Abstract
Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5 years, the use of this “VHb technology” has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Vitreoscilla hemoglobin (VHb) was first studied in the 1960s (Webster and Hackett 1966) and recognized as a hemoglobin in 1986 (Wakabayashi et al. 1986). It was the first bacterial hemoglobin discovered, but in the following 28 years, it has been recognized, particularly due to the genomics revolution, that approximately two thirds of all bacterial species encode one or more hemoglobins (Vinogradov et al. 2006).
Bacterial hemoglobins fall into three main categories (Vinogradov and Moens 2008). VHb is a single domain hemoglobin (SDHb), having a single globin domain, the structure of which is closely similar to that of vertebrate globins (Bolognesi et al. 1999; Ratakonda et al. 2013). More common are the flavohemoglobins (FHbs), which have a Vitreoscilla type globin domain fused to a flavin-binding domain (Vinogradov and Moens 2008), and the truncated hemoglobins (trHbs), which have a single domain that is about 20 % smaller than SDHbs (Wittenberg et al. 2002).
As a broad group, the functions of bacterial hemoglobins are varied. These include, particularly for the FHbs and some of the trHbs, detoxification of NO through its conversion to nitrate (Pathania et al. 2002; Vinogradov and Moens 2008); oxygen sensing; and response to oxidative stress (Anand et al. 2010). Specifically, the roles of VHb have been extensively investigated. Its main role, both in native Vitreoscilla and heterologous hosts, appears to be to bind oxygen, particularly under low O2 conditions, and deliver it to the respiratory chain by direct interaction with the terminal respiratory cytochrome (Webster 1987; Aydin et al. 2000; Ramandeep et al. 2001; Park et al. 2002; Chi et al. 2009). In this way, it likely enhances aerobic oxidative phosphorylation when O2 is scarce. Other apparent roles include delivery of O2 to oxygenases (Lin et al. 2003) and the response to oxidative stress (Anand et al. 2010). The latter function involves interactions with transcription factors, which is presumably connected to transcriptional changes reported to be mediated by VHb (Roos et al. 2004). These will be discussed in more detail below.
The cloning of the VHb gene (vgb) in 1988 (Khosla and Bailey 1988; Dikshit and Webster 1988) ushered in the engineering of heterologous organisms with vgb/VHb to improve growth (Khosla and Bailey 1988) and production of useful products (Khosravi et al. 1990). The following quarter century has seen vgb/VHb engineering applied to a wide variety of bacteria, fungi, and even higher plants and animals to improve growth and survival, production of many proteins and metabolites, and bioremediation, particularly of aromatic compounds. These applications have been reviewed by us and others (Frey and Kallio 2003; Zhang et al. 2007; Stark et al. 2008; Stark et al. 2011). We have also recently reviewed what is known of the biochemical and molecular biological mechanisms which must underlie these improvements (Stark et al. 2012).
In this review, we are concerned with the biotechnological applications of engineering using vgb/VHb. We will summarize what was examined in detail in the earlier reviews but concentrate where possible on work that has been done in the last few years. Most of the work done using genetic engineering with vgb/VHb to improve host productivity has been of the “black box” variety, that is, engineering a host to express VHb and measuring an increase in production of a particular product or useful activity such as bioremediation. Since optimization of these strategies will be aided by the most comprehensive understanding possible of VHb’s structure, biochemical activities, expression control, and interactions with other proteins, these topics are also considered.
Biochemistry and molecular biology of VHb
Structure and oxygen-binding properties
In Vitreoscilla, VHb exists as a single domain dimeric protein, having two identical subunits (15.7 kD each) and two heme b’s per dimer (Webster 1987). It forms a stable oxygenated species (kD of 7.2 μM), with an average oxygen association rate constant (kon = 78 μM−1 s−1), but a high oxygen dissociation rate (koff = 5600 s−1) (Webster 1987). This unusually high Koff correlates well with its proposed function of oxygen transfer to its host under oxygen limitation, especially to enhance respiration. Immunogold labelling studies, demonstrating localization of VHb near the cell membrane (Ramandeep et al. 2001), yeast two-hybrid studies showing direct interactions of VHb with the terminal respiratory oxidases cytochrome bo (Park et al. 2002) and cytochrome d (Duk 2007), and VHb enhancement of oxygen uptake and ubiquinol oxidase activities of respiratory membranes (Ramandeep et al. 2001) all support this role.
Recombinant VHb isolated from E. coli exists in both monomeric and dimeric forms (Giangiacomo et al. 2001; Kaur et al. 2002). The structure of the VHb monomer, although conforming to a three-over-three classical globin fold, has distal and proximal heme sites distinct from those of eukaryotic Hbs, lacking a proper E helix and E7 gate for the entry and exit of ligands (Tarricone et al. 1997; Bolognesi et al. 1999). GlnE7 does not participate in ligand stabilization and ProE8, which disrupts the structure of the E helix, does not significantly affect oxygen binding (Dikshit et al. 1998; Verma et al. 2005). In the initial crystal structure, the D region appeared disordered and flexible and could not be clearly resolved (Tarricone et al. 1997).
More recent crystal structures of VHb were able to determine the positions of most of the residues in the D region; these form a loop protruding towards the heme, which favors heme-globin interactions (Ratakonda et al. 2013). Site-directed mutants of VHb had previously identified specific residues of the D-loop (Asp44, Arg47, Glu49) that are involved in heme-globin interactions as well as binding of VHb with a flavoreductase partner (Lee et al. 2004). The proximal heme pocket of VHb also plays a key role in its function (Kaur et al. 2008).
