Next Article in Journal
Supranational Assessment of the Quality of Probiotics: Collaborative Initiative between Independent Accredited Testing Laboratories
Next Article in Special Issue
Effects of Efficient Expression of Vitreoscilla Hemoglobin on Production, Monosaccharide Composition, and Antioxidant Activity of Exopolysaccharides in Ganoderma lucidum
Previous Article in Journal
First Evidence That Nematode Communities in Deadwood Are Related to Tree Species Identity and to Co-Occurring Fungi and Prokaryotes
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 9
Issue 7
10.3390/microorganisms9071455
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin

by
Fei Yu
1,2,
Xinrui Zhao
1,2,*,
Ziwei Wang
1,
Luyao Liu
1,
Lingfeng Yi
1,
Jingwen Zhou
1,2,
Jianghua Li
1,2,
Jian Chen
1,2 and
Guocheng Du
1,2,3,*
1
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
2
Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
3
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
*
Authors to whom correspondence should be addressed.
Microorganisms 2021, 9(7), 1455; https://doi.org/10.3390/microorganisms9071455
Submission received: 27 June 2021 / Revised: 3 July 2021 / Accepted: 4 July 2021 / Published: 7 July 2021
(This article belongs to the Special Issue Microbial Hemoglobins)
Browse Figures
Versions Notes

Abstract

:
Vitreoscilla hemoglobin (VHb), the first discovered bacterial hemoglobin, is a soluble heme-binding protein with a faster rate of oxygen dissociation. Since it can enhance cell growth, product synthesis and stress tolerance, VHb has been widely applied in the field of metabolic engineering for microorganisms, plants, and animals. Especially under oxygen-limited conditions, VHb can interact with terminal oxidase to deliver enough oxygen to achieve high-cell-density fermentation. In recent years, with the development of bioinformatics and synthetic biology, several novel physicochemical properties and metabolic regulatory effects of VHb have been discovered and numerous strategies have been utilized to enhance the expression level of VHb in various hosts, which greatly promotes its applications in biotechnology. Thus, in this review, the new information regarding structure, function and expressional tactics for VHb is summarized to understand its latest applications and pave a new way for the future improvement of biosynthesis for other products.

1. Introduction

Vitreoscilla hemoglobin (VHb) is the first bacterial hemoglobin discovered in gram-negative bacterium Vitreoscilla sp. C1 [1]. Vitreoscilla was found in oxygen-limited conditions like stagnant ponds and decaying vegetable matter [2,3], but it is strictly aerobic based on the special VHb to adapt to hypoxic conditions. VHb was originally named “cytochrome o (Cyo)” because of some similar properties with cytochromes [4,5]. Subsequently, the amino acid sequencing of “Cyo” was completed and showed that it had a high homology with eukaryotic hemoglobins [2].
VHb is a single-domain hemoglobin (SDHb) that is different from the two other two kinds of bacterial hemoglobins, FHbs (flavohemoglobins, a VHb-like globin fused with flavin-binding domain) and trHbs (truncated hemoglobins, a single-domain hemoglobin approximately 20% smaller than SDHb) [6]. Based on the unique structure of VHb, it can efficiently bind and transport oxygen to the respiratory chain by interacting with terminal oxidase, especially under oxygen-limited conditions [6]. In addition, VHb also can interact with transcriptional regulators responsible for oxygen response, triggering oxidative phosphorylation in the cells [6].
Based on its powerful oxygen transport capacity, VHb has been widely applied in the field of metabolic engineering for microorganisms, plants and animals. By enhancing the regeneration of ATP and NAD+ and improving the activity of the TCA cycle [7,8], VHb can be used to promote the growth of microbial, plant and animal cells [7,9], improve the synthesis of target products under oxygen-limited conditions [10], and increase the effect of microorganisms on bioremediation [11].
In recent years, with the development of bioinformatics and synthetic biology, several novel physicochemical properties and functions of VHb were discovered and numerous strategies were utilized to enhance the expression level of VHb in various hosts, resulting in its wide application in biotechnology. Therefore, in this review, the information of structure and functions for VHb are summarized to make VHb become a promising and practical tool in metabolic engineering.

2. Biochemical Function of VHb

2.1. The Oxygen-Binding Property of VHb

VHb is a homodimer composed of two identical subunits (146 amino acids for each subunit) and two molecules of b-type heme. In the early studies, VHb was considered as a cytochrome o [1]. Subsequently, researchers gradually recognized that VHb is a kind of hemoglobin based on its primary structure, spectral properties, and oxygen binding kinetics [2]. Under different environmental conditions, VHb can present in three different states: oxidized state, reduced state, and oxygenated state. When the iron atom in the heme of VHb is in the ferrous state, it presents in a reduced state and can reversibly combine with the oxygen. The oxygenated state is the transition state between the reduced state and oxidized state and is also the most important stable state that participates in oxygen related metabolic pathways and improves the efficiency of oxidative phosphorylation in the respiratory cells [12,13].
Compared with other eukaryotic hemoglobins, the rate constant of VHb binding to oxygen (kon = 78 μM−1s−1) is in the average level, but the dissociation rate constant of VHb and oxygen (koff = 5000 s−1) is hundreds of times higher, meaning VHb is apt to release a large amount of oxygen [13]. In Vitreoscilla and Escherichia coli, the cellular VHb localizes in the periplasmic space and close to cell membrane, which facilitates it functioning as a respirator to transport oxygen to the cell membrane under hypoxic conditions [14]. Whereas in yeast, it was confirmed by two-hybrid experiments that VHb can interact with subunit I of cytochrome bo ubiquinol oxidase and enhance its activity [15].

2.2. The Activity of Terminal Oxidase and Peroxidase

On the one hand, as early as thirty years ago, it has been verified that VHb has terminal oxidase activity. After the vgb gene (Vitreoscilla hemoglobin gene) was transformed, under the condition of succinate or lactate for substrates, the E. coli mutant that lacks cytochrome o and d terminal oxidases could perform aerobic respiration and grow normally [16]. On the other hand, the activity of peroxidase was detected for VHb through in vitro experiments, and many factors (pH, temperature, etc.) can affect its activity [17,18]. In the following, in order to enhance the peroxidase activity of VHb for the application in biomedicine and dye decolorization, several mutated VHb that presenting high peroxidase activity under specific pH conditions were obtained [19]. Based on these mutants of VHb, it was found that the conserved amino acid residues 53 and 54 (glutamine and proline) in the distal pocket of VHb are closely related to its peroxide activity. Aiming for these two key catalytic sites, more and more designed mutants (P54R or Q53H/P54C) with higher peroxidase activity were obtained by site-directed mutations [20,21].

2.3. The Potential Sulfide Receptor and Storage

Different from the classic H2S-binding monomeric hemoglobin from Lucina pectinate [22], VHb exhibits unusual characteristics in its reactivity with H2S, such as steric constraints at position E11 (Leu), that play important roles in regulating the binding stability of H2S and VHb. The kinetic parameters for interaction between VHb and H2S were determined by UV-visible spectroscopic analysis and Resonance Raman (RR) spectroscopic analysis (Kon = 1.2 × 105 M−1S−1 and Koff = 2.5 × 10−4 S−1), indicating that VHb serves as a potential sulfide receptor and has a storage function in the cells [23].

2.4. Other Functions

Besides the functions mentioned above, VHb also has the properties of lipid binding. VHb not only can interact with the monolayers formed by natural phospholipids but also reversibly binds to free fatty acids [24]. Since the binding site is located in the distal pocket of the heme, combination with lipids may affect the oxygen affinity of VHb and its physiological functions [24]. In addition, VHb can also interact with other intracellular enzymes or transcriptional regulators to increase their activities or activate the downstream metabolic pathways (Table 1).

