Oxygen-binding Proteins Research Articles

Survival strategies of aerobic methanotrophs under hypoxia in methanogenic lake sediments

BackgroundMicrobial methane oxidation, methanotrophy, plays a crucial role in mitigating the release of the potent greenhouse gas methane from aquatic systems. While aerobic methanotrophy is a well-established process in oxygen-rich environments, emerging evidence suggests their activity in hypoxic conditions. However, the adaptability of these methanotrophs to such environments has remained poorly understood. Here, we explored the genetic adaptability of aerobic methanotrophs to hypoxia in the methanogenic sediments of Lake Kinneret (LK). These LK methanogenic sediments, situated below the oxidic and sulfidic zones, were previously characterized by methane oxidation coupled with iron reduction via the involvement of aerobic methanotrophs.ResultsIn order to explore the adaptation of the methanotrophs to hypoxia, we conducted two experiments using LK sediments as inoculum: (i) an aerobic "classical" methanotrophic enrichment with ambient air employing DNA stable isotope probing (DNA-SIP) and (ii) hypoxic methanotrophic enrichment with repeated spiking of 1% oxygen. Analysis of 16S rRNA gene amplicons revealed the enrichment of Methylococcales methanotrophs, being up to a third of the enriched community. Methylobacter, Methylogaea, and Methylomonas were prominent in the aerobic experiment, while hypoxic conditions enriched primarily Methylomonas. Using metagenomics sequencing of DNA extracted from these experiments, we curated five Methylococcales metagenome-assembled genomes (MAGs) and evaluated the genetic basis for their survival in hypoxic environments. A comparative analysis with an additional 62 Methylococcales genomes from various environments highlighted several core genetic adaptations to hypoxia found in most examined Methylococcales genomes, including high-affinity cytochrome oxidases, oxygen-binding proteins, fermentation-based methane oxidation, motility, and glycogen use. We also found that some Methylococcales, including LK Methylococcales, may denitrify, while metals and humic substances may also serve as electron acceptors alternative to oxygen. Outer membrane multi-heme cytochromes and riboflavin were identified as potential mediators for the utilization of metals and humic material. These diverse mechanisms suggest the ability of methanotrophs to thrive in ecological niches previously thought inhospitable for their growth.ConclusionsOur study sheds light on the ability of enriched Methylococcales methanotrophs from methanogenic LK sediments to survive under hypoxia. Genomic analysis revealed a spectrum of genetic capabilities, potentially enabling these methanotrophs to function. The identified mechanisms, such as those enabling the use of alternative electron acceptors, expand our understanding of methanotroph resilience in diverse ecological settings. These findings contribute to the broader knowledge of microbial methane oxidation and have implications for understanding and potential contribution methanotrophs may have in mitigating methane emissions in various environmental conditions.

Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

Random Forest Classifier of Heme Proteins Using Porphyrin Distortions and Axial Ligands of Heme

We achieved success in the construction of a random forest classifier that is capable of accurately classifying heme proteins for oxygen storage/transport function and redox function, with a high level of precision (>83%), by utilizing heme porphyrin distortions and axial ligands of heme as features. Our findings suggest that the heme distortions and the axial ligands are significantly correlated with oxidoreductase functions and oxygen storage/transport functions, respectively, all of which are commonly observed in heme proteins. We constructed a random forest classifier using heme porphyrin distortions and axial ligands as features to successfully classify and predict two distinct classes of heme protein functions with remarkable accuracy. Our findings revealed that heme porphyrin distortion played a significant role in the classification of oxidoreductases, while axial ligand was found to be crucial for the classification of oxygen-binding proteins.

Rapid physiological and transcriptomic changes associated with oxygen delivery in larval anemonefish suggest a role in adaptation to life on hypoxic coral reefs.

