Engineering E. coli for improved microaerobic pDNA production.

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA-, which, together with W3110recA-vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli.

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA- (W1). The plasmid DNA yields from biomass (YpDNA/X) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase YpDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The YpDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The YpDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.

Intracellular expression of Vitreoscilla haemoglobin improves lipid production in Yarrowia lipolytica.

Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in oleaginous yeast, Yarrowia lipolytica, and its effect on lipid accumulation was analysed. During fermentation, the dissolved oxygen (DO) concentration was controlled at 5, 10, 20 and 30 of full O2 saturation and also uncontrolled by varying the stirring speed and aeration rate. Yarrowia lipolytica harbouring the VHb gene (VHb+ strain) displayed more pseudomycelium than the control strain (VHb- strain), and VHb expression also enhanced the cell density. When DO level was controlled at 30%, the biomass of VHb+ strains reached about 19 g l-1 and increased by 27% compared to VHb- strains. Total fatty acid contents, although, were higher in VHb+ strains than in VHb- strains under all DO levels, and maximally increased by c. 40% (from 10·5 to 14·5% of the biomass) when the DO concentration was controlled at 30%. VHb overexpression, however, markedly suppressed citrate secretion. In addition, expression of VHb also induced significant changes in fatty acid composition and increased the oleic acid content. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic modification by overexpressing Vitreoscilla haemoglobin (VHb) is an efficient and economic method for improving O2 utilization for microbial fermentation. In this study, VHb was expressed in Yarrowia lipolytica, which is a most attractive model for microbial oil production because of its excellent lipid accumulation capacity and convenient genetic tools. Expression of VHb resulted in lipid accumulation increased and cell morphology shift, however, markedly suppressed citrate secretion. This study provided the new strategy for fermentation technology to improve lipid production in oleaginous micro-organisms.

Enhanced production of poly-γ-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is an important multifunctional biopolymer with various applications, for which adenosine triphosphate (ATP) supply plays a vital role in biosynthesis. In this study, the enhancement of γ-PGA production was attempted through various approaches of improving ATP supply in the engineered strains of Bacillus licheniformis. The first approach is to engineer respiration chain branches of B. licheniformis, elimination of cytochrome bd oxidase branch reduced the maintenance coefficient, leading to a 19.27% increase of γ-PGA yield. The second approach is to introduce Vitreoscilla hemoglobin (VHB) into recombinant B. licheniformis, led to a 13.32% increase of γ-PGA yield. In the third approach, the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed, with the AdK overexpressed strain increased γ-PGA yield by 14.69%. Our study also confirmed that the respiratory nitrate reductase, NarGHIJ, is responsible for the conversion of nitrate to nitrite, and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia. Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE, and overexpression of NarG, NasC, and ResD also improved the ATP supply and the consequent γ-PGA yield. Based on the above individual methods, a method of combining the deletion of cydBC gene and overexpression of genes vgB, adK, and resD were used to enhance ATP content of the cells to 3.53 µmol/g of DCW, the mutant WX-BCVAR with this enhancement produced 43.81 g/L of γ-PGA, a 38.64% improvement compared to wild-type strain WX-02. Collectively, our results demonstrate that improving ATP content in B. licheniformis is an efficient strategy to improve γ-PGA production.

[Co-expression of ß-glucosidase and Vitreoscilla hemoglobin in Escherichia coli].

In producing recombinant ß-glucosidase in Escherichia coli by high-cell density cultivation (HCDC), insufficient soluble oxygen is always a problem. To address it, Vitreoscilla hemoglobin (VHb) was introduced into Escherichia coli by the bicistron and T7 promoter expression systems, to improve soluble oxygen by bacterial cells and thereby to enhance the biomass and recombinant ß-glucosidase production. In the case of bicistron expression system, cell density in shaking flask reached OD600=(4.24±0.29), 35.03% higher than that of the control without VHb. Correspondingly, the maximum activity of ß-glucosidase co-expressed with VHb was (9.78±0.55) U/mL, 25.38% higher than that of the control. In a 3-L fermentor, the maximum activity of ß-glucosidase was 141.23 U/mL, 35.57% higher than that of the control. In contrast, the activity of ß-glucosidase co-expressed with VHb under T7 promoter was lower than that of the control, either in flask or in fermentor. Co-expressing ß-glucosidase with VHb using the bicistron expression system may improve the tolerance of E. coli to insufficient soluble oxygen and thus promote the bacterial biomass and the enzyme yield.

Increasing oxygen availability for improving poly(3-hydroxybutyrate) production by Halomonas.

Technologies enabling high-cell-density growth are required for economical industrial production of most biotechnological products. However, the key factor limiting cell density in bioreactors is the availability of oxygen during the late phases of fermentation. Although the expression of bacterial Vitreoscilla hemoglobin (VHb) is useful for enhanced oxygen availability, bacterial cell membrane makes efficient hemoglobin-oxygen contact a challenge. On the other hand, periplasmic spaces of Gram-negative microorganisms offer an excellent compartment for the intermittent storage of hemoglobin-bound oxygen. In this study, the cell growth was increased by a remarkable 100% using the twin-arginine translocase (Tat) pathway to export active VHb into the periplasm of Escherichia coli, Halomonas bluephagenesis TD01 and H. campaniensis LS21. Furthermore, eight low-oxygen-inducible vgb promoters were constructed in tandem to become a strong promoter cassette termed P8vgb, which better induces expression of both gene vgb encoding VHb and the PHB synthesis operon microaerobically. Both the P8vgb and VHb performed excellently in E. coli and two Halomonas spp., demonstrating their universal applicability for various organisms.