3. Structure and Bioinformatics Analysis of VHb and Its Mutants

3.1. The Structure of VHb and Its Mutants

Based on the analysis of crystal structure, VHb forms six α-helix regions (A, B, E, F, G and H), which is different from other eukaryotic hemoglobins with eight α-helix regions (A, B, C, D, E, F, G and H), and has a unique distal heme pocket [28]. In addition, there are four residues (TyrB10, GlnE7, ProE8 and LeuE11) that are closely related to the oxygen-binding property [29]. Notably, unlike most eukaryotic hemoglobins, the distal His (E7) residue in VHb is substituted by Gln residue, which cannot form hydrogen bonds with oxygen [30]. Furthermore, the Gln (E7) residue in VHb is responsible for the disorder of the D-helix region that forms between the polypeptide segment from Phe-43 (CD1) to Leu-57 (E11), leading to the weaker affinity to oxygen, higher oxygen dissociation constant (Koff) and rapid rate of oxygen transfer [30].
Apart from the wild-type VHb, the effect of key amino acid residues on the structure and function of VHb mutants was studied through site-directed mutagenesis. At first, the Tyr-29 (B10) played an important role in maintaining the stability of oxygen binding [31]. Next, the structure of the TyrB10Phe mutant is almost indistinguishable from the wild type, and the structure related to D-region ordering and E7 chain of the TyrB10Ala mutant is significantly different from the wild type [31]. Moreover, VHb also had a unique proximal heme pocket, with the structure being formed by a hydrogen-bonding network consisting of HisF8-TyrG5-GluH23 and TyrG5-TyrH12 [32]. In addition, the TyrG5Phe and TyrG5Leu mutants cannot form a stable oxygenated state and do not exhibit any nitric oxide dioxygenase activity [32]. However, the TyrH12Phe and TyrH12Leu mutants showed little effect on the oxygen-binding capacity, which is inconsistent with the previous predicted results that TyrH12Leu mutation could enhance oxygen diffusion and accumulation [32].

3.2. The Homology Analysis of VHb

It has been reported that VHb has a lower homology with eukaryotic hemoglobins and the highest homology only can reach 24% (leghemoglobin from Lupinus luteus). However, after the alignment of amino acid sequence between VHb and prokaryotic proteins, eight categories of bacterial homologous proteins were found, including bacitracin resistance protein BacA, hemoglobins (Hb), hypothetical protein (HP), NO-inducible flavohemoprotein (NOIFHP), flavohemoprotein (FHP), cytochrome o (Cyo), nitric oxide dioxygenase (NOD) and dihydropteridine reductase (DHPR).
Among eight homologous categories, the proteins with highest homology with VHb were selected, including BacA from Clostridium paraputrificum (WP_027099064.1, 73.05%), Hb from Clostridium sp. CAG:221 (CDB15533.1, 71.63%), HP from Intestinibacter bartlettii DORA_8_9 (ETI93048.1, 68.79%), NOIFHP from Ureibacillus sp. Re31 (WP_191706693.1, 66.67%), FHP from Lysinibacillus sphaericus C3-41 (ACA41869.1, 65.97%), Cyo from Clostridium sp. (SCK00776.1, 65.73%), NOD from Caryophanon latum (WP_066464548.1, 65.28%), and DHPR from Bacilli bacterium VT-13-104 (KKE77556.1, 59.86%) (Figure 1).
Based on the analysis of conserved domains, the results show that BacA from C. paraputrificum, Hb from Clostridium sp. CAG:221, and FHP from L. sphaericus C3-41 all contain heme binding sites. Particularly, there are NAD (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) binding sites on FHP from L. sphaericus C3-41, which are very similar to the structure of VHb. BacA from C. paraputrificum and Hb from Clostridium sp. CAG:221 belong to the VtHb-like_SDgb (VHb-like_SDgb) protein family. BacA has the activity of undecaprenyl pyrophosphate phosphatase that is involved in the cell wall synthesis [33]. In addition, BacA can interact with lipids, which is similar to the function of VHb [24]. HP from I. bartlettii DORA_8_9, NOIFHP from Ureibacillus sp. Re31, FHP from L. sphaericus C3-41, and DHPR from B. bacterium VT-13-104 belong to PPK13289, a member of the cl36224 protein superfamily and may span more than one domain. Although no conserved domain was obtained by alignment, the function of NO-dioxygenase (NOD) from C. latum and Cyo from Clostridium sp. is similar with VHb [16,25], indicating some potential functional domains still remain to be discovered.

4. The Heterologous Expression of VHb

4.1. The Regulation of VHb Expression by Its Native Promoter

After the vgb gene encoding VHb was identified in Vitreoscilla [2], VHb was first heterologously expressed by its native promoter in E. coli [3]. It is worth noting that the expression of VHb was induced under hypoxic conditions both in Vitreoscilla and E. coli, indicating the oxygen-sensitive regulatory mechanism for its native promoter. In the following, it is found that there are binding sites of the oxygen-responsive transcriptional regulators OxyR, Fnr, ArcA, and Crp on the vgb promoter. Under oxygen-limited conditions, Fnr, ArcA, and Crp activate the expression of the vgb gene independently or in combination to promote oxygen supply and enhance respiratory activity [34]. Under high aeration, OxyR can not only down-regulate the transcription of the vgb gene by binding to the vgb promoter, but also interacts with VHb to convert it into an oxidized state that can positively regulate the expression of genes involved in oxidative stress and enhance the ability of cells to resist oxidative stress [26].

4.2. The Strategies to Improve VHb Expression

Due to the promising effect of oxygen delivery on the growth of strains and the synthesis of useful products, the expression levels of VHb should be adjusted for different kinds of microbial hosts [35]. At present, three factors that can significantly influence VHb expression have been optimized, including the copy number of the vgb gene, the vector copy number, and the promoter strength (Table 2).
At first, based on the effect of different VHb expression levels on the growth of E. coli, the suitable copy number of the vgb gene was determined. The result showed that the increased integrated copies of the vgb gene under the control of the vgb promoter cannot improve cell growth [36]. Therefore, the single copy of vgb gene was generally adopted in the following metabolic engineered strains. Next, three different recombinant E. coli strains (harboring low, middle, and high copy numbers of vectors containing the vgb gene, respectively) were constructed to improve the titer of ethanol. The results showed that the titer of ethanol was inversely proportional to the expression level of VHb and the highest titer of ethanol was obtained by the lowest VHb co-expression [35].
At last, the efficient expression of the vgb gene was achieved by selecting appropriate promoters. The native vgb promoter works in several Gram-negative bacteria, including eight-tandem vgb promoter P8vgb in E. coli, Halomonas bluephagenesis and Halomonas campaniensis [37,38]. The specific promoters that have been chosen for other bacteria include trc promoter in E. coli [39,40], tac promoter in E. coli and Thialkalivibrio versutus [41,42,43], PphaC1-j5 promoter in Cupriavidus necator [44], and P43 promoter in Bacillus subtilis [45]. Fungal promoters that have been used for expression in fungi include: tubulin promoter in Aurantiochytrium sp. [46], constitutive ermE promoter in Streptomyces sp. [47], and AOX1 promoter in Pichia pastoris [48,49]. In addition, the CaMV35S promoter has been chosen in higher plant systems [9,50].

5. The Effect of VHb Expression on Cell Metabolism

The result of transcriptomics showed that the expression of VHb can affect hundreds of genes in E. coli, especially for the genes involved in central carbon and energy metabolism [41]. In addition, under the conditions of limited oxygen and glucose as the sole carbon in E. coli, the analysis of metabolic flux distribution further demonstrated that the expression of VHb leads to dominant carbon flux in the pentose phosphate pathway (PPP), while the remaining carbon flux is guided toward the tricarboxylic acid (TCA) cycle [51]. Further research showed that the TCA cycle in vgb+ cells of E. coli can function in a branched manner under hypoxic conditions [52]. Along with the increasing carbon flux in PPP, more NADPH was produced and a net NADH flux is generated by the NADH/NADPH transhydrogenase in vgb+ cells under microaerobic conditions [51]. Moreover, VHb delivers oxygen to the respiratory chain, the respiratory activity was enhanced, the ratio of NAD+/NADH and ATP generation was improved [7,53]. Furthermore, by-products in the fermentation process were significantly reduced (acetate ~25%, ethanol ~49%, formate ~68%, lactate ~72%, and succinate ~50%) and growth yield increased 35% in vgb+ cells [51]. Especially for acetate, the following transcriptional analysis showed that the transcriptional levels of the glyoxylate shunt genes were also decreased [54].