Connectivity of coral reef fish populations relies on successful dispersal of a pelagic larval phase. Pelagic larvae must exhibit high swimming abilities to overcome ocean and reef currents, but once settling onto the reef, larvae transition to endure habitats that become hypoxic at night. Therefore, coral reef fish larvae must rapidly and dramatically shift their physiology over a short period of time. Taking an integrative, physiological approach, using swimming respirometry, and examining hypoxia tolerance and transcriptomics, we show that larvae of cinnamon anemonefish (Amphiprion melanopus) rapidly transition between "physiological extremes" at the end of their larval phase. Daily measurements of swimming larval anemonefish over their entire early development show that they initially have very high mass-specific oxygen uptake rates. However, oxygen uptake rates decrease midway through the larval phase. This occurs in conjunction with a switch in haemoglobin gene expression and increased expression of myoglobin, cytoglobin, and neuroglobin, which may all contribute to the observed increase in hypoxia tolerance. Our findings indicate that critical ontogenetic changes in the gene expression of oxygen-binding proteins may underpin the physiological mechanisms needed for successful larval recruitment to reefs.

Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins.

Protein-based oxygen sensors exhibit a wide range of affinity values ranging from low nanomolar to high micromolar. How proteins utilize different metals, cofactors, and macromolecular structure to regulate their oxygen affinity (Kd) to a value that is appropriate for their biological function is an important question in biochemistry and microbiology. In this chapter, we describe a simple setup that integrates a UV-Vis spectrometer with an oxygen optode for direct determination of Kd of heme-containing oxygen sensors. We provide details on how to set up the assay, acquire and fit data for accurate Kd determination using Cs H-NOX (Kd=23±2nM) as an example, and also discuss tips and tricks to make the assay workfor other oxygen-binding proteins.

Structure-Function Relationships of Oxygen Transport Proteins in Marine Invertebrates Enduring Higher Temperatures and Deoxygenation.

AbstractPredictions for climate change-to lesser and greater extents-reveal a common scenario in which marine waters are characterized by a deadly trio of stressors: higher temperatures, lower oxygen levels, and acidification. Ectothermic taxa that inhabit coastal waters, such as shellfish, are vulnerable to rapid and prolonged environmental disturbances, such as heatwaves, pollution-induced eutrophication, and dysoxia. Oxygen transport capacity of the hemolymph (blood equivalent) is considered the proximal driver of thermotolerance and respiration in many invertebrates. Moreover, maintaining homeostasis under environmental duress is inextricably linked to the activities of the hemolymph-based oxygen transport or binding proteins. Several protein groups fulfill this role in marine invertebrates: copper-based extracellular hemocyanins, iron-based intracellular hemoglobins and hemerythrins, and giant extracellular hemoglobins. In this brief text, we revisit the distribution and multifunctional properties of oxygen transport proteins, notably hemocyanins, in the context of climate change, and the consequent physiological reprogramming of marine invertebrates.

Multi-descriptor approaches to oxygen binding proteins prediction and classification using deep learning

Oxygen binding proteins play a key role in the transport and storage of oxygen through the body's cells. However, costly and time consuming biological tests can only determine a very small portion of all proteins available. This has made computational approaches increasingly essential to help biologists. The key idea behind this work is to investigate the effect of using several descriptors and deep learning to achieve better prediction. Three kinds of descriptors to represent protein sequences are considered namely amino acid composition (AAC), dipeptide composition (DC) and conjoint triad feature (CTF). Firstly, we applied deep neural networks (DNN) to single descriptor and then to multi-descriptor by investigation of the combination of descriptors. Secondly, DNN was developed to classify the oxygen binding proteins into six different classes. The experimental results show that the proposed prediction models outperform the existing methods where we obtained a Matthews correlation coefficient (MCC) of 0.9677.

Multi-descriptor approaches to oxygen binding proteins prediction and classification using deep learning

Multi-descriptor approaches to oxygen binding proteins prediction and classification using deep learning

Meta-Transcriptomic Analysis of RNAseq Data Reveals Pacu and Loach Fish with Unusually High Levels of Myoglobin Expression in Skeletal Muscles.

