VHb Expression Research Articles

Overexpression of Shinorhizobium meliloti flavohemoglobin improves cell growth and fatty acid biosynthesis in oleaginous fungus Mucor circinelloides.

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which deliver oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. The VHb and SHb genes were integrated into the M. circinelloides MU402 genome by homologous recombination. VHb and SHb protein expression was verified by carbon monoxide difference spectrum analysis. The biomass was increased by ~ 50% in the strain expressing SHb compared with VHb. The total fatty acid (TFA) content of the strain expressing SHb reached 15.7% of the dry cell weight (~ 40% higher than that of the control strain) during flask cultivation. The biomass and TFA content were markedly increased (12.1g/L and 21.1% dry cell weight, respectively) in strains expressing SHb than strains expressing VHb during fermenter cultivation. VHb and SHb expression also increased the proportion of polyunsaturated fatty acids. Overexpressed bacterial hemoglobins, especially SHb, increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb improves fatty acid production more effectively than VHb in oleaginous microorganisms.

Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene

Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it’s also of great importance for the industrial applications of welan gum under harsh conditions.

Enhancing nosiheptide production in Streptomyces actuosus by heterologous expression of haemoprotein from Sinorhizobium meliloti.

Oxygen deficiency is a critical limiting factor for nosiheptide production in Streptomyces actuosus during fermentation. To alleviate oxygen limitation and enhance the yield of nosiheptide, haemoprotein from Sinorhizobium meliloti (SmHb) was overexpressed in S.actuosus with overexpression of haemoglobin from Vitreoscilla (VHb) as a positive control. The expression of SmHb and VHb in S.actuosus was confirmed by SDS-PAGE and CO-difference spectra analysis. The results showed that S.actuosus recombinant strain with SmHb expression had higher nosiheptide production (increased by 151%) than the wild strain (WT) under the low aeration conditions, which was similar with S.actuosus mutant strain with VHb expression. Furthermore, two copies of SmfHb gene were integrated in S.actuosus, which further increased the nosiheptide production by approx. 1·9-fold compared with original strain, and final nosiheptide yield was up to 2352μgml(-1) . These results suggested that engineering of SmHb expression could be used as an efficient method for constructing a high nosiheptide-accumulating strain. The significant improvement of nosiheptide production was found in recombinant strain with overexpressed sm gene. And further improvement was obtained in the two copies of sm overexpressing strain. These results suggested that engineering of SmHb expression could be used as an efficient method for constructing a high nosiheptide-accumulating strain.

Efficient ethanol production from potato and corn processing industry waste using E. coli engineered to express Vitreoscilla haemoglobin

Engineering of ethanologenic E. coli to express the haemoglobin (VHb) from the bacterium Vitreoscilla has been shown to enhance ethanol production by fermentation of pure sugars, sugars from hydrolysis of lignocellulose, components of whey, and sugars from wastewater produced during potato processing. Here, these studies were extended to see whether the same effect could be seen when a mixture of waste materials from processing of potatoes and corn into potato and corn chips were used as sugar sources. Consistent increases in ethanol production coincident with VHb expression were seen in shake flasks at both low aeration and high aeration conditions. The ethanol increases were due almost entirely to increases in the amount of ethanol produced per unit of cell mass. The VHb strategy for increasing fermentation to ethanol (and perhaps other valuable fermentation products) may be of general use, particularly regarding conversion of otherwise discarded materials into valuable commodities.

Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation.

Since its first use in 1990 to enhance production of α-amylase in E. coli, engineering of heterologous hosts to express the hemoglobin from the bacterium Vitreoscilla (VHb) has become a widely used strategy to enhance production of a variety of bioproducts, stimulate bioremediation, and increase growth and survival of engineered organisms. The hosts have included a variety of bacteria, yeast, fungi, higher plants, and even animals. The beneficial effects of VHb expression are presumably the result of one or more of its activities. The available evidence indicates that these include oxygen binding and delivery to the respiratory chain and oxygenases, protection against reactive oxygen species, and control of gene expression. In the past 4 to 5years, the use of this "VHb technology" has continued in a variety of biotechnological applications in a wide range of organisms. These include enhancement of production of an ever wider array of bioproducts, new applications in bioremediation, a possible role in enhancing aerobic waste water treatment, and the potential to enhance growth and survival of both plants and animals of economic importance.

Improved ethanol production from cheese whey, whey powder, and sugar beet molasses by “Vitreoscilla hemoglobin expressing” Escherichia coli

This work investigated the improvement of ethanol production by engineered ethanologenic Escherichia coli to express the hemoglobin from the bacterium Vitreoscilla (VHb). Ethanologenic E. coli strain FBR5 and FBR5 transformed with the VHb gene in two constructs (strains TS3 and TS4) were grown in cheese whey (CW) medium at small and large scales, at both high and low aeration, or with whey powder (WP) or sugar beet molasses hydrolysate (SBMH) media at large scale and low aeration. Culture pH, cell growth, VHb levels, and ethanol production were evaluated after 48 h. VHb expression in TS3 and TS4 enhanced their ethanol production in CW (21-419%), in WP (17-362%), or in SBMH (48-118%) media. This work extends the findings that "VHb technology" may be useful for improving the production of ethanol from waste and byproducts of various sources.