6. Applications of VHb in Biotechnology

Dependent on the robust capacity of oxygen transport under hypoxic conditions, VHb has been widely used for the improvement of biosynthesis, cell growth and bioremediation (Figure 2).

6.1. VHb in Biotechnological Productions

VHb has been used for some important value-added products such as acetoin, butanediol and L-asparaginase under hypoxic conditions. For acetoin and butanediol, the vgb+ engineered Enterobacter aerogenes showed an enhancement of 83% in accumulation of acetoin and butanediol compared to the control strain without vgb [59]. In addition, an increase of 70% on the production of L-asparaginase was achieved in Pseudomonas aeruginosa by introducing the vgb gene [60]. Furthermore, many processes of ethanol production from pure sugars and industrial waste (corn, molasses, whey, whey powder, etc.) can be enhanced by VHb expression [61]. By combining immobilization with VHb, the immobilized engineered E. coli increased by 47% in the medium with an intermediate concentration of lactose from whey powder [62]. After the optimization of immobilization conditions (10% bead inoculation) and the medium composition (8% lactose from whey powder), the vgb+ immobilized E. coli displayed a higher titer of ethanol to 4.64% [63].
Besides the above-mentioned products, the production of other target products, including antibiotics, enzymes, organic acids and polysaccharides, can also be significantly increased through heterologous expression of VHb (Table 3). For antibiotics, the synthesis of pyocyanin and rifamycin B can be increased 3-fold in E. coli [64] and 2.2-fold in Amycolatopsis mediterranei [65], respectively. For enzymes, the expression of lipase 2, coenzyme Q10 and xylanase can be enhanced by 87.84% in P. pastoris [48], 71% in Rhodobacter sphaeroides [66] and 31% in P. pastoris [49], respectively. For organic acids, the VHb expression has a greater effect on arachidonic acid (8-fold) in Mortierella alpina [67], docosahexaenoic acid (2.74-fold) in Aurantiochytrium sp. [68], and ganoderic acid (1.4-fold) in G. lucidum [69]. For polysaccharides, several complex compounds, including bacterial cellulose (58.6%) in Gluconacetobacter xylinus [70], pullulan (42.08%) in Aureobasidium melanogenum [71], β-glucan (12.9–24.0%) in Lentinula edodes [72], and 6-(N-hydroxyethyl)-amino-6-deoxy-alpha-l-sorbofuranose (11.89%) in Gluconobacter oxydans [10] can be efficiently obtained, respectively.

6.2. VHb in Plants

The expression of VHb was also used to improve the waterlogging tolerance of higher plants. Under waterlogging conditions, VHb expressed in Zea mays L. seedlings can induce a higher activity of peroxidase and alcohol dehydrogenase 1 that are correlation with tolerance to oxidative stress and enhance the growth performance of plants (seedling height, root dry weight, primary root length, etc.) [9]. In addition, the expression of VHb can regulate the transcription of endogenous genes that refer to antioxidant biosynthesis and oxygen metabolism in plant cells, and protect cells from oxidative damage [57]. Furthermore, the biosynthesis of ascorbate and the tolerance to photo-oxidative stress were enhanced in VHb-expressing Arabidopsis cells [57]. VHb also plays a positive role in some other higher plants, including the enhancement of productivity and resistance to the herbicide glyphosate in Oryza sativa L. [78], and the improvement of seed germination and tolerance to submergence stress in cabbage (Brassica oleracea var. capitata L.) [79]. However, the expression of VHb does not always have a positive effect on plants. In the case of Hordeum vulgare, it exhibited a slower germination rate and impaired rooting of seedlings, which may be attributed to the removal of a significant signaling molecule (NO) related to seed germination and root formation by VHb [80]. In addition, the introduction of the vgb gene into Populus alba L. did not improve tolerance to submergence, oxidative and nitrosative stresses [81]. Therefore, it is necessary to carefully investigate the influence of the genetic manipulation of oxygen metabolism of higher plants on the physiological and biochemical characteristics of cells in order to evaluate the true value of VHb application for higher plants.

6.3. VHb in Mammalian Cells

There are also several successful reports on the application of VHb engineering in animal cells, including increased tissue plasminogen activator (~40–100%) in Chinese hamster ovary cells [82], increased survival rate (vgb+-34.57~92% and vgb−-15.69~65%) in Danio rerio [58], and increased biomass yields by 60% and lactate decrease of 40% in Chinese hamster ovary cells [7]. Since the metabolism of animals is much more complicated than that of microorganisms, the application of VHb in the field of animals has been relatively slow in recent years.

6.4. VHb in Biodegradation Applications

VHb expression was frequently applied in biodesulfurization, degradation of pesticides, and wastewater treatment (Table 4). Dependent on the function of sulfide receptor and storage [23], the expression of VHb in Rhodococcus erythropolis (desulfurization bacterium) presented a higher desulfurization ratio than the control (vgb+-37.5% and vgb−-20.5%) under hypoxic conditions [83]. In the following, an increase of 11.7 ± 1.8% on the rate of thiosulfate scavenge was achieved in T. versutus by introducing the vgb gene [42]. In addition, the co-culture of desulfurization bacteria is also an effective sulfur degradation method [84]. For example, co-culture of Paenibacillus strains (vgb+) showed a stronger growth than the control (vgb−) under the conditions of dibenzothiophene [11]. Moreover, introduction of the vgb gene into Pseudomonas putida can improve its pesticide degradation function under oxygen-limited conditions, including simultaneous degradation of methyl γ-hexachlorocyclohexane and parathion [85], the removal of 1,2,3-trichloropropane [86], and the simultaneous elimination of carbamates, pyrethroids, and organophosphates [87]. As for wastewater treatment, the vgb+ engineered Burkholderia cepacia strain was first applied with a parallel membrane bioreactors system and displayed a significant increase in the degradation efficiency of 2-chlorobenzoic acid (vgb+ ~94–97% and vgb− ~67–85%) [88]. In addition, based on the activity of peroxidase, one of the VHb variants (Q53H/P54C) shows excellent prospects for treating wastewater contaminated by textile dyes [21]. Furthermore, the b-type heme derived from VHb is quite beneficial for the activity of hemoglobin in activated sludge, which functions as oxidase or peroxidase and plays an important role in traditional aerobic wastewater treatment [89].

7. Conclusions and Future Perspectives

VHb is a special bacterial hemoglobin that can interact with terminal oxidase to provide enough oxygen for cell growth. Based on analyses of its properties and crystal structures, VHb has been applied in the field of metabolic engineering for microorganisms, plants, and animals to achieve high-cell-density fermentation and to enhance product synthesis and stress tolerance under oxygen-limited conditions. By the optimization of its expression strategies, the effect of VHb was further improved, allowing VHb technology to be used for more and more products.
In the future, there are four possible directions for the development of VHb application. Firstly, the precursors of heme (5-aminolevulinic acid) could be supplemented or the biosynthesis of heme could be enhanced to increase the activity of VHb because many microorganisms cannot supply enough heme for VHb expression. In the case of eukaryotic hemoglobins, the active Arenicola Marina globin chains were efficiently expressed by the addition of 5-aminolevulinic acid in E. coli [92]. In addition, an improvement of human hemoglobin production was obtained in S. cerevisiae with an enhanced heme synthesis pathway [93]. Secondly, the addition of iron and transport of iron over cell membranes also have a positive effect on hemoglobin production. The hemoglobin of β-thalassemic mice was increased with the exogenous addition of iron [94]. Thirdly, more and more VHb mutants with improved characteristics can be selected by protein engineering and high throughput screening. Furthermore, the expression of VHb will also contribute extra metabolic burden, but the optimization of promoter, substrate and inducer can significantly relieve this adverse effect on the host [95,96]. Finally, more research on the regulatory mechanism of VHb on oxygen-response is needed to expand its application in other areas.