Simple SummaryOxygen-binding proteins that mediate oxygen-binding for storage and consumption, to reduce energy, are very diverse in fish, depending on their habitats. In the present study, oxygen-binding protein gene expression in the skeletal muscle of 25 diverse fish species was examined by a meta-transcriptomic approach. By using RNAseq data, this is the first study to examine the high level of myoglobin, one of the oxygen-binding proteins, transcripts in pacu and loach fish that might be related to their high tolerance for the oxygen-deficient environment. In addition, this study presents the power of the current method to compare the fish oxygen-binding protein expression and its putative gene expansion event.Oxygen-binding proteins, such as myoglobin, hemoglobin, neuroglobin, and cytoglobin, play a role in oxygen binding and delivery to tissues. In icefish, the loss of myoglobin and hemoglobin genes has been reported to be an adaptive evolution event. This interesting finding prompted us to exam oxygen-binding protein expression in diverse fish species. Taking advantage of substantial RNAseq data deposited in the NCBI (National Center for Biotechnology Information) database, we adopted a meta-transcriptomic approach to explore and compare four oxygen-binding protein gene expression levels in the skeletal muscle of 25 diverse fish species for the first time. RNAseq data were downloaded from the NCBI Sequence Read Archive (SRA) database, and de novo assembly was performed to generate transcript contigs. The genes encoding oxygen-binding proteins were then identified by the BLAST search, and the relative expression level of oxygen-binding protein genes was normalized by the RPKM (Reads per Kilobase Million) method. By performing expression profiling, hierarchy clustering, and principal component analysis, pacu and loach fish were noticed by their high myoglobin expression levels in skeletal muscle tissues among 25 diverse fish species. In conclusion, we demonstrated that meta-transcriptomic analysis of RNAseq data is an informative approach to compare the oxygen-binding protein expression and putative gene expansion event in fish.

The association of HFE gene H63D polymorphism with endurance athlete status and aerobic capacity: novel findings and a meta-analysis

PurposeIron is an important component of the oxygen-binding proteins and may be critical to optimal athletic performance. Previous studies have suggested that the G allele of C/G rare variant (rs1799945), which causes H63D amino acid replacement, in the HFE is associated with elevated iron indexes and may give some advantage in endurance-oriented sports. The aim of the present study was to investigate the association between the HFE H63D polymorphism and elite endurance athlete status in Japanese and Russian populations, aerobic capacity and to perform a meta-analysis using current findings and three previous studies.MethodsThe study involved 315 international-level endurance athletes (255 Russian and 60 Japanese) and 809 healthy controls (405 Russian and 404 Japanese). Genotyping was performed using micro-array analysis or by PCR. VO2max in 46 male Russian endurance athletes was determined using gas analysis system.ResultsThe frequency of the iron-increasing CG/GG genotypes was significantly higher in Russian (38.0 vs 24.9%; OR 1.85, P = 0.0003) and Japanese (13.3 vs 5.0%; OR 2.95, P = 0.011) endurance athletes compared to ethnically matched controls. The meta-analysis using five cohorts (two French, Japanese, Spanish, and Russian; 586 athletes and 1416 controls) showed significant prevalence of the CG/GG genotypes in endurance athletes compared to controls (OR 1.96, 95% CI 1.58–2.45; P = 1.7 × 10–9). Furthermore, the HFE G allele was associated with high V̇O2max in male athletes [CC: 61.8 (6.1), CG/GG: 66.3 (7.8) ml/min/kg; P = 0.036].ConclusionsWe have shown that the HFE H63D polymorphism is strongly associated with elite endurance athlete status, regardless ethnicities and aerobic capacity in Russian athletes.

The haemoglobin subunits alpha and beta: Old and new genetic variants in the Italian Mediterranean buffalo