Lipid-binding properties of VHb
Our immunogold labelling studies have shown that VHb remains associated with the cell membrane in recombinant E. coli and also in its native host, Vitreoscilla (Ramandeep et al. 2001). The membrane-associating properties of VHb have been studied by investigating the interaction of VHb with an artificial monolayer formed by phospholipids; this demonstrated that VHb is able to bind and penetrate the monolayer. Additionally, VHb displays reversible binding with phospholipids and free fatty acids in solution. UV-visible spectroscopy indicated the possibility of lipid binding at the distal site, which changes the five-coordinated state of VHb into a six-coordinated state (Rinaldi et al. 2006).
Such a possibility is supported by the crystal structure of VHb, which revealed that it has a unique distal site and a flexible DE loop region that can allow binding of other molecules in the distal pocket in addition to dioxygen. Thus, lipid binding at the distal site may interfere with VHb ligand binding ability, in turn, perhaps regulating the oxygen binding and physiological function of VHb in vivo. This is supported by a more than 20-fold decrease in oxygen affinity of VHb after lipid binding in vitro (Rinaldi et al. 2006).
VHb isolated from its native host, Vitreoscilla, displays several-fold lower oxygen affinity than what has been reported for the recombinant VHb, purified from E. coli (Giangiacomo et al. 2001). This difference might be due to differences in the lipid-ligated states of the protein isolated from the two different hosts. Recombinant VHb has been purified to a very high level and might be lacking lipid-bound species, whereas VHb isolated from Vitreoscilla might retain the bound lipids as a result of its strong affinity to the cell membrane of its native host. These findings suggested that lipid/ligand-induced changes in the oxygen affinity of VHb may occur during its expression in native/heterologous hosts and may contribute to its functional diversity.
Regulation of VHb biosynthesis
Early work in Vitreoscilla showed that VHb expression is induced under low oxygen conditions (Webster 1987). Subsequent studies showed that oxygen-responsive regulation was transcriptional and operates similarly in Vitreoscilla and E. coli (Khosla and Bailey 1989; Dikshit et al. 1990). The bulk of this work has been done in E. coli, showing control by the oxygen sensitive regulators Fnr and ArcA, as well as by Crp (Joshi and Dikshit 1994; Yang et al. 2005). An OxyR binding site has also been identified within the vgb promoter, suggesting that multiple circuits may provide fine-level control of vgb expression in response to different oxygen levels and environmental stimuli (Anand et al. 2010).
VHb: one protein with many functions
As mentioned above, in addition to its proposed role in delivery of oxygen to the respiratory apparatus under hypoxia, VHb appears to have a number of other functions. These are likely related to its ability to exist in different oligomeric states (Giangiacomo et al. 2001; Kaur et al. 2002) and alter its oxygen-binding properties via interacting with cellular components, e.g., lipids, fatty acids, and especially “partner proteins”. These functions, as they are likely to occur in bacteria, are summarized in Table 1 and Fig. 1 and discussed in more detail below.
Electron transfer
VHb’s ability to interact directly with terminal respiratory oxidases may generate an alternate and/or efficient electron transfer pathway in the cell for facilitating energy generation. This may be related to its ability to act as an alternate terminal oxidase itself (Dikshit et al. 1992).
Nitric oxide dioxygenase (NOD)
VHb closely resembles the heme domain of bacterial and yeast flavohemoglobins and carries conserved residues that are known to interact with the FAD-binding domain that may allow VHb to interact transiently with a suitable reductase partner(s) (Lee et al. 2004). In fact, a flavin-binding reductase was found associated with VHb when it was purified from its native host (Gonzales-Prevatt and Webster 1980), and a VHb chimera with the reductase domain of a flavohemoglobin acquired nitric oxide dioxygenase activity and was able to detoxify NO (Kaur et al. 2002). Thus, VHb may function as an NOD under certain conditions, if associated with a suitable partner protein.
Antioxidant
A peroxidase like activity has been detected in VHb (Kvist et al. 2007), and it functions as an antioxidant in many heterologous hosts by conferring protection from oxidative stress (Geckil et al. 2003; Wang et al. 2009). This occurs through VHb interaction with OxyR and Fnr, modulating their activities in a redox dependent manner (Anand et al. 2010).
Oxygenase enhancement
Several studies have shown that VHb appears to be able to stimulate oxygenase activities (Fish et al. 2000; Urgun-Demirtas et al. 2004), perhaps via direct oxygen delivery by VHb (Lin et al. 2003).
Transcriptional control
Transcriptome and metabolic flux analyses showed that VHb expression may have significant effects on host gene expression and thus metabolism (Roos et al. 2004; Isarankura-Na-Ayudhya et al. 2008), for example, shifting metabolism to an energetically more efficient aerobic state (Ramachandran et al. 2012). VHb interaction with OxyR and Fnr (see above) transmits signals to OxyR to activate the oxidative stress regulon of the cell, simultaneously auto-regulating its own biosynthesis (Anand et al. 2010); this sustains aerobic metabolism via a steady supply of oxygen (especially under hypoxia) while simultaneously balancing the toxic effects of reactive oxygen species. This study provided an insight into the mode of the protective effect conferred by VHb during expression in different heterologous hosts (Geckil et al. 2003; Wang et al. 2009).
Protein engineering of VHb and engineering using the vgb promoter
VHb fusion proteins and use of the vgb promoter
A report by Park et al. (2003) described construction of a plasmid vector for use in E. coli which provides for fusion of a cloned gene downstream of a His-tag-vgb-protease cleavage site sequence. An improved version of the vector was described in Kwon et al. (2005). The resulting fusion proteins can be purified in one step using His-tag affinity chromatography and easily visualized during purification due to the red color of VHb. In addition, the VHb fusion can increase the solubility of proteins which, on their own, are sparingly soluble, and thus aid in increasing the yields of recombinant proteins. The protease cleavage site then can be used to remove the His-tag-VHb sequence. A related strategy used VHb fusions with a flavoreductase domain to enhance conversion of nitric oxide to nitrate (Kaur et al. 2002) and with D-amino acid oxidase, enhancing metabolism of cephalosporin C (Khang et al. 2003).
