Enhancement of Biodesulfurization in Two-Liquid Systems by Heterogeneous Expression of Vitreoscilla Hemoglobin

The vgb gene, encoding Vitreoscilla hemoglobin (VHb), was introduced into a specific desulfurization bacterium, Rhodococcus erythropolis LSSE8-1. The VHb-specific spectrum was observed for the recombinant. Compared to the wild type, the strain bearing vgb showed a higher biomass yield and desulfurizing activity.     

α-Hemoglobin-stabilizing Protein (AHSP) Perturbs the Proximal Heme Pocket of Oxy-α-hemoglobin and Weakens the Iron-Oxygen Bond

α-Hemoglobin (αHb)-stabilizing protein (AHSP) is a molecular chaperone that assists hemoglobin assembly. AHSP induces changes in αHb heme coordination, but how these changes are facilitated by interactions at the αHb·AHSP interface is not well understood. To address this question we have used NMR, x-ray absorption spectroscopy, and ligand binding measurements to probe αHb conformational changes induced by AHSP binding. NMR chemical shift analyses of free CO-αHb and CO-αHb·AHSP indicated that the seven helical elements of the native αHb structure are retained and that the heme Fe(II) remains coordinated to the proximal His-87 side chain. However, chemical shift differences revealed alterations of the F, G, and H helices and the heme pocket of CO-αHb bound to AHSP. Comparisons of iron-ligand geometry using extended x-ray absorption fine structure spectroscopy showed that AHSP binding induces a small 0.03 Å lengthening of the Fe-O₂ bond, explaining previous reports that AHSP decreases αHb O₂ affinity roughly 4-fold and promotes autooxidation due primarily to a 3–4-fold increase in the rate of O₂ dissociation. Pro-30 mutations diminished NMR chemical shift changes in the proximal heme pocket, restored normal O₂ dissociation rate and equilibrium constants, and reduced O₂-αHb autooxidation rates. Thus, the contacts mediated by Pro-30 in wild-type AHSP promote αHb autooxidation by introducing strain into the proximal heme pocket. As a chaperone, AHSP facilitates rapid assembly of αHb into Hb when βHb is abundant but diverts αHb to a redox resistant holding state when βHb is limiting.     

Development of Intergeneric Conjugal Gene Transfer System in Streptomyces diastatochromogenes 1628 and Its Application for Improvement of Toyocamycin Production

Streptomyces diastatochromogenes 1628, capable of producing toyocamycin (TM), has exhibited a potential biocontrol effect in inhibiting the development of phytopathogens in the agriculture field. In this study, an efficient transformation system was developed using the intergeneric conjugation. This was achieved by optimization of experimental parameters. Under optimal conditions, a maximal conjugation frequency of 4.1 × 10^?4 per recipient was obtained. In order to heterologously express the gene vgb encoding Vitreoscilla hemoglobin in S. diastatochromogenes 1628, we placed vgb under the control of the constitutive promoter PermE^* and constructed plasmid pIB139-vgb. This plasmid was integrated into the chromosome of S. diastatochromogenes 1628 using intergeneric conjugation established above. Finally, strain 1628-VHB-23 with the highest TM production was screened. Results indicated that expression of vgb gene had always significantly promoted the cell growth and TM production in S. diastatochromogenes 1628 under different dissolved oxygen conditions. In particular, under the limited aerobic condition, strain 1628-VHB-23 obtained 33.3 % more DCW and produced 210 % more TM in 7-l fermentor as compared with the wild-type strain.     

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (Fe^IIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower Fe^IIINO dissociation rate (k_d) and NO association rate (k_on) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO k_on and k_d by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O₂ catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O₂ intermediate, but instead were due to the slower NO k_on and k_d and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS.     

Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene (vgb) integrated into their chromosomes

Using genetic engineering, the Vitreoscilla (bacterial) hemoglobin gene (vgb) was integrated stably into the chromosomes of Pseudomonas aeruginosa and Burkholderia sp. strain DNT. This was done for both wild type vgb and two site-directed mutants of vgb that produce Vitreoscilla hemoglobin (VHb) with lowered oxygen affinities; in all cases functional VHb was expressed. Similar to previous results, the wild type VHb improved growth for both species and degradation of 2,4-dinitrotoluene (Burkholderia sp.) or benzoic acid (P. aeruginosa) under both normal and low aeration conditions. Both mutant vgbs enhanced these parameters compared to wild type vgb, and the improvement was seen in both species. The enhancements were generally greater at low aeration than at normal aeration. The results demonstrate the possibility that the positive effects provided by VHb may be augmented by protein engineering.     

Enhancement of natamycin production on Streptomyces gilvosporeus by chromosomal integration of the Vitreoscilla hemoglobin gene (vgb)

Oxygen deficiency is a critical factor during the fermentation production of natamycin. In order to alleviate oxygen limitation and enhance the yield of natamycin, the vgb gene, encoding Vitreoscilla hemoglobin (VHb) was inserted into pSET152 with its native promoter and integrated into the chromosome of Streptomyces gilvosporeus (S. gilvosporeus). The expression of VHb was determined by Western blotting. 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 recombinant. Integration of the empty plasmid pSET152 did not affect natamycin production of S. gilvosporeus. While the vgb-harboring strain exhibited high natamycin productivity, reaching 3.31 g/L in shake flasks and 8.24 g/L in 1-L fermenters. Compared to the wild strain, expression of VHb, increased the natamycin yield of the strain bearing vgb by 131.3 % (jar fermenter scale) and 175 % (shake flask scale), respectively, under certain oxygen-limiting condition. Addition of an extra copy of the vgb gene in S. gilvosporeus-vgb2 did not enhance the natamycin production obviously. These results provided a superior natamycin-producing strain which can be directly used in industry and a useful strategy for increasing yields of other metabolites in industrial strains.     

Crystal structures of two hemoglobin components from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera)

The polymorphic components of hemoglobin (Hb) of the midge larva Propsilocerus akamusi were classified into two distinct types dependent on their spectroscopic properties, normal absorption (N) and low absorption (L). Analyses of the amino acid sequences of component VII (N-type Hb) and component V (L-type Hb) from P. akamusi indicated that one remarkable difference is the replacement of the distal histidine (His) with isoleucine (Ile) in component V. To clarify the structural differences between the two Hb components, we determined the crystal structures of components V and VII at resolutions of 1.64 A and 1.50 A, respectively. These crystal structures indicated a short additional helix comprising three amino acid residues at the C-terminal region in component V, and a typical globin fold including eight helices in component VII. Comparison of the heme regions of the Hb components suggests that the structural changes of the heme region in component V on ligation differ from that of usual Hb.     

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P_vgb of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P_vgb was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P_vgb provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P_vgb, while a construct containing P_vgb, the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P_vgb, vgb operon) or pKVpPV (P_vgb, vgb operon, par) slightly attenuated (30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P_vgb, egfp) or pKVpP-E (P_vgb, par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P_vgb, vgb operon, par, tod) or pKVpP-T (P_vgb, par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.     

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33. Received 20 October 2000/ Accepted in revised form 04 May 2001     

Effect of Growth Conditions on Yield and Heme Content of Vitreoscilla

Vitreoscilla, a gliding bacterium in the Beggiatoaceae, is an obligate aerobe in which cytochrome o functions as the terminal oxidase. Protoheme IX is the only heme type present in this organism. The yield and heme content of Vitreoscilla cells grown in yeast extract, peptone, and acetate were dependent on growth conditions. Cells harvested in early stationary phase contained roughly three times as much heme as cells in early log phase. There was an optimal shaking rate for maximum heme content of cells harvested in stationary phase at fixed initial nutrient concentration. The heme content of cells grown at a fixed shaking rate increased from 5 nmol/g (wet weight) in media which had low nutrient concentration to a maximum of 45 nmol/g (wet weight) in media which had high nutrient concentration, and there was a corresponding sixfold increase in cytochrome o content and an eightfold increase in respiratory rate, evidence that some of the additional heme was incorporated into respiratory pigments. Heme content may be controlled jointly by competition for oxygen and availability of nutrients. Temperature and initial pH affected the growth rate but not the final yield or heme content. Growth rate was optimal at pH 8.0 to 8.5. A defined medium for Vitreoscilla, which is based on glutamate as the carbon source, is described; the other organic components of this medium are acetate, tryptophan, thiamine, biotin, and riboflavin.     

Combining co-culturing of Paenibacillus strains and Vitreoscilla hemoglobin expression as a strategy to improve biodesulfurization

Enhancement of the desulfurization activities of Paenibacillus strains 32O-W and 32O-Y were investigated using dibenzothiophene (DBT) and DBT sulfone (DBTS) as sources of sulphur in growth experiments. Strains 32O-W, 32O-Y and their co-culture (32O-W plus 32O-Y), and Vitreoscilla hemoglobin (VHb) expressing recombinant strain 32O-Yvgb and its co-culture with strain 32O-W were grown at varying concentrations (0·1–2 mmol l⁻¹) of DBT or DBTS for 96 h, and desulfurization measured by production of 2-hydroxybiphenyl (2-HBP) and disappearance of DBT or DBTS. Of the four cultures grown with DBT as sulphur source, the best growth occurred for the 32O-Yvgb plus 32O-W co-culture at 0·1 and 0·5 mmol l⁻¹ DBT. Although the presence of vgb provided no consistent advantage regarding growth on DBTS, strain 32O-W, as predicted by previous work, was shown to contain a partial 4S desulfurization pathway allowing it to metabolize this 4S pathway intermediate.     

Enhanced production of acetoin and butanediol in recombinant Enterobacter aerogenes carrying Vitreoscilla hemoglobin gene

Microbial production of butanediol and acetoin has received increasing interest because of their diverse potential practical uses. Although both products are fermentative in nature, their optimal production requires a low level of oxygen. In this study, the use of a recombinant oxygen uptake system on production of these metabolites was investigated. Enterobacter aerogenes was transformed with a pUC8-based plasmid carrying the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). The presence of vgb and production of VHb by this strain resulted in an increase in viability from 72 to 96 h in culture, but no overall increase in cell mass. Accumulation of the fermentation products acetoin and butanediol were enhanced (up to 83%) by the presence of vgb/VHb. This vgb/VHb related effect appears to be due to an increase of flux through the acetoin/butanediol pathway, but not at the expense of acid 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.     

Effects of<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of<Emphasis Type="Italic"> Burkholderia</Emphasis> and on 2,4-DNT degradation in two-phase bioreactors

Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.     

Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.     

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236?±?3?mV in the absence of PEG, which was negatively shifted to +200?±?4?mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0?Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.     

Effects of Carbon Source and Vitreoscilla Hemoglobin (VHb) on the Production of β-Galactosidase in Enterobacter aerogenes

At fixed concentration (0.5%), lactose and galactose acted as inducers while glucose and other tested carbon sugars showed repression effects on β-galactosidase production in Enterobacter aerogenes strain. The expression of Vitreoscilla hemoglobin gene (vgb) in this bacterial strain managed to overcome the repression effects as well as improving the induction of β-galactosidase formation by carbon sources. In parallel, the bacterial O₂ consumption was increased correspondingly to the vgb induction of β-galactosidase synthesis. When Enterobacter aerogenes strains were grown at the incubation temperature 42°C, about 5-fold higher enzyme productivity was obtained than with a similar incubation at 37°C. The bacterial growth expressed as biomass yield had a different optimum temperature and was not influenced to the same extent by variations in the carbon sources. These data are discussed in terms of proposed enhancement in β-galactosidase productivity by vgb expression as well as its significance to improve the technology of whey processing.     

Hb Villaverde [β 89 (F5) Ser → Thr]: the structural modification of an intrasubunit contact is responsible for a high oxygen affinity

Hb Villaverde [β89 (F5) Ser → Thr], identified in a Spanish patient, is a new human hemoglobin variant, electrophoretically silent, responsible for a severe erythrocytosis. This abnormal hemoglobin displays a very high oxygen affinity and a markedly reduced cooperativity that is partly restored in the presence of IHP. Determination of the structural abnormality was achieved on a mixture of the normal and abnormal β-chains. After isolation of the abnormal tryptic peptide by RP-HPLC, its sequence was determined by mass spectrometry. The structural abnormality disturbs the intrasubunit interaction between helices F and H and, thus, may weaken the C-terminal bonds of the deoxy conformation and the heme contacts of several hydrophobic residues. Hb Villaverbe demonstrates that this intrasubunit contact between helices F and H is essential for the cohesion of the hemoglobin molecule.     

Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli

Biosynthetic threonine deaminase (TD) is a key enzyme for the synthesis of isoleucine which is allosterically inhibited and activated by Ile and Val, respectively. The binding sites of Ile and Val and the mechanism of their regulations in TD are not clear, but essential for a rational design of efficient productive strain(s) for Ile and related amino acids. In this study, structure-based computational approach and site-directed mutagenesis were combined to identify the potential binding sites of Ile and Val in Escherichia coli TD. Our results demonstrated that each regulatory domain of the TD monomer possesses two nonequivalent effector-binding sites. The residues R362, E442, G445, A446, Y369, I460, and S461 only interact with Ile while E347, G350, and F352 are involved not only in the Ile binding but also in the Val binding. By further considering enzyme kinetic data, we propose a concentration-dependent mechanism of the allosteric regulation of TD by Ile and Val. For the construction of Ile overproducing strain, a novel TD mutant with double mutation of F352A/R362F was also created, which showed both higher activity and much stronger resistance to Ile inhibition comparing to those of wild-type enzyme. Overexpression of this mutant TD in E. coli JW3591 significantly increased the production of ketobutyrate and Ile in comparison to the reference strains overexpressing wild-type TD or the catabolic threonine deaminase (TdcB). This work builds a solid basis for the reengineering of TD and related microorganisms for Ile production.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.