Haemoglobin (HB), the most widely distributed respiratory pigment in the animal kingdom, is among the best characterized oxygen-binding proteins, both at functional and molecular level. However, very little information is available about the genomic features of HB in river buffalo (Bubalus bubalis), even though there are reports in literature confirming the presence of interesting polymorphisms at the protein level in Mediterranean buffalo. We hence address the characterization of exonic as well as intronic nucleotide polymorphism in the haemoglobin subunit alpha and beta in a set of nine Italian Mediterranean buffaloes exhibiting different HB phenotypes. The nine buffaloes were selected from a random set of 398 samples, previously analysed for their HB protein polymorphism, in order to account for both globin variants and the evolution of intron variability within the most common domesticated species of the family Bovidae. All four sequenced clones of the subunit alpha were 1311 bp, whereas the length of the five different sequenced clones of the subunit beta ranged from 1841 to 1960 bp, due to an insertion of 119 nucleotides. Six polymorphic sites were detected in the four amplicons of alpha subunit. Among them, two variations concern exclusively haplotype A, while four sequence variations were found to be specific to haplotype B. Several variations, both in exonic and intronic regions, were detected in the B. bubalis subunit beta. In conclusion, the nucleotide sequence variants observed in this work substantiate the known haemoglobin protein polymorphisms, and an updated protein nomenclature is provided here. In addition, we observed a high sequence similarity in the overall pattern of variation in the haemoglobin subunits, possibly the results of a concerted evolution, with relatively more extensive gene homogenization in river buffalo than in other ruminant species.

Resonant inelastic X-ray scattering determination of the electronic structure of oxyhemoglobin and its model complex

Hemoglobin and myoglobin are oxygen-binding proteins with S = 0 heme {FeO2}8 active sites. The electronic structure of these sites has been the subject of much debate. This study utilizes Fe K-edge X-ray absorption spectroscopy (XAS) and 1s2p resonant inelastic X-ray scattering (RIXS) to study oxyhemoglobin and a related heme {FeO2}8 model compound, [(pfp)Fe(1-MeIm)(O2)] (pfp = meso-tetra(α,α,α,α-o-pivalamido-phenyl)porphyrin, or TpivPP, 1-MeIm = 1-methylimidazole) (pfpO2), which was previously analyzed using L-edge XAS. The K-edge XAS and RIXS data of pfpO2 and oxyhemoglobin are compared with the data for low-spin FeII and FeIII [Fe(tpp)(Im)2]0/+ (tpp = tetra-phenyl porphyrin) compounds, which serve as heme references. The X-ray data show that pfpO2 is similar to FeII, while oxyhemoglobin is qualitatively similar to FeIII, but with significant quantitative differences. Density-functional theory (DFT) calculations show that the difference between pfpO2 and oxyhemoglobin is due to a distal histidine H bond to O2 and the less hydrophobic environment in the protein, which lead to more backbonding into the O2 A valence bond configuration interaction multiplet model is used to analyze the RIXS data and show that pfpO2 is dominantly FeII with 6-8% FeIII character, while oxyhemoglobin has a very mixed wave function that has 50-77% FeIII character and a partially polarized Fe-O2 π-bond.

Specific Proteins in Relation with Iron Overload in Experimental Study

Iron is an essential mineral for organisms and due to environment iron deficiency and pollution it is very important to understand and monitor the iron homeostasis. Thus, the concentration of iron is very important in hematological and biochemical parameters because iron is component of hemoglobin, erythrocytes, and myoglobin -being part of oxygen-binding proteins. The aim of our experiment was to try a natural protection with fresh, organic plants administration � of iron overload in rabbits. Thereby, we administrated ferrous gluconate (hydro-solution, 15g Fe2+/body weight) as intraperitoneal injection for 43 days to German Lop Eared breed rabbits, and in the same time we tried to protect the animals with a diet consisting in black cumin seeds; clover, chives, leek, parsley, coriander, rucola, radish, fenugreek - leaves; cucumber and carrots. At the end of experiment we analyzed the protein electrophoresis (albumin, a1-globulin, a2-globulin, b1-globulin, b2-globulin and g-globulin), total iron binding capacity, and transferrin concentration. The analytical results demonstrated that plant administration was beneficial for iron homeostasis and the tested blood parameters did not record significant variations between control and experimental group, which proves that diet is very important in health and homeostatic mechanisms in living organisms.

Discovery of Novel Hemocyanin-Like Genes in Metazoans.

