimeric hemoglobin under hypoxic conditions. Expression of VHb in heterologous bacteria often enhances growth and increases yields of recombinant proteins and production of antibiotics, especially under oxygen-limiting conditions. There is evidence that VHb interacts with bacterial respiratory membranes and cytochrome bo proteoliposomes. We have examined whether there are binding sites for VHb on the cytochrome, using the yeast two-hybrid system with VHb as the bait and testing every Vitreoscilla cytochrome bo subunit as well as the soluble domains of subunits I and II. A significant interaction was observed only between VHb and intact subunit I. We further examined whether there are binding sites for VHb on cytochrome bo from Escherichia coli and Pseudomonas aeruginosa, two organisms in which stimulatory effects of VHb have been observed. Again, in both cases a significant interaction was observed only between VHb and subunit I. Because subunit I contains the binuclear center where oxygen is reduced to water, these data support the function proposed for VHb of providing oxygen directly to the terminal oxidase; it may also explain its positive effects in Vitreoscilla as well as in heterologous organisms.     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Effect of the gene for bacterial hemoglobin vhb on the effectiveness of the process of Escherichia coli-Streptomyces interspecies conjugation and production of antibiotics in streptomycetes

The bacterial hemoglobin vhb gene was cloned from sliding bacterium Vitreoscilla sp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhb gene was transferred from Escherichia coli to some Streptomyces strains, producers of antibiotics, by the method of intergeneric conjugation using conjugative-integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhb gene on processes of conjugation and antibiotic production in a number of examined strains was shown.     

Role of hemoglobin in improving biodegradation of aromatic contaminants under hypoxic conditions

Genetic engineering of bacteria using the Vitreoscilla (bacterial) hemoglobin gene has been used to enhance bioremediation of several compounds which are models for, or are themselves, toxic chemicals which may contaminate soil and water. Initial experiments, done mostly in shake flasks, with Escherichia coli, Burkholderia sp. DNT and Pseudomonas aeruginosa demonstrated that expression of Vitreoscilla hemoglobin in heterologous hosts can enhance biodegradation of several aromatic compounds as well as an organophosphorus compound. These studies concentrated for the most part on enhancement of endogenous catabolic capabilities of the hosts; the presence of vgb/VHb enhanced both growth and biodegradation. The initial studies were followed by experiments in systems which more closely approximated conditions that would exist in field applications. These included soil columns, continuous flow reactors and membrane bioreactors. The latter work also enabled calculation of the effects of the presence of vgb/VHb on kinetic parameters such as growth rate, substrate and oxygen utilization rate, and degradation rate of pollutants, etc. Although not always the case, for the most part, and particularly in bioreactors, the advantages due to vgb/VHb were greater under conditions of limited aeration or hypoxic conditions.     

透明颤菌血红蛋白基因在产PHB重组大肠杆菌中的引入

将透明颤菌血红蛋白基因 (vgb)采用插入染色体的方式引入产聚 β 羟基丁酸酯(PHB)重组大肠杆菌VG1 (pTU1 4)中 ,以从分子水平上提高克隆菌对氧的利用率 ,解决PHB发酵生产过程中的供氧矛盾 ,透明颤菌血红蛋白的一氧化碳差光谱分析明表 ,vgb基因可以在VG1 (pTU1 4)中成功表达 ,且其表达量受溶氧水平的调控。Vgb基因的引入可以同时促进菌体生长和PHB产品的积累 ,且溶氧水平越低 ,VHB表达量越高 ,这种促进作用就越明显     

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 greater chemical reactivity, protect 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 VHb counterparts.     

Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies.

The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).     