Author Contributions

Conceptualization, F.Y. and X.Z.; writing—original draft preparation, F.Y., Z.W., L.L. and L.Y.; writing—review and editing, F.Y. and X.Z.; supervision, X.Z., J.Z., J.L., J.C. and G.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (2021YFC2101400), the National First-Class Discipline Program of Light Industry Technology and Engineering (LITE2018-08), the National Natural Science Foundation of China (31900067), and the Fundamental Research Funds for the Central Universities (JUSRP52021).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Webster, D.A.; Hackett, D.P. The Purification and Properties of Cytochrome o from Vitreoscilla. J. Biol. Chem. 1966, 241, 3308–3315. [Google Scholar] [CrossRef]
  2. Wakabayashi, S.; Matsubara, H.; Webster, D.A. Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla. Nature 1986, 322, 481–483. [Google Scholar] [CrossRef]
  3. Khosla, C.; Bailey, J.E. The Vitreoscilla hemoglobin gene: Molecular cloning, nucleotide sequence and genetic expression in Escherichia coli. Mol. Gen. Genet. 1988, 214, 158–161. [Google Scholar] [CrossRef]
  4. Webster, D.A.; Liu, C.Y. Reduced nicotinamide adenine dinucleotide cytochrome o reductase associated with cytochrome o purified from Vitreoscilla. Evidence for an intermediate oxygenated form of cytochrome o. J. Biol. Chem. 1974, 249, 4257–4260. [Google Scholar] [CrossRef]
  5. Tyree, B.; Webster, D.A.; Tyree, B. The binding of cyanide and carbon monoxide to cytochrome o purified from Vitreoscilla. Evidence for subunit interaction in the reduced protein. J. Biol. Chem. 1978, 253, 6988–6991. [Google Scholar] [CrossRef]
  6. Stark, B.C.; Pagilla, K.R.; Dikshit, K.L. Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. Appl. Microbiol. Biotechnol. 2015, 99, 1627–1636. [Google Scholar] [CrossRef]
  7. Juárez, M.; La Rosa, C.H.G.-D.; Memún, E.; Sigala, J.-C.; Lara, A.R. Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells. Biotechnol. J. 2017, 12, 1600438. [Google Scholar] [CrossRef]
  8. Chen, Y.; Chen, X.-Y.; Du, H.-T.; Zhang, X.; Ma, Y.-M.; Chen, J.-C.; Ye, J.-W.; Jiang, X.-R.; Chen, G.-Q. Chromosome engineering of the TCA cycle in Halomonas bluephagenesis for production of copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV). Metab. Eng. 2019, 54, 69–82. [Google Scholar] [CrossRef]
  9. Du, H.; Shen, X.; Huang, Y.; Huang, M.; Zhang, Z. Overexpression of Vitreoscilla hemoglobin increases waterlogging tolerance in Arabidopsis and maize. BMC Plant Biol. 2016, 16, 35. [Google Scholar] [CrossRef] [Green Version]
  10. Liu, D.; Ke, X.; Hu, Z.-C.; Zheng, Y.-G. Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose. J. Biosci. Bioeng. 2021, 131, 518–524. [Google Scholar] [CrossRef]
  11. Sar, T.; Chen, Y.; Bai, Y.; Liu, B.; Agarwal, P.; Stark, B.C.; Akbas, M.Y. Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization. Lett. Appl. Microbiol. 2021, 72, 484–494. [Google Scholar] [CrossRef]
  12. Orii, Y.; Webster, D.A. Photodissociation of oxygenated cytochrome o(s) (Vitreoscilla) and kinetic studies of reassociation. J. Biol. Chem. 1986, 261, 3544–3547. [Google Scholar] [CrossRef]
  13. Webster, D.A. Structure and function of bacterial hemoglobin and related proteins. Adv. Inorg. Biochem. 1988, 7, 245–265. [Google Scholar]
  14. Ramandeep; Hwang, K.W.; Raje, M.; Kim, K.J.; Stark, B.C.; Dikshit, K.L.; Webster, D.A. Vitreoscilla hemoglobin. Intracellular localization and binding to membranes. J. Biol. Chem. 2001, 276, 24781–24789. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Park, K.-W.; Kim, K.-J.; Howard, A.J.; Stark, B.C.; Webster, D.A. Vitreoscilla Hemoglobin Binds to Subunit I of Cytochrome bo Ubiquinol Oxidases. J. Biol. Chem. 2002, 277, 33334–33337. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Dikshit, R.P.; Dikshit, K.L.; Liu, Y.; Webster, D.A. The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Arch. Biochem. Biophys. 1992, 293, 241–245. [Google Scholar] [CrossRef]
  17. Kvist, M.; Ryabova, E.S.; Nordlander, E.; Bülow, L. An investigation of the peroxidase activity of Vitreoscilla hemoglobin. J. Biol. Inorg. Chem. 2007, 12, 324–334. [Google Scholar] [CrossRef] [PubMed]
  18. Li, W.; Zhang, Y.; Xu, H.; Wu, L.; Cao, Y.; Zhao, H.; Li, Z. pH-induced quaternary assembly of Vitreoscilla hemoglobin: The monomer exhibits better peroxidase activity. Biochim. Biophys. Acta. 2013, 1834, 2124–2132. [Google Scholar] [CrossRef]
  19. Suwanwong, Y.; Kvist, M.; Isarankura-Na-Ayudhya, C.; Tansila, N.; Bulow, L.; Prachayasittikul, V. Chimeric Antibody-Binding Vitreoscilla Hemoglobin (vhb) Mediates Redox-Catalysis Reaction: New Insight into the Functional Role of VHb. Int. J. Biol. Sci. 2006, 2, 208–215. [Google Scholar] [CrossRef] [Green Version]
  20. Isarankura-Na-Ayudhya, C.; Tansila, N.; Worachartcheewan, A.; Bülow, L.; Prachayasittikul, V. Biochemical and cellular investigation of Vitreoscilla hemoglobin (vhb) variants possessing efficient peroxidase activity. J. Microbiol. Biotechnol. 2010, 20, 532–541. [Google Scholar]
  21. Zhang, Z.; Li, W.; Li, H.; Zhang, J.; Zhang, Y.; Cao, Y.; Ma, J.; Li, Z. Construction and Characterization of Vitreoscilla Hemoglobin (vhb) with Enhanced Peroxidase Activity for Efficient Degradation of Textile Dye. J. Microbiol. Biotechnol. 2015, 25, 1433–1441. [Google Scholar] [CrossRef]
  22. Ramos-Alvarez, C.; Yoo, B.K.; Pietri, R.; Lamarre, I.; Martin, J.L.; Lopez-Garriga, J.; Negrerie, M. Reactivity and dynamics of H2S, NO, and O2 interacting with hemoglobins from Lucina pectinata. Biochemistry 2013, 52, 7007–7021. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, D.; Liu, L.; Wang, H.; Xu, H.; Chen, L.; Ma, L.; Li, Z. Clues for discovering a new biological function of Vitreoscilla hemoglobin in organisms: Potential sulfide receptor and storage. FEBS Lett. 2016, 590, 1132–1142. [Google Scholar] [CrossRef] [Green Version]
  24. Rinaldi, A.C.; Bonamore, A.; Macone, A.; Boffi, A.; Bozzi, A.; Di Giulio, A. Interaction of Vitreoscilla Hemoglobin with Membrane Lipids. Biochemistry 2006, 45, 4069–4076. [Google Scholar] [CrossRef]
  25. Kaur, R.; Pathania, R.; Sharma, V.; Mande, S.C.; Dikshit, K.L. 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. 2002, 68, 152–160. [Google Scholar] [CrossRef] [Green Version]