Among animals, two major groups of oxygen-binding proteins are found: proteins that use iron to bind oxygen (hemoglobins and hemerythrins) and two non-homologous hemocyanins that use copper. Although arthropod and mollusc hemocyanins bind oxygen in the same manner, they are distinct in their molecular structures. In order to better understand the range of natural variation in hemocyanins, we searched for them in a diverse array of metazoan transcriptomes by using bioinformatics tools to examine hemocyanin evolutionary history and to consequently revive the discussion about whether all metazoan hemocyanins shared a common origin with frequent losses or whether they originated separately after the divergence of Lophotrochozoa and Ecdysozoa. We confirm that the distribution of hemocyanin-like genes is more widespread than previously reported, including five putative novel mollusc hemocyanin genes in two annelid species from Chaetopteridae. For arthropod hemocyanins, 16 putative novel genes were retained, and the presence of arthropod hemocyanins in 11 annelid species represents a novel observation. Interestingly, Annelida is the lineage that presents the greatest repertoire of oxygen transport proteins reported to date, possessing all the main superfamily proteins, which could be explained partially by the immense variability of lifestyles and habitats. Work presented here contradicts the canonical view that hemocyanins are restricted to molluscs and arthropods, suggesting that the occurrence of copper-based blood pigments in metazoans has been underestimated. Our results also support the idea of the presence of oxygen carrier hemocyanins being widespread across metazoans with an evolutionary history characterized by frequent losses.

Hemerythrins are widespread and conserved for methanotrophic guilds

Hemerythrins (Hrs) are non-heme oxygen-binding proteins which act as carriers of di-oxygen. Hrs were earlier thought to be present only in marine invertebrates. Bacteriohemerythrins were discovered for the first time in two prokaryotes, of which one was a methanotroph, Methylococcus capsulatus and later found to be present in many bacterial and archaeal families. The role of hemerythrin in Methylococcus had been predicted to be in transporting oxygen to the particulate methane monooxygenase enzyme (pMMO). In our present study, we investigated the presence of Hrs as well as Hr-like domains in the sequenced genomes of methanotrophs. We found that most of the methanotrophs (80%) have a single and up to three variants of Hr and a few possess additional or exclusively Hr containing domain/s. Phylogenetic analysis showed that Hr from all the methanotrophic guilds (i.e. alpha-, gamma-proteobacterial and Verrucomicrobial) grouped together. The phylogeny of Hr showed two main clusters and two small branches. The phylogeny of pMMO and Hr Cluster I showed congruence. Three novel methanotrophic genera isolated from rice fields showed the presence of multiple and divergent copies of Hrs. Hr and Hr domain was found to be absent in Methylocella silvestris a facultative methanotroph which lacks pMMO and intracytoplasmic membranes (ICM). Single domain Hr may have relevance with the pink or brown coloration seen in methanotrophs as almost all of the colored methanotrophs showed single domain Hr in their genomes.

Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

Broad Phylogenetic Occurrence of the Oxygen-Binding Hemerythrins in Bilaterians.

Animal tissues need to be properly oxygenated for carrying out catabolic respiration and, as such, natural selection has presumably favored special molecules that can reversibly bind and transport oxygen. Hemoglobins, hemocyanins, and hemerythrins (Hrs) fulfill this role, with Hrs being the least studied. Knowledge of oxygen-binding proteins is crucial for understanding animal physiology. Hr genes are present in the three domains of life, Archaea, Bacteria, and Eukaryota; however, within Animalia, Hrs has been reported only in marine species in six phyla (Annelida, Brachiopoda, Priapulida, Bryozoa, Cnidaria, and Arthropoda). Given this observed Hr distribution, whether all metazoan Hrs share a common origin is circumspect. We investigated Hr diversity and evolution in metazoans, by employing in silico approaches to survey for Hrs from of 120 metazoan transcriptomes and genomes. We found 58 candidate Hr genes actively transcribed in 36 species distributed in 11 animal phyla, with new records in Echinodermata, Hemichordata, Mollusca, Nemertea, Phoronida, and Platyhelminthes. Moreover, we found that “Hrs” reported from Cnidaria and Arthropoda were not consistent with that of other metazoan Hrs. Contrary to previous suggestions that Hr genes were absent in deuterostomes, we find Hr genes present in deuterostomes and were likely present in early bilaterians, but not in nonbilaterian animal lineages. As expected, the Hr gene tree did not mirror metazoan phylogeny, suggesting that Hrs evolutionary history was complex and besides the oxygen carrying capacity, the drivers of Hr evolution may also consist of secondary functional specializations of the proteins, like immunological functions.