Enhancement of l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin.

l-Phenylalanine is an important amino acid that is widely used in the production of food flavors and pharmaceuticals. Generally, l-phenylalanine production by engineered Escherichia coli requires a high rate of oxygen supply. However, the coexpression of Vitreoscilla hemoglobin gene (vgb), driven bya tac promoter, with the genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase (aroF) and feedback-resistant chorismate mutase/prephenate dehydratase (pheAfbr ), led to increased productivity and decreased demand for aeration by E. coli CICC10245. Shake-flask studies showed that vgb-expressing strains displayed higher rates of oxygen uptake, and l-phenylalanine production under standard aeration conditions was increased. In the aerobic fermentation process, cell growth, l-phenylalanine production, and glucose consumption by the recombinant E. coli strain PAPV, which harbored aroF, pheAfbr , and tac-vgb genes, were increased compared to that in the strain harboring only aroF and pheAfbr (E. coli strain PAP), especially under oxygen-limited conditions. The vgb-expressing strain PAPV produced 21.9% more biomass and 16.6% more l-phenylalanine, while consuming only approximately 5% more glucose after 48 H of fermentation. This study demonstrates a method to enhance the l-phenylalanine production by E. coli using less intensive and thus more economical aeration conditions.

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO-K1 cells.

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO-K1 cell culture was investigated. For this purpose, CHO-K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP-expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.

Improved polysaccharide production in a submerged culture of Ganoderma lucidum by the heterologous expression of Vitreoscilla hemoglobin gene.

Expression of Vitreoscilla hemoglobin (VHb) gene was used to improve polysaccharide production in Ganoderma lucidum. The VHb gene, vgb, under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase gene promoter was introduced into G. lucidum. The activity of expressed VHb was confirmed by the observation of VHb specific CO-difference spectrum with a maximal absorption at 419 nm for the transformant. The effects of VHb expression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including phosphoglucomutase (PGM), uridine diphosphate glucose pyrophosphorylase (UGP), and ß-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in the vgb-bearing G. lucidum were 26.4 mg/100mg dry weight and 0.83 g/L, respectively, which were higher by 30.5% and 88.2% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were up-regulated by 1.51-, 1.55- and 3.83-fold, respectively, in the vgb-bearing G. lucidum. This work highlights the potential of VHb to enhance G. lucidum polysaccharide production by large scale fermentation.

Construction of a Genetic System for Streptomyces albulus PD-1 and Improving Poly(ε-L-lysine) Production Through Expression of Vitreoscilla Hemoglobin.

Poly(ε-L-lysine) (ε-PL) is a novel bioactive polymer secreted by filamentous bacteria. Owing to lack of a genetic system for most ε-PL-producing strains, very little research on enhancing ε-PL biosynthesis by genetic manipulation has been reported. In this study, an effective genetic system was established via intergeneric conjugal transfer for Streptomyces albulus PD-1, a famous ε-PL-producing strain. Using the established genetic system, the Vitreoscilla hemoglobin (VHb) gene was integrated into the chromosome of S. albulus PD-1 to alleviate oxygen limitation and to enhance the biosynthesis of ε-PL in submerged fermentation. Ultimately, the production of ε-PL increased from 22.7 g/l to 34.2 g/l after fed-batch culture in a 5 L bioreactor. Determination of the oxygen uptake rate, transcriptional level of ε-PL synthetase gene, and ATP level unveiled that the expression of VHb in S. albulus PD-1 enhanced ε-PL biosynthesis by improving respiration and ATP supply. To the best of our knowledge, this is the first report on enhancing ε-PL production by chromosomal integration of the VHb gene in an ε-PL-producing strain, and it will open a new avenue for ε-PL production.

Expression profiling of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis in acid-nitrosative multi-stress displays defined regulatory networks.

Several studies regarding the transcriptome of Mycobacterium tuberculosis following the exposure to various in vitro simulated phagosomal stressors, have already tried to elucidate the bacterium behavior during the intracellular infection. An in vitro acid-nitrosative multi-stress was carried out for M. tuberculosis H37Rv and Mycobacterium smegmatis MC(2)155 in order to analyze by DNA-microarray the gene expression changes associated respectively to pathogenic and non-pathogenic mycobacterial species. During acid-nitrosative multi-stress both mycobacteria shift their transcriptome to allow the anaerobic respiratory state and energy pathways characteristic of starvation. M. tuberculosis counteracts the combined acid-nitrosative stress more efficiently than M. smegmatis as also shown by the up-regulation of glbN and hmp genes, that are specifically directed to NO detoxification. Moreover, the down-regulation of some virulence factors involved in phthiocerol dimycocerosates synthesis strengthens the hypothesis that these major virulence determinants may be attenuated by M. tuberculosis in the presence of reactive nitrogen species. In fact, it down-regulates other genes implicated in the synthesis of membrane structural lipids but in contrast to M. smegmatis, M. tuberculosis up-regulates many genes annotated for the synthesis of peptidoglycan. Results suggest a gene regulation of M. tuberculosis which reveals a distinctive expression pattern under stressful environment.

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

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

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

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

Enhanced production of total flavones and exopolysaccharides viaVitreoscilla hemoglobin biosynthesis in Phellinus igniarius.

The Vitreoscilla hemoglobin gene (vgb) was expressed by chromosomal integration in Phellinus igniarius to alleviate oxygen limitation and improve metabolites yields during submerged fermentation. Firstly, an expression vector containing vgb was constructed, and transformed into protoplast from P. igniarius. Carbon monoxide difference spectrum absorbance assay showed that vgb was successfully expressed and had biological activity. In shake flasks, the vgb expression enhanced dry mycelial weight 1.32-fold and increased total flavones and exopolysaccharides production 1.78- and 1.33-fold, respectively. When P. igniarius (vgb+) and P. igniarius (vgb-) strains were cultured in bioreactor, Vitreoscilla hemoglobin in P. igniarius promoted the mycelia growth from 5.40 to 10.90 g/L and stimulated total flavones and exopolysaccharides synthesis; their maximum productions reached to 11.43 and 1.33 g/L. Furthermore, compared to P. igniarius (vgb-), the acetic acid accumulation in P. igniarius (vgb+) cultures decreased from 1.54 and 1.78 to 1.19 and 1.27 g/L in flask and bioreactor, respectively.

[Expression of Vitreoscilla hemoglobin improves recombinant lipase production in Pichia pastoris].

Yarrowia lipolytica lipase Lip2 (YlLip2) is an important industrial enzyme with many potential applications. To alleviate the dissolved oxygen (DO) limitation and improve YlLip2 production during high-cell density fermentation, the YlLip2 gene lip2 and Vitreoscilla hemoglobin (VHb) gene vgb were co-expressed in Pichiapastoris under the control of AOX1 and PsADH2 promoter, respectively. The PsADH2 promoter from Pichia stipitis could be activated under oxygen limitation. The SDS-PAGE and CO-difference spectrum analysis indicated that VHb and YlLip2 had successfully co-expressed in recombinant strains. Compared with the control cells (VHb-, GS115/9Klip2), the expression levels of YlLip2 in VHb-expressing cells (VHb+, GS115/9Klip2-pZPVT) under oxygen limitation were improved 25% in shake-flask culture and 83% in a 10 L fermentor. Moreover, the VHb+ cells displayed higher biomass than VHb- cells at lower DO levels in a 10 L fermentor. In this study, we also achieved a VHb-expressing clone harboring multicopy lip2 gene (GS115/9Klip2-pZPVTlip2 49#), which showed the maximum lipolytic activity of 33 900 U/mL in a 10 L fermentor under lower DO conditions. Therefore, it can be seen that expression of VHb with PsADH2 promoter in P. pastoris combined with increasing copies of lip2 gene is an effective strategy to improve YlLip2 production.

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose.

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.

Nitric oxide and oxygen regulate truncated hemoglobin gene expression in Frankia strain CcI3.

The Frankia genome contains two truncated hemoglobin genes (hboN and hboO) whose functions remain to be determined. Nitric oxide (NO) generated by the addition of 400 microM SNAP (S-nitroso-N-acetylpenicillamine) caused a 10-fold increase in hboN gene expression but had no effect on hboO expression. The addition of the NO scavenger, carboxy-PT10, reduced the effect of SNAP. hboO gene expression increased under low-oxygen conditions, while hboN expression was unaffected. These results suggest that HboN may function in protection from nitrosative stress and that HboO may act as an oxygen transport molecule for increased respiration in hypoxic environments.

[Fusion expression of D-amino acid oxidase from Trignoposis variabilis with maltose binding protein and Vitreoscilla hemoglobin].

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.

Responses of Mycobacterium tuberculosis hemoglobin promoters to in vitro and in vivo growth conditions.

The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H(2)O(2), but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced approximately 2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased approximately 2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host.

Assessment of biotechnologically relevant characteristics of heterologous hemoglobins in E. coli.

The use of the heterologous bacterial hemoglobin (VHb) from Vitreoscilla to enhance growth and productivity of Escherichia coli under conditions of oxygen limitation has been one of the foremost examples of metabolic engineering. Although VHb has earned its merits during the last two decades by providing enhanced physiological enhancements to organisms from all kingdoms of life, it has been the candidate of choice primarily for historical reasons. Findings made during the last years, however, suggest that hemoglobin and flavohemoglobin proteins from bacterial species other than Vitreoscilla or artificially generated mutant proteins or fusion variants of hemoglobins and flavohemoglobins may be better suited for use in biotechnological processes. This account provides guidelines for the assessment of biotechnologically relevant characteristics conferred by such novel heterologous hemoglobins and flavohemoglobins in E. coli.