  26. Anand, A.; Duk, B.T.; Singh, S.; Akbas, M.Y.; Webster, D.A.; Stark, B.C.; Dikshit, K.L. Redox-mediated interactions of VHb (Vitreoscilla haemoglobin) with OxyR: Novel regulation of VHb biosynthesis under oxidative stress. Biochem. J. 2010, 426, 271–280. [Google Scholar] [CrossRef] [Green Version]
  27. Lin, J.-M.; Stark, B.C.; Webster, D.A. Effects of Vitreoscilla hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of Burkholderia and on 2,4-DNT degradation in two-phase bioreactors. J. Ind. Microbiol. Biotechnol. 2003, 30, 362–368. [Google Scholar] [CrossRef]
  28. Tarricone, C.; Galizzi, A.; Coda, A.; Ascenzi, P.; Bolognesi, M. Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp. Structure 1997, 5, 497–507. [Google Scholar] [CrossRef] [Green Version]
  29. Bolognesi, M.; Boffi, A.; Coletta, M.; Mozzarelli, A.; Pesce, A.; Tarricone, C.; Ascenzi, P. Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: A thermodynamic, kinetic and X-ray crystallographic study. J. Mol. Biol. 1999, 291, 637–650. [Google Scholar] [CrossRef]
  30. Dikshit, K.L.; Orii, Y.; Navani, N.; Patel, S.; Huang, H.Y.; Stark, B.C.; Webster, D.A. Site-directed mutagenesis of bacterial hemoglobin: The role of glutamine (E7) in oxygen-binding in the distal heme pocket. Arch. Biochem. Biophys. 1998, 349, 161–166. [Google Scholar] [CrossRef]
  31. Ratakonda, S.; Anand, A.; Dikshit, K.; Stark, B.C.; Howard, A.J. Crystallographic structure determination of B10 mutants of Vitreoscilla hemoglobin: Role of Tyr29 (B10) in the structure of the ligand-binding site. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2013, 69, 215–222. [Google Scholar] [CrossRef] [Green Version]
  32. Kaur, R.; Ahuja, S.; Anand, A.; Singh, B.; Stark, B.C.; Webster, D.A.; Dikshit, K.L. Functional implications of the proximal site hydrogen bonding network in Vitreoscilla hemoglobin (vhb): Role of Tyr95 (G5) and Tyr126 (H12). FEBS Lett. 2008, 582, 3494–3500. [Google Scholar] [CrossRef] [Green Version]
  33. El Ghachi, M.; Bouhss, A.; Blanot, D.; Mengin-Lecreulx, D. The bacA Gene of Escherichia coli Encodes an Undecaprenyl Pyrophosphate Phosphatase Activity. J. Biol. Chem. 2004, 279, 30106–30113. [Google Scholar] [CrossRef] [Green Version]
  34. Yang, J.; Webster, D.A.; Stark, B.C. ArcA works with Fnr as a positive regulator of Vitreoscilla (bacterial) hemoglobin gene expression in Escherichia coli. Microbiol. Res. 2005, 160, 405–415. [Google Scholar] [CrossRef] [PubMed]
  35. Sanny, T.; Arnaldos, M.; Kunkel, S.A.; Pagilla, K.; Stark, B.C. Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose. Appl. Microbiol. Biotechnol. 2010, 88, 1103–1112. [Google Scholar] [CrossRef]
  36. Khosla, C.; Curtis, J.E.; DeModena, J.; Rinas, U.; Bailey, J.E. Expression of Intracellular Hemoglobin Improves Protein Synthesis in Oxygen-Limited Escherichia coli. Nat. Biotechnol. 1990, 8, 849–853. [Google Scholar] [CrossRef]
  37. Wu, H.; Wang, H.; Chen, J.; Chen, G.-Q. Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically. Appl. Microbiol. Biotechnol. 2014, 98, 10013–10021. [Google Scholar] [CrossRef]
  38. Ouyang, P.; Wang, H.; Hajnal, I.; Wu, Q.; Guo, Y.; Chen, G.-Q. Increasing oxygen availability for improving poly(3-hydroxybutyrate) production by Halomonas. Metab. Eng. 2018, 45, 20–31. [Google Scholar] [CrossRef] [PubMed]
  39. Lara, A.R.; Velázquez, D.; Penella, I.; Islas, F.; La Rosa, C.H.G.-D.; Sigala, J.-C. Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli. Bioprocess Biosyst. Eng. 2019, 42, 1391–1397. [Google Scholar] [CrossRef]
  40. Jaén, K.E.; Velazquez, D.; Delvigne, F.; Sigala, J.-C.; Lara, A.R. Engineering E. coli for improved microaerobic pDNA production. Bioprocess Biosyst. Eng. 2019, 42, 1457–1466. [Google Scholar] [CrossRef]
  41. Roos, V.; Andersson, C.I.; Bülow, L. Gene expression profiling of Escherichia coli expressing double Vitreoscilla haemoglobin. J. Biotechnol. 2004, 114, 107–120. [Google Scholar] [CrossRef] [PubMed]
  42. Mu, T.; Yang, M.; Zhao, J.; Sharshar, M.M.; Tian, J.; Xing, J. Improvement of desulfurizing activity of haloalkaliphilic Thialkalivibrio versutus SOB306 with the expression of Vitreoscilla hemoglobin gene. Biotechnol. Lett. 2016, 39, 447–452. [Google Scholar] [CrossRef]
  43. Wu, W.; Guo, X.; Zhang, M.; Huang, Q.; Qi, F.; Huang, J. Enhancement of l -phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin. Biotechnol. Appl. Biochem. 2017, 65, 476–483. [Google Scholar] [CrossRef] [PubMed]
  44. Tang, R.; Weng, C.; Peng, X.; Han, Y. Metabolic engineering of Cupriavidus necator H16 for improved chemoautotrophic growth and PHB production under oxygen-limiting conditions. Metab. Eng. 2020, 61, 11–23. [Google Scholar] [CrossRef]
  45. Vyas, R.; Pandya, M.; Pohnerkar, J.; Kumar, G.N. Vitreoscilla hemoglobin promotes biofilm expansion and mitigates sporulation in Bacillus subtilis DK1042. 3 Biotech 2020, 10, 1–7. [Google Scholar] [CrossRef]
  46. Suen, Y.L.; Tang, H.; Huang, J.; Chen, F. Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin. J. Agric. Food Chem. 2014, 62, 12392–12398. [Google Scholar] [CrossRef]
  47. Wang, T.; Bai, L.; Zhu, D.; Lei, X.; Liu, G.; Deng, Z.; You, D. Enhancing macrolide production in Streptomyces by coexpressing three heterologous genes. Enzym. Microb. Technol. 2012, 50, 5–9. [Google Scholar] [CrossRef]
  48. Zhou, Q.; Su, Z.; Jiao, L.; Wang, Y.; Yang, K.; Li, W.; Yan, Y. High-Level Production of a Thermostable Mutant of Yarrowia lipolytica Lipase 2 in Pichia Pastoris. Int. J. Mol. Sci. 2019, 21, 279. [Google Scholar] [CrossRef] [Green Version]
  49. Wang, J.; Li, Y.; Liu, D. Improved production of Aspergillus usamii endo-beta-1,4-Xylanase in Pichia Pastoris via combined strategies. Biomed. Res. Int. 2016, 2016, 3265895. [Google Scholar]
  50. Guo, Z.; Tan, H.; Lv, Z.; Ji, Q.; Huang, Y.; Liu, J.; Chen, D.; Diao, Y.; Si, J.; Zhang, L. Targeted expression of Vitreoscilla hemoglobin improves the production of tropane alkaloids in Hyoscyamus niger hairy roots. Sci. Rep. 2018, 8, 17969. [Google Scholar] [CrossRef]
  51. Tsai, P.S.; Hatzimanikatis, V.; Bailey, J.E. Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism. Biotechnol. Bioeng. 1996, 49, 139–150. [Google Scholar] [CrossRef]