Expression of double Vitreoscilla hemoglobin enhances growth and alters ribosome and tRNA levels in Escherichia coli

In several organisms, expression of a gene encoding dimeric hemoglobin (VHb) from the obligate aerobic bacterium Vitreoscilla stercoraria has been shown to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism. In an attempt to further improve these effects of VHb, a gene encoding two vhb genes connected by a short linker of six base pairs was constructed and expressed in Escherichia coli(double VHb). Escherichia coli cells expressing double VHb reached a cell density 19% higher than that of cells expressing native VHb. The protein production per cell remained constant since the increase in cell growth was accompanied by an increase in protein content by 16%. Investigation of ribosome and tRNA content revealed that cells expressing double VHb reached their maximal capacity of protein synthesis later during cultivation than cells expressing native VHb, and furthermore they reached considerably higher levels of ribosome and tRNA compared to that of the VHb-expressing cells.     

Relationships between membrane-bound cytochrome o from Vitreoscilla and that of Escherichia coli

The cytochrome o terminal oxidases from the bacteria Vitreoscilla and Escherichia coli are structurally and functionally related. They have similar optical spectra, both exhibit ubiquinol-1 oxidase activity and are inhibited similarly. Both enzymes contain four subunits by SDS-polyacrylamide gel electrophoresis analysis and contain protoheme IX and Cu2+ prosthetic groups. Antibodies raised against the oxidase purified from E. coli crossreact with the Vitreoscilla oxidase.     

Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin

Mussel adhesive proteins (MAPs) have been considered as potential underwater and medical bioadhesives. Previously, we reported a functional expression of recombinant MAP hybrid fp-151, which is a fusion protein with six type 1 (fp-1) decapeptide repeats at each type 5 (fp-5) terminus, with practical properties in Escherichia coli. In the present work, we introduced the Vitreoscilla hemoglobin (VHb) co-expression strategy to enhance the production levels of hybrid fp-151 since VHb has been successfully used for efficient oxygen utilization in several expression systems, including E. coli. In both batch-type flask and fed-batch-type bioreactor cultures, we found that co-expression of VHb conferred higher cell growth and hybrid fp-151 production. Its positive effects were significantly increased in high cell density bioreactor cultures as the microaerobic environment was more quickly and severely formed. We obtained a approximately 1.9-fold higher (approximately 1 g/L) production of MAP fp-151 from VHb co-expressing cells in fed-batch bioreactor cultures as compared to that from VHb non-expressing cells. Collectively and regardless of the culture type, VHb co-expression strategy was successful in enhancing the production of recombinant mussel adhesive proteins in the E. coli expression system.     

透明颤菌血红蛋白的表达对酵母中麦角固醇合成的影响

构建了含透明颤菌（Vistreoscilla）血红蛋白基因vgb和酵母遗传霉素（G418）抗性基因的重组质粒pVgbkanMX4,转化至酿酒酵母Saccharomyces cerevisiae 1190中,经过分析,基因vgb在酵母细胞中得到表达。对重组菌和野生菌进行了摇瓶培养及5 L发酵罐培养的研究。在摇瓶实验中,重组菌的麦角固醇产量比野生菌有显著提高,在野生菌中的含量为0.573%、而在重组菌中的产量为1.07%。 经过30 h发酵罐培养的实验,野生菌中麦角固醇含量为0.9%,重组菌中其含量为1.38%,验证了摇瓶实验的结果。结果证明vgb基因有利于酵母中麦角固醇的合成。     

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.     

Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering

In hypoxic conditions, bacteria express a kind of hemoglobin, which is proposed to enhance respiration and energy metabolism by promoting oxygen delivery. Bacteria hemoglobin from Vitreoscilla stercoraria - Vitreoscilla hemoglobin (VHb), when expressed in various hosts in oxygen-limited conditions, has been shown to improve growth, protein secretion, metabolite productivity and stress resistance of hosts, thus rendering the protein promising in metabolic engineering, especially in plant metabolism optimization. In this review, many well-studies areas are presented to illustrate the potential of VHb application in biotechnology industry, to discuss the cellular mechanisms of VHb function and to show the wide variety of approaches taken within the field.