A recent report (Wu et al. 2014) investigated the use of the vgb promoter, which is inducible under low oxygen conditions, to enhance production of poly(hydroxybutyrate) (PHB) by recombinant E. coli. The rationale is that high density bacterial cultures will be starved for oxygen, and placing the genes of the PHB pathway under control of the vgb promoter will thus induce their expression under these conditions. In this case, a substantial increase in PHB production occurred using a promoter containing eight repeats of the vgb promoter organized in tandem.
VHb site-directed mutants
Several studies have investigated the effects of site-directed mutations in vgb on VHb structure and properties, with the secondary goal of producing a mutant VHb with improved ability to enhance the productivity of VHb-engineered organisms. Targeting two sites in the distal heme pocket resulted in mutant VHbs that were not better than wild-type VHb regarding oxygen binding (Dikshit et al. 1998; Verma et al. 2005). Lee et al. (2004) introduced a number of mutations into the (relatively disordered) D region of VHb, but again, none had improved ligand binding properties compared to those of wild-type VHb. The proximal heme site was investigated similarly and, while not true for all mutants, two VHbs with mutations in proximal site Tyr126 provided both improved growth properties and degradation of 2,4-dinitrotoluene (Kim et al. 2005) or production of recombinant protein (Kaur et al. 2008) for bacterial cells expressing them compared to those expressing wild-type VHb.
Enhancement of bioproduct synthesis using vgb/VHb
Earlier work
As mentioned above, the first case in which engineering of a heterologous host using vgb/VHb was used to increase production of a useful biomolecule (in this case, recombinant α-amylase in E. coli) was reported in 1990. In the following years, the same strategy was used in a wide variety of both prokaryotes and eukaryotes to enhance the production of many different biological molecules of practical importance. The recombinant organisms used included a variety of bacteria, a number of fungi, and several plants. The biomolecules produced included enzymes, amino acids, biofuels, and polymers that can replace petrochemical feed stocks (mostly in bacteria); and antimicrobials (in fungi). The applications in plants focused on improvements in growth and resistance to stress (improving, for example, tolerance to submergence in water). Detailed lists of these applications through about 2010 can be found in Stark et al. (2011). More recent work (discussed below) is summarized in Table 2. Most of the recent studies have been performed in various laboratories in China.
Recent work
Bacteria
As mentioned above, through 2010 vgb/VHb engineering had been used successfully to enhance production of a wide variety of biomolecules in a wide variety of bacterial species. Such applications have continued since then as well. Although they have been produced using conventional genetic engineering/expression strategies, the bioproducts in question have varied and important uses. Production by VHb-expressing Pseudomonas aeruginosa of rhamnolipid (a surfactant produced endogenously by P. aeruginosa) was studied by Kahraman and Erenler (2012), although no comparison with wild-type P. aeruginosa was reported. Luo et al. (2012) reported enhancement of production of the natural insecticide spinosad by Saccharopolyspora spinosa grown under both normal and limited aeration conditions. The production by Bacillus amyloliquefaciens of endogenous poly-γ-glutamic acid (a biomolecule used in a wide variety of commercial applications (Shih and Van 2001)) was increased by 30 % coincident with expression of VHb from vgb stably integrated into the host chromosome (Zhang et al. 2013), and VHb was correlated with 11 % higher biomass in E. coli cultures grown under conditions of limiting oxygen (Pablos et al. 2011).
Other recent applications in which vgb/VHb engineering has been correlated with increases in production of biochemicals include: hydroxyalkanoates by Aeromonas hydrophila (Liu et al. 2011); L-arginine by Corynebacterium crenatum (17 %; Xu et al. 2011); gellan gum by Sphingomonas elodea (up to 27 %; Wu et al. 2011); the antifungal medicinal natamycin by Streptomyces gilvosporeus (up to 175 %; Wang et al. 2014); and the antifungal and anticancer drug toyocamycin by Streptomyces diastatochromogenes (up to 210 %; Ma et al. 2014).
Expression of VHb in several bacterial species under a variety of growth conditions resulted in most cases in little or no increase in production of the anti-leukemia enzyme, L-asparaginase, although substantial increases (up to 2-fold) occurred in a few cases (Erenler and Geckil 2014); 2–3.1-fold increases in the production by Citrobacter freundii of the antileukemic methionine γ-lyase has also been reported (Kahraman et al. 2011). Engineering of Streptomyces sp. FR-008 contributed to increases in the production of the antifungal candicidin D (Wang et al. 2012).
A revisiting of whether VHb expression and consequent delivery of small amounts of oxygen to bacteria could increase the yield of biofuels via fermentation, although counter intuitive, showed promise (Sanny et al. 2010). Several studies using controlled supply of small amounts of oxygen to fermenting cultures have yielded similar results (Jansen et al. 1984; Okuda et al. 2007; Nieves et al. 2011). Cost-effective production of bioethanol and other biofuels will most likely require the use of lignocellulosic material and other waste products as carbon sources. Several recent studies in which rich media have been supplemented with such material for growth of ethanologenic E. coli have shown that substantial increases in ethanol production are correlated with vgb/VHb expression, although the amount of the increases varied with growth conditions. The supplements include hydrolyzed corn fiber (Arnaldos et al. 2012), hydrolyzed potato processing waste (Abanoz et al. 2012), and cheese whey and molasses (Akbas et al. 2014).