  52. Frey, A.; Fiaux, J.; Szyperski, T.; Wüthrich, K.; Bailey, J.E.; Kallio, P.T. Dissection of Central Carbon Metabolism of Hemoglobin-Expressing Escherichia coli by 13C Nuclear Magnetic Resonance Flux Distribution Analysis in Microaerobic Bioprocesses. Appl. Environ. Microbiol. 2001, 67, 680–687. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Cai, D.; Chen, Y.; He, P.; Wang, S.; Mo, F.; Li, X.; Wang, Q.; Nomura, C.T.; Wen, Z.; Ma, X.; et al. Enhanced production of poly-gamma-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis. Biotechnol. Bioeng. 2018, 115, 2541–2553. [Google Scholar] [CrossRef] [PubMed]
  54. Lara, A.R.; Galindo, J.; Jaén, K.E.; Juárez, M.; Sigala, J.-C. Physiological Response of Escherichia coli W3110 and BL21 to the Aerobic Expression of Vitreoscilla Hemoglobin. J. Microbiol. Biotechnol. 2020, 30, 1592–1596. [Google Scholar] [CrossRef]
  55. Frey, A.D.; Koskenkorva, T.; Kallio, P.T. Vitreoscilla hemoglobin promoter is not responsive to nitrosative and oxidative stress in Escherichia coli. FEMS Microbiol. Lett. 2003, 224, 127–132. [Google Scholar] [CrossRef] [Green Version]
  56. Kallio, P.T.; Bollinger, C.J.; Koskenkorva, T.; Frey, A.D. Assessment of Biotechnologically Relevant Characteristics of Heterologous Hemoglobins in E. coli. Methods Enzymol. 2008, 436, 255–272. [Google Scholar] [CrossRef]
  57. Wang, Z.; Xiao, Y.; Chen, W.; Tang, K.; Zhang, L. Functional expression of Vitreoscilla hemoglobin (vhb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism. Plant Sci. 2009, 176, 66–77. [Google Scholar] [CrossRef]
  58. Guan, B.; Ma, H.; Wang, Y.; Hu, Y.; Lin, Z.; Zhu, Z.; Hu, W. Vitreoscilla Hemoglobin (vhb) Overexpression Increases Hypoxia Tolerance in Zebrafish (Danio rerio). Mar. Biotechnol. 2010, 13, 336–344. [Google Scholar] [CrossRef]
  59. Geckil, H.; Barak, Z.; Chipman, D.M.; Erenler, S.O.; Webster, D.A.; Stark, B.C. Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene. Bioprocess Biosyst. Eng. 2004, 26, 325–330. [Google Scholar] [CrossRef]
  60. Geckil, H.; Gencer, S.; Ates, B.; Ozer, U.; Uckun, M.; Yilmaz, I. Effect of Vitreoscilla hemoglobin on production of a chemotherapeutic enzyme, L-asparaginase, by Pseudomonas aeruginosa. Biotechnol. J. 2006, 1, 203–208. [Google Scholar] [CrossRef]
  61. Akbas, M.Y.; Stark, B.C. Recent trends in bioethanol production from food processing byproducts. J. Ind. Microbiol. Biotechnol. 2016, 43, 1593–1609. [Google Scholar] [CrossRef]
  62. Sar, T.; Stark, B.C.; Akbas, M.Y. Effective ethanol production from whey powder through immobilized E. coli expressing Vitreoscilla hemoglobin. Bioengineered 2016, 8, 171–181. [Google Scholar] [CrossRef] [Green Version]
  63. Sar, T.; Stark, B.C.; Akbas, M.Y. Bioethanol production from whey powder by immobilized E. coli expressing Vitreoscilla hemoglobin: Optimization of sugar concentration and inoculum size. Biofuels 2019. [Google Scholar] [CrossRef]
  64. da Silva, A.J.; Cunha, J.S.; Hreha, T.; Micocci, K.C.; Selistre-De-Araujo, H.S.; Barquera, B.; Koffas, M.A. Metabolic engineering of E. coli for pyocyanin production. Metab. Eng. 2021, 64, 15–25. [Google Scholar] [CrossRef] [PubMed]
  65. Mejía, A.; Luna, D.; Fernández, F.J.; Barrios-González, J.; Gutierrez, L.H.; Reyes, A.G.; Absalón, A.E.; Kelly, S. Improving rifamycin production in Amycolatopsis mediterranei by expressing a Vitreoscilla hemoglobin (vhb) gene fused to a cytochrome P450 monooxygenase domain. 3 Biotech. 2018, 8, 456. [Google Scholar] [CrossRef] [PubMed]
  66. Zhu, Y.; Ye, L.; Chen, Z.; Hu, W.; Shi, Y.; Chen, J.; Wang, C.; Li, Y.; Li, W.; Yu, H. Synergic regulation of redox potential and oxygen uptake to enhance production of coenzyme Q 10 in Rhodobacter sphaeroides. Enzym. Microb. Technol. 2017, 101, 36–43. [Google Scholar] [CrossRef] [PubMed]
  67. Zhang, H.; Feng, Y.; Cui, Q.; Song, X. Expression of Vitreoscilla hemoglobin enhances production of arachidonic acid and lipids in Mortierella alpina. BMC Biotechnol. 2017, 17, 68. [Google Scholar] [CrossRef]
  68. Ye, J.; Liu, M.; He, M.; Ye, Y.; Huang, J. Illustrating and Enhancing the Biosynthesis of Astaxanthin and Docosahexaenoic Acid in Aurantiochytrium sp. SK4. Mar. Drugs 2019, 17, 45. [Google Scholar] [CrossRef] [Green Version]
  69. Li, H.-J.; He, Y.-L.; Zhang, D.-H.; Yue, T.-H.; Jiang, L.-X.; Li, N.; Xu, J.-W. Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum. J. Biotechnol. 2016, 227, 35–40. [Google Scholar] [CrossRef]
  70. Liu, M.; Li, S.; Xie, Y.; Jia, S.; Hou, Y.; Zou, Y.; Zhong, C. Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation. Appl. Microbiol. Biotechnol. 2018, 102, 1155–1165. [Google Scholar] [CrossRef]
  71. Xue, S.-J.; Jiang, H.; Chen, L.; Ge, N.; Liu, G.-L.; Hu, Z.; Chi, Z.-M.; Chi, Z. Over-expression of Vitreoscilla hemoglobin (vhb) and flavohemoglobin (FHb) genes greatly enhances pullulan production. Int. J. Biol. Macromol. 2019, 132, 701–709. [Google Scholar] [CrossRef] [PubMed]
  72. Wang, X.; Ding, Y.; Gao, X.; Liu, H.; Zhao, K.; Gao, Y.; Qiu, L. Promotion of the growth and plant biomass degrading enzymes production in solid-state cultures of Lentinula edodes expressing Vitreoscilla hemoglobin gene. J. Biotechnol. 2019, 302, 42–47. [Google Scholar] [CrossRef] [PubMed]
  73. Akbas, M.Y.; Sar, T.; Ozcelik, B. Improved ethanol production from cheese whey, whey powder, and sugar beet molasses by “Vitreoscilla hemoglobin expressing” Escherichia coli. Biosci. Biotechnol. Biochem. 2014, 78, 687–694. [Google Scholar] [CrossRef] [PubMed]
  74. Arnaldos, M.; Kunkel, S.A.; Wang, J.; Pagilla, K.; Stark, B.C. Vitreoscilla hemoglobin enhances ethanol production by Escherichia coli in a variety of growth media. Biomass Bioenergy 2012, 37, 1–8. [Google Scholar] [CrossRef]
  75. Mirończuk, A.M.; Kosiorowska, K.E.; Biegalska, A.; Rakicka-Pustułka, M.; Szczepańczyk, M.; Dobrowolski, A. Heterologous overexpression of bacterial hemoglobin VHb improves erythritol biosynthesis by yeast Yarrowia lipolytica. Microb. Cell Factories 2019, 18, 1–8. [Google Scholar] [CrossRef] [Green Version]
  76. Chen, Y.; Tan, T. Enhanced S-Adenosylmethionine Production by Increasing ATP Levels in Baker’s Yeast (Saccharomyces cerevisiae). J. Agric. Food Chem. 2018, 66, 5200–5209. [Google Scholar] [CrossRef]
  77. Zhang, X.; Xu, C.; Liu, Y.; Wang, J.; Zhao, Y.; Deng, Y. Enhancement of glucaric acid production in Saccharomyces cerevisiae by expressing Vitreoscilla hemoglobin. Biotechnol. Lett. 2020, 42, 2169–2178. [Google Scholar] [CrossRef]