The loss of hemoglobin and myoglobin does not minimize oxidative stress in Antarctic icefishes

The unusual pattern of expression of hemoglobin (Hb) and myoglobin (Mb) among Antarctic notothenioid fishes provides an exceptional model system for assessing the impact of these proteins on oxidative stress. We tested the hypothesis that the lack of oxygen-binding proteins may reduce oxidative stress. Levels and activity of pro-oxidants and small-molecule and enzymatic antioxidants, and levels of oxidized lipids and proteins in the liver, oxidative skeletal muscle and heart ventricle were quantified in five species of notothenioid fishes differing in the expression of Hb and Mb. Levels of ubiquitinated proteins and rates of protein degradation by the 20S proteasome were also quantified. Although levels of oxidized proteins and lipids, ubiquitinated proteins, and antioxidants were higher in red-blooded fishes than in Hb-less icefishes in some tissues, this pattern did not persist across all tissues. Expression of Mb was not associated with oxidative damage in the heart ventricle, whereas the activity of citrate synthase and the contents of heme were positively correlated with oxidative damage in most tissues. Despite some tissue differences in levels of protein carbonyls among species, rates of degradation by the 20S proteasome were not markedly different, suggesting either alternative pathways for eliminating oxidized proteins or that redox tone varies among species. Together, our data indicate that the loss of Hb and Mb does not correspond with a clear pattern of either reduced oxidative defense or oxidative damage.

New Lessons from an Old Fish: What Antarctic Icefishes May Reveal about the Functions of Oxygen-Binding Proteins.

The loss of expression of the oxygen-binding protein hemoglobin (Hb) in the family Channichthyidae (suborder Notothenioidei) of Antarctic fishes is considered a disaptation that has persisted because of the unusual conditions prevailing in the Southern Ocean during the evolution of the family. The loss of expression of the intracellular oxygen-binding protein myoglobin (Mb) in heart ventricles is more of a conundrum because it occurred at four points during the radiation of the family, suggesting weakened selective pressure maintaining expression of the protein. Yet, studies have shown that when present, Mb enhances function. Here, I discuss potential reasons for weakened selective pressure maintaining Mb expression in light of the multiple functions proposed for Mb. Additionally, I discuss results from recent studies exploring the possibility that the loss of Hb and Mb may be advantageous because it reduces the production of reactive oxygen species, levels of oxidized proteins, and the energetic costs associated with replacing oxidatively damaged proteins.

Recombinant Escherichia coli strains with inducible Campylobacter jejuni single domain hemoglobin CHb expression exhibited improved cell growth in bioreactor culture.

Maintaining an appropriate concentration of dissolved oxygen in aqueous solution is critical for efficient operation of a bioreactor, requiring sophisticated engineering design and a system of regulation to maximize oxygen transfer from the injected air bubbles to the cells. Bacterial hemoglobins are oxygen-binding proteins that transfer oxygen from the environment to metabolic processes and allow bacteria to grow even under microaerophilic conditions. To improve the oxygen utilization efficiency of cells and overcome the oxygen shortage in bioreactors, the gene coding for the Campylobacter jejuni single domain hemoglobin (CHb) gene was artificially synthesized and functionally expressed under the control of inducible expression promoters PT7 and Pvgh in Escherichia coli. The effects of the recombinants PT7-CHb and Pvgh-CHb on cell growth were evaluated in aerobic shake flasks, anaerobic capped bottles and a 5-L bioreactor, and a pronounced improvement in cell biomass was observed for CHb-expressing cells. To determine the growth curves, CHb gene expression, and CHb oxygen-binding capacity of specific recombinants with different promoters, we determined the time course of CHb gene expression in the two recombinants by semi-quantitative RT-PCR and CO differential spectrum assays. Based on the growth patterns of the two recombinants in the bioreactor, we proposed different recombinant types with optimal performance under specific culture conditions.