Yeast and fungi
In the last several years, engineering of yeast and fungi to express vgb/VHb with the aim of enhancing production of useful biomolecules of various types and functions has continued. Shen et al. (2012), for example, increased the production of amorpha-4,11-diene (a precursor to the antimalarial agent artemisinin) in S. cerevisiae by 2–3-fold by the concomitant expression of VHb, while Li and Zhang (2014) reported a VHb-correlated increase of 3.2-fold in S. cerevisiae production of the cancer and HIV drug betulinic acid. Of special interest in this area, however, is the use of the fungus Pichia pastoris.
P. pastoris has become a particularly useful host for production of recombinant enzymes because of its abilities to produce recombinant proteins at high levels and modify proteins in a manner similar to that of higher eukaryotes, its vigorous growth in simple media, and its robust genetic system (Macauley-Patrick et al. 2005). Because of the high cell densities achieved by P. pastoris, oxygen transfer to the cultures can be a problem, and several recent studies have addressed this issue using the vgb/VHb strategy.
Wu and Fu (2012) increased production of recombinant β-galactosidase in P. pastoris by 9.9 % under low aeration conditions by simultaneous expression of VHb. Growth was also increased substantially as was oxygen uptake (by about 28 %). Wu et al. (2012) also looked at the effects of expression of vgb/VHb on recombinant β-galactosidase production in P. pastoris and found that the positive effects of VHb were enhanced when cultures were grown at 23 °C compared with 30 °C. Expression of VHb in P. pastoris was correlated with an increase in production of Yarrowia lipolytica lipase by 22–84 % (depending on the culture aeration rate; greatest relative increase at low aeration); again, growth and oxygen uptake were also increased in the VHb-expressing strain (Wang et al. 2012).
Eukaryotes other than fungi
A particularly interesting application of vgb/VHb technology has been the engineering of higher plants with vgb under the transcriptional control of higher plant promoters. Between 2004 and 2009, several such studies were reported (reviewed in Stark et al. 2011). The early studies focused mostly on plants of agricultural or biofuel use (rice, cabbage, and white poplar) and on improving characteristics of direct practical importance (growth; productivity; resistance to submergence, nitrosative, and oxidative stress).
Recent work on aspen has determined that VHb expression had little effect on the stress response of the tree to herbivory (in this case, by Lepidopteran larvae) or survival of these herbivores (Sutela et al. 2013). Vgb/VHb was used to engineer the legume Astragalus membranaceus, an important herb in traditional Chinese medicine, which produces astragaloside IV, a biochemical with various medicinal activities. Vgb was transferred to A. membranaceus by Agrobacterium tumefaciens, and the production of astragaloside IV in the resulting hairy roots increased by 5–6-fold compared to hairy roots not expressing VHb (Wang et al. 2011).
The eukaryotic microalga Aurantiochytrium, a source of fatty acids with potential for biodiesel and the antioxidant food supplement astaxanthin, was engineered to express VHb, resulting in an up to 44 % increase in the former product and a 9-fold increase in the latter product (Suen 2013). In another interesting pilot study, in this case related to aquaculture farming, zebrafish were engineered to express VHb and the resulting transgenics had greater survival rates than non-VHb-expressing fish when grown under the stress of low dissolved oxygen levels (Guan et al. 2011).
Applications of VHb for environmental biotechnology
Earlier work
Environmental biotechnology applications of vgb/VHb include systems to remediate pollution of air, water, and soil by biological treatment. The earliest reported use of vgb/VHb-engineered bacteria for bioremediation involved simulated studies with benzoic acid as a model pollutant using Xanthomonas maltophilia; in this case, there was limited enhancement compared to the wild type (Liu et al. 1996). Subsequent bioremediation work with vgb-engineered bacterial strains involved other organic contaminants, namely, 2,4-dinitrotoluene using Burkholderia strain DNT (Patel et al. 2000; Fish et al. 2000; Nasr et al. 2001; Lin et al. 2003; So et al. 2004); organophosphorus using E. coli (Kang et al. 2002); 2-chlorobenzoic acid using Burkholderia cepacia (Urgun-Demirtas et al. 2003; 2004; 2005; 2006); benzene, toluene, xylene (BTX) using Pseudomonas aeruginosa (Kahraman and Geckil, 2005); and benzoic acid using Pseudomonas aeruginosa (Chung et al. 2001; Kim et al. 2005).
The lab scale experimental systems used in these studies included batch shake flasks, continuous flow chemostat bioreactors, continuous flow membrane bioreactors, and continuous flow sand column bioreactors using vgb-bearing and wild-type strains of the respective species. The key findings included improvement in the extent and rate of contaminant degradation under hypoxic conditions, pathway modification to enable complete mineralization of the contaminant, improvement in the growth rate of the bacteria used, and improved oxygen utilization rates under hypoxic conditions. The mechanistic basis for such improvements were found/reasoned to be due to VHb-related increases in the supply of oxygen to the oxygenases in the reaction pathways and/or increased expression of degradative enzymes as a result of increases in oxygen supply to the respiratory chain.
Recent work
The recent work on investigations and applications of hemoglobin technology involve enhancements under microaerobic or hypoxic conditions in the transformation of inorganics such as ammonia nitrogen, sequestering of metals, and solubilization of phosphate by increased production of organic acids (Table 3).
Nitrification of ammonia to nitrite/nitrate is one of the most important biochemical reactions in mineralization of naturally occurring organic nitrogen and in control of N pollution. Arnaldos et al. (2013; 2014) gave a new direction to bacterial hemoglobin technology by promoting/enriching the ability of native bacteria in mixed cultures to express hemoglobin type proteins under hypoxic conditions. The first step in oxidation of ammonia to nitrite/nitrate involves ammonia monooxygenase (AMO), the oxygen substrate requirements of which could theoretically be met, under low bulk dissolved oxygen (DO) concentrations, through delivery of oxygen by hemoglobin. This, in turn, could greatly reduce the oxygen supply requirements of nitrification and, hence, the energy needed to provide oxygen by conventional aeration.
Arnaldos et al. (2013) investigated mixed culture nitrifying activated sludge from a biological wastewater treatment plant and enriched it to nitrify at low DO concentration (∼0.1 mg O2/L) in a lab scale sequencing batch reactor (SBR). The low DO SBR completely nitrified the influent ammonia after a prolonged acclimation period (greater than 140 days) and performed comparably to a high DO SBR (near saturation DO). Achievement of complete nitrification in the low DO reactor coincided with the increased specific oxygen uptake rate of the biomass compared to the high DO reactor biomass and expression of a CO-binding soluble heme protein. The heme protein expressed was linked to ammonia oxidizing bacteria (AOB), which were dominant in the biomass, and was excluded as any one of the enzymes or proteins in the conventional account of ammonia oxidation by AOB.
Arnaldos et al. (2014) further investigated this heme protein by conducting bioassays targeting its function and activity, location in the cell, and the organisms that express it. It was shown that the heme protein is preferentially expressed in the cytoplasm by AOB, with heme c as its prosthetic group. Activity assays indicated that it is neither a peroxidase nor oxidase and none of the known heme proteins involved in ammonia oxidation. Whether this heme protein is a hemoglobin or not has not been established, but if it is, it would open a new area of research into methods to enhance wastewater treatment under low oxygen conditions.
Earlier, it was shown that VHb expression by Gordonia amarae enhanced production of extracellular biosurfactants (Dogan et al. 2006). The ability of such extracellular polymers to sequester heavy metals was studied by Kahraman et al. (2014). They showed that engineering Pseudomonas aeruginosa with vgb enhanced Pb, Co, and Cu uptake by the cells. Liao et al. (2014) showed that a Bacillus sp. expressing vgb had enhanced Mn oxidation in Mn (II)-contaminated water under oxygen-restricted conditions. It was hypothesized that VHb increases the concentration of intracellular oxygen needed to oxidize Mn (II).
Yadav et al. (2014) studied VHb enhanced solubilization of phosphate, as in soils, under hypoxic conditions through the increased expression of citrate, a chelating agent. An artificial citrate operon comprised of genes encoding citrate synthase and engineered to contain vgb was constructed and transformed into Enterobacter hormaechei. The transformant secreted citric acid and released soluble phosphate from rock phosphate, whereas the native strain could not. This may have applications in enhancing mineral phosphate solubilization under buffered, microaerobic conditions, such as in the rhizospheric environment.
Conclusions, insights, future directions
To date, the number and variety of bioproducts enhanced by VHb expression in heterologous hosts seems limited only by the imagination of the investigators, and we can expect these applications to grow in number in the future (Table 4). Specifically, regarding biofuel (e.g., ethanol, butanol) production, enhancement by VHb expression will need to be shown to occur both at large scale and in media in which low cost carbon sources such as food processing wastes and lignocellulosic material do not need to be supplemented with the expensive components of traditional rich microbiological media. The uses of VHb technology regarding bioremediation (including its potential in normal waste water treatment) are fewer in number, but future applications might also be expected in novel areas. Finally, as with any practical application in biology, continuing investigation into the biochemical activities of VHb, which seem to be many and varied and involve interaction with a variety of partner proteins with varied functions, will certainly aid in developing more systematic and successful employment of VHb technology.
References
Abanoz K, Stark BC, Akbas MY (2012) Enhancement of ethanol production from potato processing waste water by engineering Escherichia coli using Vitreoscilla hemoglobin. Lett Appl Microbiol 55:436–443
Akbas MY, Sar T, Ozcelik B (2014) Improved ethanol production from cheese whey, whey powder, and sugar beet molasses by “Vitreoscilla hemoglobin expressing” Escherichia coli. Biosci Biotechnol Biochem 78:687–694
Anand A, Duk BT, Singh S, Akbas MY, Webster DA, Stark BC, Dikshit KL (2010) Redox mediated interactions of Vitreoscilla hemoglobin [VHb] with OxyR: novel regulation of VHb biosynthesis under oxidative stress. Biochem J 426:271–280
Arnaldos M, Kunkel SA, Stark BC, Pagilla KR (2013) Enhanced heme protein expression by ammonia-oxidizing communities acclimated to low dissolved oxygen conditions. Appl Microb Biotech 97:10211–10221
Arnaldos M, Kunkel SA, Stark BC, Pagilla KR (2014) Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions. Appl Microb Biotech 98:3231–3239
Arnaldos M, Kunkel SA, Wang J, Pagilla KR, Stark BC (2012) Vitreoscilla hemoglobin enhances ethanol production by Escherichia coli in a variety of growth media. Biomass Bioenergy 37:1–8
Aydin S, Webster DA, Stark BC (2000) Nitrite inhibition of Vitreoscilla hemoglobin (VHb) in recombinant E. coli: evidence that VHb enhances recombinant protein production. Biotechnol Prog 16:917–921
Bolognesi M, Boffi A, Coletta M, Mozzarelli A, Pesce A, Tarricone C, Ascenzi P (1999) Anti co-operative ligand binding properties of recombinant ferric Vitreoscilla homodimeric haemoglobin: a thermodynamic, kinetic and X-Ray crystallographic study. J Mol Biol 291:637–650
Chi P, Webster DA, Stark BC (2009) Vitreoscilla hemoglobin aids respiration under hypoxic conditions in its native host. Microbiol Res 164:267–275
Chung JW, Webster DA, Pagilla KR, Stark BC (2001) Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability. J Ind Microbiol Biotechnol 27:27–33
Dikshit RP, Dikshit KL, Liu XY, Webster DA (1992) The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Arch Biochem Biophys 293:241–245
Dikshit KL, Dikshit RP, Webster DA (1990) Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. Nucl Acids Res 18:4149–4155
Dikshit KL, Orii Y, Navani N, Patel S, Huang H-Y, Stark BC, Webster DA (1998) Site-directed mutagenesis of bacterial hemoglobin: the role of glutamine (E7) in oxygen-binding in the distal heme pocket. Arch Biochem Biophys 349:161–166
Dikshit KL, Webster DA (1988) Cloning, characterization and expression of the bacterial globin gene from Vitreoscilla in Escherichia coli. Gene 70:377–386
Dogan I, Pagilla KR, Webster DA, Stark BC (2006) Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production. J Indust Microbiol Biotechnol 33:693–700
Duk BT (2007) Exploration of protein-protein interactions between Vitreoscilla hemoglobin (VHb) and E. coli Fnr, OxyR, and Cyda. PhD thesis, Illinois Institute of Technology, Chicago, IL USA
Erenler SO, Geckil H (2014) Effect of Vitreoscilla hemoglobin and culture conditions on production of bacterial L-asparaginase, an oncolytic enzyme. Appl Biochem Biotechnol 173:2140–2151
Fish PA, Webster DA, Stark BC (2000) Vitreoscilla hemoglobin enhances the first step in 2,4-dinitrotoluene degradation in vitro and at low aeration in vivo. J Mol Cat B: Enzymatic 9:75–82
Frey AD, Kallio PT (2003) Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology. FEMS Microbiol Rev 27:525–45
Geckil H, Gencer S, Kahraman H, Erenler SO (2003) Genetic engineering of Enterobacter aerogenes with Vitreoscilla hemoglobin gene: cell growth, survival and antioxidant enzyme status under oxidative stress. Res in Microbiol 154:425–431
Giangiacomo L, Mattu M, Arcovito A, Bellenchi G, Bolognesi M, Ascenzi P, Boffi A (2001) Monomer-dimer equilibrium and oxygen binding properties of ferrous Vitreoscilla hemoglobin. Biochemistry 40:9311–9216
Gonzales-Prevatt V, Webster DA (1980) Purification and properties of NADH cytochrome o reductase from Vitreoscilla. J Biol Chem 255:1478–1482
Guan B, Ma H, Wang Y, Hu Y, Lin Z, Zhu Z, Hu W (2011) Vitreoscilla hemoglobin (VHb) overexpression increases hypoxia tolerance in zebrafish (Danio rerio). Mar Biotechnol (NY) 13:336–344
Isarankura-Na-Ayudhya C, Panpumthong P, Tangkosakul T, Boonpangrak S, Prachayasittikul V (2008) Shedding light on the role of Vitreoscilla hemoglobin on cellular catabolic regulation by proteomic analysis. Int J Biol Sci 3:71–80
Jansen NB, Flickinger MC, Tsao GT (1984) Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724. Biotechnol Bioeng 26:362–369
Joshi M, Dikshit KL (1994) Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochem Biophys Res Comm 202:535–542
Kahraman H, Aytan E, Kurt AG (2011) Production of methionine γ-lyase in recombinant Citrobacter freundii bearing the hemoglobin gene. BMB Rep 44:590–594
Kahraman H, Erenler SO (2012) Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene. Prikl Biokhim Mikrobiol 48:212–217
Kahraman H, Geckil H (2005) Degradation of benzene, toluene, and xylene with Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene. Eng Life Sci 5:363–368
Kahraman H, Kurt AG, Aytan E (2014) Effect of heavy metals on recombinant Pseudomonas aeruginosa carrying Vitreoscilla hemoglobin gene. Electron J Biol 10:40–45
Kang DG, Kim JYH, Cha HJ (2002) Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorous hydrolase and bacterial hemoglobin. Biotechnol Lett 24:879–883
Kaur R, Ahuja S, Anand A, Singh B, Stark BC, Webster DA, Dikshit KL (2008) Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (VHb): role of Tyr95(G5) and Tyr126(H12). FEBS Lett 582:3494–3500
Kaur R, Pathania R, Sharma V, Mande SC, Dikshit KL (2002) Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb. Appl Environ Microbiol 68:152–160
Khang YH, Kim IW, Hah YR, Hwangbo JH, Kang KK (2003) Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C. Biotechnol Bioeng 82:480–488
Khosla C, Bailey JE (1988) Heterologous expression of a bacterial hemoglobin improves the growth properties of recombinant Escherichia coli. Nature 331:633–635
Khosla C, Bailey JE (1989) Characterization of the oxygen dependent promoter of the Vitreoscilla haemoglobin gene in Escherichia coli. J Bacteriol 171:5995–6004
Khosravi M, Webster DA, Stark BC (1990) Presence of the bacterial hemoglobin gene improves alpha-amylase production of a recombinant E. coli strain. Plasmid 24:190–194
Kim Y, Webster DA, Stark BC (2005) Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene (vgb) integrated into their chromosomes. J Ind Microbiol Biotechnol 32:148–154
Kvist M, Ryabova ES, Nordlander E, Bulow L (2007) An investigation of the peroxidase activity of Vitreoscilla hemoglobin. J Biol Inorg Chem 12:324–334
Kwon S-Y, Choi Y-J, Kang T-H, Lee K-H, Cha S-S, Kim G-H, Lee H-S, Kim K-T, Kim K-J (2005) Highly efficient protein expression and purification using bacterial hemoglobin fusion vector. Plasmid 53:274–282
Lee SY, Stark BC, Webster DA (2004) Structure-function of the Vitreoscilla hemoglobin D-region. Biochem Biophys Res Comm 316:1101–1106
Li J, Zhang Y (2014) Modulating betulinic acid production in Saccharomyces cerevisiae by managing the intracellular supplies of the cofactor NADPH and oxygen. J Biosci Bioeng. doi:10.1016/j.biosc.2014.06.013
Liao B, Wang Y, Su J, Liu F, He J (2014) Expression of Vitreoscilla hemoglobin in Bacillus thuringiensis BMB171 can promote manganese (II) oxidation under oxygen-restricted conditions. Annals Microbiol. doi:10.1007/s13213-014-0825-z
Lin JM, Stark BC, Webster DA (2003) Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (DNT) dioxygenase activity of Burkholderia and on DNT degradation in two-phase bioreactors. J Indust Microbiol Biotechnol 30:362–368
Liu F, Jian J, Shen X, Chung A, Chen J, Chen G-Q (2011) Metabolic engineering of Aeromonas hydrophila 4AK4 for production of copolymers of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoate. Bioresource Technol 102:8123–8129
Liu SC, Liu XY, Webster DA, Stark BC (1996) Genetic engineering to contain the Vitreoscilla hemoglobin gene enhances degradation of benzoic acid by Xanthomonas maltophilia. Biotechnol Bioeng 49:101–105
Luo Y, Kou X, Ding X, Hu S, Tang Y, Li W, Huang F, Yang O, Chen H, Xia L (2012) Promotion of spinosad biosynthesis by chromosomal integration of the Vitreoscilla hemoglobin gene in Saccharopolyspora spinosa. Sci China Life Sci 55:172–180
Ma Z, Liu J, Bechthold A, Tao L, Shentu X, Bian Y, Yu X (2014) Development of intergeneric conjugal gene transfer system in Streptomyces diastatochromogenes 1628 and its application for improvement of toyocamycin production. Curr Microbiol 68:180–185
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Nasr MA, Hwang K-W, Akbas M, Webster DA, Stark BC (2001) Effects of culture conditions on enhancement of 2,4-dinitrotoluene degradation by Burkholderia engineered with the Vitreoscilla hemoglobin gene. Biotechnol Prog 17:359–361
Nieves IU, Geddes CC, Mullinnix MT, Hoffman RW, Tong Z, Castro E, Shanmugam KT, Ingram LO (2011) Injection of air into the headspace improves fermentation of phosphoric acid pretreated sugarcane bagasse by Escherichia coli MM170. Bioresource Technol 102:6959–65
Okuda N, Ninomiya K, Takao M, Katakura Y, Shioya S (2007) Microaeration enhances productivity of ethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11. J Biosci Bioeng 103:350–57
Pablos TE, Mora EM, Le Borgne S, Ramirez OT, Gosset G, Lara AR (2011) Vitreoscilla hemoglobin expression in engineered Escherichia coli: improved performance in high cell-density batch cultivations. Biotechnol J 6:993–1002
Park KW, Kim KJ, Howard AJ, Stark BC, Webster DA (2002) Vitreoscilla hemoglobin binds to subunit I of cytochrome bo ubiquinol oxidases. J Biol Chem 277:33334–33337
Park KW, Webster DA, Stark BC, Howard AJ, Kim KJ (2003) Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli. Plasmid 50:169–175
Patel SM, Stark BC, Hwang K-W, Dikshit KL, Webster DA (2000) Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation. Biotechnol Prog 16:26–30
Pathania R, Navani NK, Gardner AM, Gardner PR, Dikshit KL (2002) Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli. Mol Microbiol 45:1303–1314
Ramachandran B, Dikshit KL, Dharmlingam K (2012) Recombinant E. coli expressing Vitreoscilla haemoglobin prefers aerobic metabolism under microaerobic conditions: a proteome-level study. J Biosci 37:617–633
Ramandeep HK-W, Raje M, Kim KJ, Stark BC, Dikshit KL, Webster DA (2001) Vitreoscilla hemoglobin: intracellular localization and binding to membranes. J Biol Chem 276:24781–24789
Ratakonda S, Anand A, Dikshit KL, Stark BC, Howard AJ (2013) Crystallographic structure determination of B10 mutants of Vitreoscilla hemoglobin: role of Tyr29 (B10) in the structure of the ligand binding site. Acta Crystallograp Sect F: Struct Biol Crystalliz Comm 69:215–222
Rinaldi AC, Bonamore A, Macone A, Boffi A, Bozzi A, DiGiulio A (2006) Interaction of Vitreoscilla hemoglobin with membrane lipids. Biochemistry 45:4069–4076
Roos V, Andersson CI, Bülow L (2004) Gene expression profiling of Escherichia coli expressing double Vitreoscilla haemoglobin. J Biotechnol 114:107–120
Sanny T, Arnaldos M, Kunkel SA, Pagilla KR, Stark BC (2010) Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl Microbiol Biotechnol 88:1103–1112
Shen J, Zheng H, Zhi X, Shi Y, Huang Y, Wang W, Chen Y, Kong J, Zhu P (2012) Improvement of amorpha-4,11-diene production by a yeast-conform variant of Vitreoscilla hemoglobin. Z Naturforsch C 67:195–207
Shih IL, Van YT (2001) The production of poly-(γ-glutamic acid) from microorganisms and its various applications. Biores Technol 79:207–22
So J, Stark BC, Webster DA, Pagilla KR (2004) Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp. in sand bioreactors using bacterial hemoglobin technology. Biodegradation 15:161–171
Stark BC, Dikshit KL, Pagilla KR (2011) Recent advances in understanding the structure, function, and biotechnological usefulness of the hemoglobin from the bacterium Vitreoscilla. Biotechnol Lett 33:1705–1714
Stark BC, Dikshit KL, Pagilla KR (2012) The Biochemistry of Vitreoscilla hemoglobin. Computat Struct Biotechnol J 3:e201210002
Stark BC, Urgun-Demirtas M, Pagilla KR (2008) Role of hemoglobin in improving biodegradation of aromatic contaminants under hypoxic conditions. J Mol Microbiol Biotechnol 15:181–189
Suen Y (2013) Expression of Vitreoscilla hemoglobin in Aurantiochytrium sp. enhances the production of fatty acids and astaxanthin. PhD thesis, The University of Hong Kong (Pokfulam, Hong Kong)
Sutela S, Ylioja T, Jokipii-Lukkari S, Anttila A, Julkunen-Tiito R, Niemi K, Mollari T, Kallio P, Haggman H (2013) The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves. J Plant Res 126:795–809
Tarricone C, Galizzi A, Coda A, Ascenzi P, Bolognesi M (1997) Unusual structure of the oxygen binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 5:497–507
Urgun-Demirtas M, Pagilla KR, Stark BC (2004) Enhanced kinetics of genetically engineered Burkholderia cepacia: role of vgb in the hypoxic metabolism of 2-CBA. Biotechnol Bioeng 87:110–118
Urgun-Demirtas M, Pagilla KR, Stark BC (2006) Comparison of 2-chlorobenzoic acid biodegradation in a membrane bioreactor by B. cepacia and B. cepacia bearing the bacterial hemoglobin gene. Water Res 40:3123–3130
Urgun-Demirtas M, Pagilla KR, Stark BC, Webster DA (2003) Biodegradation of 2-chlorobenzoate by recombinant Burkholderia cepacia expressing Vitreoscilla hemoglobin under hypoxic conditions. Biodegradation 14:357–365
Urgun-Demirtas M, Stark BC, Pagilla KR (2005) 2-Chlorobenzoate biodegradation by recombinant Burkholderia cepacia under hypoxic conditions in a membrane bioreactor. Water Env Res 77:511–518
Verma S, Patel S, Kaur R, Chung YT, Duk BT, Dikshit KL, Stark BC, Webster DA (2005) Mutational study of the bacterial hemoglobin distal pocket. Biochem Biophys Res Comm 326:290–297
Vinogradov SN, Hoogewijs D, Bailey X, Arredondo-Peter R, Gough J, Dewilde S, Moens L, Vanfleteren JR (2006) A phylogenomic profile of globins. BMC Evol Biol 6:31
Vinogradov SN, Moens L (2008) Diversity of globin function: enzymatic, transport, storage, and sensing. J Biol Chem 283:8773–8777
Wakabayashi S, Matsubara H, Webster DA (1986) Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322:481–483
Wang S, Liu F, Hou Z, Zong G, Zhu X, Ling P (2014) Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb). World J Microbiol Biotechnol 30:1369–1376
Wang T, Bai L, Zhu D, Lei X, Liu G, Deng Z, You D (2012) Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes. Enz Microb Technol 50:5–9
Wang Z, Xiao Y, Chen W, Tang K, Zhang L (2009) Functional expression of Vitreoscilla hemoglobin (VHb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism. Plant Sci 176:66–77
Wang ZY, Hu ZB, Wang ZT (2011) Regulation of Vitreoscilla hemoglobin on biosynthesis of astragaloside IV. Yao Xue Xue Bao 46:355–360
Webster DA (1987) Structure and function of bacterial hemoglobin and related proteins. In: Eichorn GC, Marzilli LG (eds) Advances in inorganic biochemistry, vol 7. Elsevier, New York, pp 245–265
Webster DA, Hackett DP (1966) The purification and properties of cytochrome o from Vitreoscilla. J Biol Chem 241:3308–3315
Wittenberg JB, Bolognesi M, Wittenberg BA, Guertin M (2002) Truncated hemoglobins: a new family of hemoglobins widely distributed in bacteria, unicellular eukaryotes, and plants. J Biol Chem 277:871–874
Wu H, Wang H, Chen J, Chen G-Q (2014) Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically. Appl Microbiol Biotechnol 98:10013–10021
Wu JM, Fu WC (2012) Intracellular co-expression of Vitreoscilla hemoglobin enhances cell performance and β-galactosidase production in Pichia pastoris. J Biosci Bioeng 113:332–337
Wu JM, Wang SY, Fu WC (2012) Lower temperature cultures enlarge the effects of Vitreoscilla hemoglobin expression on recombinant Pichia pastoris. Int J Molec Sci 13:13212–13226
Wu XC, Chen YM, Li YD, Li O, Zhu L, Qian CD, Tao XL, Teng Y (2011) Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production. J Appl Microbiol 110:422–430
Xu M, Rao Z, Xu H, Lan C, Dou W, Zhang X, Xu H, Jin J, Xu Z (2011) Enhanced production of L-arginine by expression of Vitreoscilla hemoglobin using a novel expression system in Corynebacterium crenatum. Appl Biochem Biotechnol 163:707–719
Yadav K, Kumar C, Archana G, Kumar NG (2014) Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS. Appl Microb Biotech 98:8327–8336
Yang J, Webster DA, Stark BC (2005) ArcA works with Fnr as a positive regulator of Vitreoscilla (bacterial) hemoglobin gene expression in Escherichia coli. Microbiol Res 160:405–415
Zhang L, Li Y, Wang Z, Xia Y, Chen W, Tang K (2007) Recent developments and future prospects of Vitreoscilla hemoglobin applications in metabolic engineering. Biotechnol Adv 25:123–136
Zhang W, Xie H, He Y, Feng J, Gao W, Gu Y, Wang S, Song C (2013) Chromosome integration of the Vitreoscilla hemoglobin gene (vgb) mediated by temperature-sensitive plasmid enhances γ-PGA production in Bacillus amyloliquefaciens. FEMS Microbiol Lett 343:127–134
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Stark, B.C., Pagilla, K.R. & Dikshit, K.L. Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Appl Microbiol Biotechnol 99, 1627–1636 (2015). https://doi.org/10.1007/s00253-014-6350-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-014-6350-y