  78. Cao, M.X.; Huang, J.Q.; Wei, Z.M.; Yao, Q.H.; Wan, C.Z.; Lu, J.A. Engineering Higher Yield and Herbicide Resistance in Rice by Agrobacterium-Mediated Multiple Gene Transformation. Crop. Sci. 2004, 44, 2206–2213. [Google Scholar] [CrossRef]
  79. Li, X.; Peng, R.-H.; Fan, H.-Q.; Xiong, A.-S.; Yao, Q.-H.; Cheng, Z.-M.; Li, Y. Vitreoscilla hemoglobin overexpression increases submergence tolerance in cabbage. Plant Cell Rep. 2005, 23, 710–715. [Google Scholar] [CrossRef]
  80. Wilhelmson, A.; Kallio, P.T.; Oksman-Caldentey, K.-M.; Nuutila, A.M. Heterologous expression of Vitreoscilla haemoglobin in barley (Hordeum vulgare). Plant Cell Rep. 2007, 26, 1773–1783. [Google Scholar] [CrossRef]
  81. Zelasco, S.; Reggi, S.; Calligari, P.; Balestrazzi, A.; Bongiorni, C.; Quattrini, E.; Delia, G.; Bisoffi, S.; Fogher, C.; Confalonieri, M. Expression of the Vitreoscilla Hemoglobin (VHb)-Encoding Gene in Transgenic White Poplar: Plant Growth and Biomass Production, Biochemical Characterization and Cell Survival under Submergence, Oxidative and Nitrosative Stress Conditions. Mol. Breed. 2006, 17, 201–216. [Google Scholar] [CrossRef]
  82. Pendse, G.J.; Bailey, J.E. Effect of Vitreoscilla hemoglobin expression on growth and specific tissue plasminogen activator productivity in recombinant Chinese hamster ovary cells. Biotechnol. Bioeng. 1994, 44, 1367–1370. [Google Scholar] [CrossRef] [PubMed]
  83. Xiong, X.; Xing, J.; Li, X.; Bai, X.; Li, W.; Li, Y.; Liu, H. Enhancement of biodesulfurization in two-liquid systems by heterogeneous expression of Vitreoscilla hemoglobin. Appl. Environ. Microbiol. 2007, 73, 2394–2397. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Martínez, I.; Mohamed, M.E.-S.; Rozas, D.; García, J.L.; Díaz, E. Engineering synthetic bacterial consortia for enhanced desulfurization and revalorization of oil sulfur compounds. Metab. Eng. 2016, 35, 46–54. [Google Scholar] [CrossRef] [PubMed]
  85. Gong, T.; Liu, R.; Zuo, Z.; Che, Y.; Yu, H.; Song, C.; Yang, C. Metabolic engineering of Pseudomonas putida KT2440 for complete mineralization of methyl parathion and gamma-hexachlorocyclohexane. ACS. Synth. Biol. 2016, 5, 434–442. [Google Scholar] [CrossRef] [PubMed]
  86. Gong, T.; Xu, X.; Che, Y.; Liu, R.; Gao, W.; Zhao, F.; Yu, H.; Liang, J.; Xu, P.; Song, C.; et al. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane. Sci. Rep. 2017, 7, 7064. [Google Scholar] [CrossRef] [Green Version]
  87. Gong, T.; Xu, X.; Dang, Y.; Kong, A.; Wu, Y.; Liang, P.; Wang, S.; Yu, H.; Xu, P.; Yang, C. An engineered Pseudomonas putida can simultaneously degrade organophosphates, pyrethroids and carbamates. Sci. Total. Environ. 2018, 628–629, 1258–1265. [Google Scholar] [CrossRef]
  88. Urgun-Demirtas, M.; Stark, B.C.; Pagilla, K.R. Comparison of 2-chlorobenzoic acid biodegradation in a membrane bioreactor by B. cepacia and B. cepacia bearing the bacterial hemoglobin gene. Water Res. 2006, 40, 3123–3130. [Google Scholar] [CrossRef]
  89. Kunkel, S.A.; Pagilla, K.; Stark, B.C. Directed evolution to produce sludge communities with improved oxygen uptake abilities. Appl. Microbiol. Biotechnol. 2015, 99, 10725–10734. [Google Scholar] [CrossRef]
  90. Kahraman, H.; Geckil, H. Degradation of Benzene, Toluene and Xylene by Pseudomonas aeruginosa Engineered with the Vitreoscilla Hemoglobin Gene. Eng. Life Sci. 2005, 5, 363–368. [Google Scholar] [CrossRef]
  91. Khleifat, K.M.; Abboud, M.M.; Al-Mustafa, A.H. Effect of Vitreoscilla hemoglobin gene (vgb) and metabolic inhibitors on cadmium uptake by the heterologous host Enterobacter aerogenes. Process. Biochem. 2006, 41, 930–934. [Google Scholar] [CrossRef]
  92. Harnois, T.; Rousselot, M.; Rogniaux, H.; Zal, F. High-level Production of Recombinant Arenicola marina Globin Chains in Escherichia coli: A New Generation of Blood Substitute. Artif. Cells Blood Substit. Biotechnol. 2009, 37, 106–116. [Google Scholar] [CrossRef]
  93. Liu, L.; Martínez, J.L.; Liu, Z.; Petranovic, D.; Nielsen, J. Balanced globin protein expression and heme biosynthesis improve production of human hemoglobin in Saccharomyces cerevisiae. Metab. Eng. 2014, 21, 9–16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Ginzburg, Y.Z.; Rybicki, A.C.; Suzuka, S.M.; Hall, C.B.; Breuer, W.; Cabantchik, Z.I.; Bouhassira, E.E.; Fabry, M.E.; Nagel, R.L. Exogenous iron increases hemoglobin in beta-thalassemic mice. Exp. Hematol. 2009, 37, 172–183. [Google Scholar] [CrossRef] [PubMed]
  95. Shen, Q.; Yu, Z.; Zhou, X.-T.; Zhang, S.-J.; Zou, S.-P.; Xiong, N.; Xue, Y.-P.; Liu, Z.-Q.; Zheng, Y.-G. Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris. Appl. Microbiol. Biotechnol. 2021, 105, 3211–3223. [Google Scholar] [CrossRef] [PubMed]
  96. Malakar, P.; Venkatesh, K.V. Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins. Appl. Microbiol. Biotechnol. 2012, 93, 2543–2549. [Google Scholar] [CrossRef]
Microorganisms 09 01455 g001 550
Figure 1. The homology analysis of VHb. The accession numbers in GenBank of eight homologous proteins and their homology with VHb: BacA from C. paraputrificum (WP_027099064.1, 73.05%), Hb from Clostridium sp. CAG:221 (CDB15533.1, 71.63%), HP from I. bartlettii DORA_8_9 (ETI93048.1, 68.79%), NOIFHP from Ureibacillus sp. Re31 (WP_191706693.1, 66.67%), FHP from L. sphaericus C3-41 (ACA41869.1, 65.97%), Cyo from Clostridium sp. (SCK00776.1, 65.73%), NOD from C. latum (WP_066464548.1, 65.28%), and DHPR from B. bacterium VT-13-104 (KKE77556.1, 59.86%). Hb: hemoglobin; BacA: bacitracin resistance protein; HP: hypothetical protein; NOIFHP: NO-inducible flavohemoprotein; FHP: flavohemoprotein; Cyo: cytochrome o; NOD: nitric oxide dioxygenase; DHPR: dihydropteridine reductase; VtHb: VHb, Vitreoscilla hemoglobin; SDgb: single-domain globin; PPK13289: belongs to the superfamily cl36224 and may span more than one domain. FAD: flavin adenine dinucleotide; NAD: nicotinamide adenine dinucleotide; Yhb1-globin_like: a globin domain such as the globin domain of the Saccharomyces cerevisiae flavohemoglobin (Yhb1p). Different colors represent different proteins or domains.