The effect of heterologous VHb expression to the functioning of stress-related genes in hybrid aspen lines exposured to biotic stress

Background The hemoglobin produced by Vitreoscilla bacterium, VHb, is capable to promote the respiratory activity and ATP production under hypoxic conditions [1]. In micro-organisms grown under oxygen-limited conditions, the heterologous vhb expression improves the production of primary and secondary compounds, growth as well as stress tolerance [2,3]. VHb has also been shown to protect plants against deleterious effects of nitric oxide (NO), the important signalling molecule produced during pathogen challenges [4]. We have previously studied the vhb expressing hybrid aspen lines (Populus tremula L. x tremuloides Michx.) under elevated UV-B illumination and showed that the VHb lines had increased secondary metabolite production and also enhanced accumulation of starch in chloroplasts when grown in standard greenhouse conditions [5]. In order to reveal the effect of VHb on the expression of stressrelated genes in hybrid aspen exposed to biotic stress, we conducted experiments with pathogenic fungus and larvae of insect herbivore.

Functional expression of Vitreoscilla hemoglobin (VHb) in Arabidopsis relieves submergence, nitrosative, photo-oxidative stress and enhances antioxidants metabolism

A chimeric construct consisting of the double CaMV35S promoter fused to the Myc-vhb gene encoding Vitreoscilla (bacterial) hemoglobin (VHb) was introduced into Arabidopsis thaliana and transgenic plants were cultured under various growth and stress conditions with the aim of studying the generality phenomena. Molecular analysis indicated that the exogenous vhb was stably integrated into the Arabidopsis genome and successfully expressed. Constitutive vhb expression improved the germination rate of transgenic seeds. Under submergence conditions, in vitro and in vivo bioassays revealed that the vhb expression protected transgenic plants against prolonged waterlogging. Leaf discs from the transgenic plants maintained in standard growth conditions reveal increased tolerance to nitrosative stress induced by sodium nitroprusside (SNP) treatment. Significant differences in chlorophyll a, b, total protein content and conductivity were observed among the transgenic, wild type (WT) and blank transformation (CK) plants. No significant differences were observed between the tested VHb, WT and CK lines in the presence of oxidative stresses by paraquat treatment. VHb expression also affected the levels of some Arabidopsis endogenesis genes that are involved in oxygen metabolism and antioxidants biosynthesis; up-regulating their expression at transcriptional level. LC–MS revealed that VHb expression shifted plant secondary metabolism, which may reflect the availability of extra metabolite production for possible enhanced protection ability. VHb transgenic plants were more tolerant to photo-oxidative damage due to the increased antioxidants, such as ascorbate. These results suggested that vhb gene might have the potential use in molecular breeding for the improvement of stress resistance and fine chemicals production in plants.

Heterologous expression of Vitreoscilla haemoglobin in barley (Hordeum vulgare)

The vhb gene encoding Vitreoscilla haemoglobin (VHb) was transferred to barley with the aim of studying the role of oxygen availability in germination and growth. Previous findings indicate that VHb expression improves the efficiency of energy generation during oxygen-limited growth, and germination is known to be an energy demanding growth stage during which the embryos also suffer from oxygen deficiency. When subjected to oxygen deficiency, the roots of vhb-expressing barley plants showed a smaller increase in alcohol dehydrogenase (ADH) activity than those of the control plants. This indicates that VHb plants experienced less severe oxygen deficiency than the control plants, possibly due to the ability of VHb to substitute ADH for recycling NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels in any of the conditions studied. In some cases, vhb expression even slowed down germination slightly. VHb production also appeared to restrict root formation in young seedlings. The adverse effects of VHb on germination and root growth may be related to its ability to scavenge nitric oxide (NO), an important signal molecule in both seed germination and root formation. Because NO has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. VHb production also affected the levels of endogenous barley haemoglobin, which may explain the relatively moderate effects of VHb in this study.

Intracellular expression of Vitreoscilla hemoglobin improves S-adenosylmethionine production in a recombinant Pichia pastoris

To develop an efficient way to produce S-adenosylmethionine (SAM), methionine adenosyltransferase gene (mat) from Streptomyces spectabilis and Vitreoscilla hemoglobin gene (vgb) were coexpressed intracellularly in Pichia pastoris, both under control of methanol-inducible promoter. Expression of mat in P. pastoris resulted in about 27 times higher specific activity of methionine adenosyltransferase (SMAT) and about 19 times higher SAM production relative to their respective control, suggesting that overexpression of mat could be used as an efficient method for constructing SAM-accumulating strain. Under induction concentration of 0.8 and 2.4% methanol, coexpression of vgb improved, though to different extent, cell growth, SAM production, and respiratory rate. However, the effects of VHb on SAM content (specific yield of SAM production) and SMAT seemed to be methanol concentration-dependent. When cells were induced with 0.8% methanol, no significant effects of VHb expression on SAM content and specific SMAT could be detected. When the cells were induced with 2.4% methanol, vgb expression increased SAM content significantly and depressed SMAT remarkably. We suggested that under our experimental scheme, the presence of VHb might improve ATP synthesis rate and thus improve cell growth and SAM production in the recombinant P. pastoris.

Expression ofVitreoscillaHemoglobin Enhances Growth ofHyoscyamus muticusHairy Root Cultures

The Vitreoscilla hemoglobin gene (vhb) was introduced into Hyoscyamus muticus with the aim of investigating its effect on growth and alkaloid production of Agrobacterium rhizogenes-induced hairy root cultures. We were able to generate several VHb-expressing hairy root lines with different integration patterns. Substantial somaclonal variation was observed in growth and hyoscyamine production amongst both VHb-expressing lines and controls. Despite this variation, the growth properties of single lines remained stable over time. Expression of VHb was found to improve growth of H. muticus hairy roots in shake-flask cultures. The dry weights of the root cultures expressing Vitreoscilla hemoglobin were on average 18 % higher than those of the controls. VHb expression also increased the volumetric hyoscyamine production, mainly due to the improved growth properties. However, this difference was not statistically significant due to the wide somaclonal variation and fluctuations over time in both VHb and control hairy root lines.