Figure 1. The homology analysis of VHb. The accession numbers in GenBank of eight homologous proteins and their homology with VHb: BacA from C. paraputrificum (WP_027099064.1, 73.05%), Hb from Clostridium sp. CAG:221 (CDB15533.1, 71.63%), HP from I. bartlettii DORA_8_9 (ETI93048.1, 68.79%), NOIFHP from Ureibacillus sp. Re31 (WP_191706693.1, 66.67%), FHP from L. sphaericus C3-41 (ACA41869.1, 65.97%), Cyo from Clostridium sp. (SCK00776.1, 65.73%), NOD from C. latum (WP_066464548.1, 65.28%), and DHPR from B. bacterium VT-13-104 (KKE77556.1, 59.86%). Hb: hemoglobin; BacA: bacitracin resistance protein; HP: hypothetical protein; NOIFHP: NO-inducible flavohemoprotein; FHP: flavohemoprotein; Cyo: cytochrome o; NOD: nitric oxide dioxygenase; DHPR: dihydropteridine reductase; VtHb: VHb, Vitreoscilla hemoglobin; SDgb: single-domain globin; PPK13289: belongs to the superfamily cl36224 and may span more than one domain. FAD: flavin adenine dinucleotide; NAD: nicotinamide adenine dinucleotide; Yhb1-globin_like: a globin domain such as the globin domain of the Saccharomyces cerevisiae flavohemoglobin (Yhb1p). Different colors represent different proteins or domains.
Microorganisms 09 01455 g001
Microorganisms 09 01455 g002 550
Figure 2. The potential applications of VHb in the field of metabolic engineering for bacteria [55,56], fungi [48,49], plants [57] and animals [58]. ROS: reactive oxygen species; RNS: reactive nitrogen species.
Figure 2. The potential applications of VHb in the field of metabolic engineering for bacteria [55,56], fungi [48,49], plants [57] and animals [58]. ROS: reactive oxygen species; RNS: reactive nitrogen species.
Microorganisms 09 01455 g002
Table
Table 1. Interactions between VHb and intracellular enzymes or regulators.
Table 1. Interactions between VHb and intracellular enzymes or regulators.
Enzymes/RegulatorsFunctionsReferences
FlavoreductaseRelieve nitrosative stress[25]
Transcriptional regulators (OxyR, Fnr, ArcA, Crp)Transcriptional regulation[26]
2,4-dinitrotoluene dioxygenaseEnhance dioxygenase activity[27]
OxyR: oxidative stress regulator; Fnr: fumarate and nitrate reductase; ArcA: aerobic respiration control A; Crp: catabolic repressor protein.
Table
Table 2. The strategies of VHb expression.
Table 2. The strategies of VHb expression.
StrainExpression StrategiesReferences
Escherichia coliFree; inducible; vgb promoter[36]
E. coliFree; inducible; vgb promoter[35]
E. coliFree; inducible; P8vgb[37]
E. coli, Halomonas bluephagenesis and Halomonas campaniensisFree; inducible; P8vgb[38]
E. coliIntegrative; constitutive; trc promoter[39]
E. coliIntegrative; inducible; trc promoter[40]
E. coliFree; inducible; tac promoter[41]
Thialkalivibrio versutusFree; constitutive; tac promoter[42]
E. coliFree; constitutive; tac promoter[43]
Cupriavidus necatorFree; constitutive; PphaC1-j5 promoter[44]
Bacillus subtilisFree; constitutive; P43 promoter[45]
Aurantiochytrium sp.Integrative; constitutive; tubulin promoter[46]
Streptomyces sp.Integrative; constitutive; ermE promoter[47]
Pichia pastorisIntegrative; inducible; AOX1 promoter[48]
P. pastorisIntegrative; inducible; AOX1 promoter[49]
Arabidopsis and Zea mays L.Integrative; constitutive; CaMV35S promoter[9]
Hyoscyamus nigerIntegrative; constitutive; CaMV35S promoter[50]
Free: intracellular free expression by plasmid; Integrative: intracellular integrative expression by chromosomally integration; Inducible: intracellular inducible expression by the addition of inducers; Constitutive: intracellular constitutive expression that do not need inducers. P8vgb: eight-tandem vgb promoter; trc promoter: trp and lac UV5 promoter hybridized; tac promoter: a hybrid between the trp and lac promoters; PphaC1-j5 promoter: a hybrid between PphaC1 and Pj5 promoter; tubulin promoter: a promoter amplified from the genome of Aurantiochytrium sp.; ermE promoter: a strong constitutive promoter commonly used in Streptomyces sp.; AOX1 promoter: methanol-inducible promoter commonly used in P. pastoris; CaMV35S promoter: the 35S promoter from the plant pathogen cauliflower mosaic virus.
Table
Table 3. The titer of products increased by the expression of VHb.
Table 3. The titer of products increased by the expression of VHb.
ProductsEnhancementStrainReferences
AlcoholsEthanol~362%E. coli[73]
~118%E. coli[35]
~60%E. coli[74]
~47%E. coli[62]
~(41-83%)E. coli[63]
Butanediol~83%Enterobacter aerogenes[59]
Erythritol~26.13%Yarrowia lipolytica[75]
AntibioticsPyocyanin~3-foldE. coli[64]
Rifamycin B~2.2-foldAmycolatopsis mediterranei[65]
EnzymesLipase 2~87.84%P. pastoris[48]
Coenzyme Q10~71%Rhodobacter sphaeroides[66]
Xylanase~31%P. pastoris[49]
L-asparaginase~70%Pseudomonas aeruginosa[60]
AcidsArachidonic acid~8-foldMortierella alpina[67]
Docosahexaenoic acid~2.74-foldAurantiochytrium sp.[68]
Ganoderic acid~1.4-foldGanoderma lucidum[69]
S-adenosylmethionine~67%S. cerevisiae[76]
Glucaric acid~28.76%S. cerevisiae[77]
L-phenylalanine~16.6%E. coli[43]
PolysaccharidesBacterial cellulose~58.6%Gluconacetobacter xylinus[70]
Pullulan~42.08%Aureobasidium melanogenum[71]
β-glucan~(12.9–24.0%)Lentinula edodes[72]
6-(N-hydroxyethyl)-amino-6-deoxy-alpha-l-sorbofuranose~11.89%Gluconobacter oxydans[10]
OthersPolyhydroxybutyrate~71.5%C. necator[44]
Acetoin~83%Enterobacter aerogenes[59]
Enhancement: the ratio of the increase in the product titer of vgb+ strain relative to the control (% or fold).
Table
Table 4. The applications of VHb in biodegradation.
Table 4. The applications of VHb in biodegradation.
CompoundsStrainReferences
DibenzothiopheneRhodococcus erythropolis[83]
ThiosulfateT. versutus[42]
DibenzothiophenePaenibacillus[11]
PesticidesPseudomonas putida[85,86,87]
2-chlorobenzoic acidBurkholderia cepacia[88]
Benzene, toluene and xylenePseudomonas aeruginosa[90]
CadmiumEnterobacter aerogenes[91]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Yu, F.; Zhao, X.; Wang, Z.; Liu, L.; Yi, L.; Zhou, J.; Li, J.; Chen, J.; Du, G. Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin. Microorganisms 2021, 9, 1455. https://doi.org/10.3390/microorganisms9071455

AMA Style

Yu F, Zhao X, Wang Z, Liu L, Yi L, Zhou J, Li J, Chen J, Du G. Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin. Microorganisms. 2021; 9(7):1455. https://doi.org/10.3390/microorganisms9071455

Chicago/Turabian Style

Yu, Fei, Xinrui Zhao, Ziwei Wang, Luyao Liu, Lingfeng Yi, Jingwen Zhou, Jianghua Li, Jian Chen, and Guocheng Du. 2021. "Recent Advances in the Physicochemical Properties and Biotechnological Application of Vitreoscilla Hemoglobin" Microorganisms 9, no. 7: 1455. https://doi.org/10.3390/microorganisms9071455

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop