VHb-expressing Cells Research Articles

Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli

• VHb reduced overflow metabolism. • VHb improved pDNA production under oxygen limitations. • VHb improved pDNA yields even at lower RNAII/RNAI ratios. • Higher lactate production may be caused by stronger NAD + demand. Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In this study, Vitreoscilla hemoglobin (VHb) was expressed in the chromosome of E. coli as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the wild type strain. The culture was shifted from aerobic to microaerobic conditions during the fed-batch phase. pDNA yields increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the wild type under microaerobic conditions. Unexpectedly, the ratio of positive/negative replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the wild type strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the fluxes in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD + in glycolysis under microaerobic conditions.

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.

Influence of Vitreoscilla hemoglobin gene expression on 2,3-butanediol production in Klebsiella oxytoca

A hemoglobin-like protein is a protein that aids in the delivery of oxygen in microbes in a similar fashion as hemoglobins in plants and animals. In this study, expression of Vitreoscilla hemoglobin (VHb) was used to improve the growth of Klebsiella oxytoca, and consequently, the 2,3-butanediol (2,3-BDO) production. The Vitreoscilla hemoglobin gene (vgb) was cloned into the expression vector pET-24a, and the recombination plasmid was then transformed in Klebsiella oxytoca ATCC43863. The efficiency of 2,3-BDO production and cell growth was analyzed by placing VHb expression under the control of the nar and luxS promoters instead of the T7 in Klebsiella oxytoca. Fermentation experiments were performed for comparison with the wild type under conditions of high/low aeration for the optimization of VHb-expressing cells in a 500 mL shaking flask and a 5 L fermenter. Moreover, cultivations were performed with addition of acetic acid to the culture media to study the effects of acetic acid on 2,3-BDO production, as acetic acid is known to induce 2,3-BDO production. The results indicated a 10% increase of cell growth in the presence of VHb, while 2,3-BDO production was further improved by the addition of acetic acid. In particular, the fed-batch fermentation of Klebsiella oxytoca LV with acetic acid added exhibited increase of the dry cell weight from 11.9 to 14.0 g/L, and improvement of the 2,3-BDO production from 34.9 to 49.4 g/L in 40 h.

Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

Abstract H2 production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H2 production by fermentative bacteria by maximizing the number of electrons that are available for H2. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O2)-binding protein on oxic H2 production in a closed batch system that was supplemented with glucose. The H2 yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O2 tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H2 yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H2 production and not by working as an O2-scavenger during H2 production. This new approach allowed more substrate to remain to be further utilized for the production of more H2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H2-producing microbial systems to enhance the overall H2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H2.

Intracellular expression of Vitreoscilla hemoglobin improves production of Yarrowia lipolytica lipase LIP2 in a recombinant Pichia pastoris

The Yarrowia lipolytica lipase LIP2 (YlLIP2) gene lip2 and Vitreoscilla hemoglobin gene vgb were co-expressed in Pichia pastoris, both under the control of AOX1 promoter, in order to alleviate respiration limitation under conditions of high cell-density fermentation and enhance YlLIP2 production. The results showed that recombinant P. pastoris strains harboring the lip2 and vgb genes (VHb +) displayed higher biomass and YlLIP2 activity than control strains (VHb −). Compared with VHb − cells, the expression levels of YlLIP2 in VHb-expressing cells when oxygen was not a limiting factor were improved 31.5% in shake-flask culture and 22% in a 10-L fermentor. Under non-limiting dissolved oxygen (DO) conditions, the maximum YlLIP2 activity of VHb + in a 10-L fermentor reached 33,000 U/mL. Oxygen limitation had a more negative effect on YlLIP2 productivity in VHb − cells than in VHb + cells. The highest YlLIP2 activity of VHb + cells was approximately 1.84-fold higher than that of VHb − cells at lower DO levels. Moreover, the recombinant strain VHb + exhibited a higher specific oxygen uptake rate and achieved higher cell viability under oxygen limiting and non-limiting conditions compared with VHb − cells. Therefore, the above results suggest that intracellular expression of VHb in recombinant P. pastoris has the potential to improve cell growth and industrial enzyme production.

Analysis of novel hemoglobins during microaerobic growth of HMP-negative Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology leading to reduced growth and productivity. Vitreoscilla hemoglobin (VHb) has been extensively studied showing positive and energetically favorable characteristics. Species-specific differences also suggest that VHb is not optimal for all applications. Therefore, mutant and fusion proteins were generated with improved characteristics. Hemoglobins of various microorganisms also provide phenotypes eliciting positive properties. Therefore, two new globin genes, encoding Novosphingobium aromaticivorans hemoglobin (NHb) and Bacillus halodurans flavohemoglobins (HmpBh), were isolated. Genes encoding two mutant flavohemoglobins of Pseudomonas aeruginosa PAO1 and a clinical isolate were also analyzed. The proteins were expressed to study the physiological effects of globin expression relative to controls, carrying either the parental plasmid or expressing VHb, during oxygen limited fed-batch cultivations of Escherichia coli BO6, lacking the endogenous flavohemoglobin. HmpBh-expressing cells reached 25 and 10% higher final optical densities relative to the plasmid carrying or VHb-expressing cells, respectively. HmpBh-expressing cells also showed 34% higher yield of biomass on glucose relative to control. Sequence comparison indicates that hemoglobin domains of Bacillus species are closely related to VHb sequence. Thus, suggesting that proteins having a globin domain similar to VHb could be important in biotechnology.

Expression of Vitreoscilla hemoglobin enhances growth and levels of α-amylase in Schwanniomyces occidentalis

A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.

Correlation Between Bacterial Hemoglobin and Carbon Sources: Their Effect on Copper Uptake by Transformed E. coli Strain αDH5

Escherichia coli alphaDH5 harboring Vitreoscilla hemoglobin gene, vgb (Escherichia:pUC8:16), and control strains (parental and vgb-lacking transformed strains, E. coli:pUC9) were employed for studying the potential ability of copper uptake. The strains were grown on a minimal medium, 5XM9, that contained different carbon (mannitol, fructose, lactose, and maltose) and nitrogen (casein and yeast extract) sources. A clear type-response relationship between the level of copper uptake and the type of carbon and nitrogen sources was observed. Copper uptake ability was better for vgb-bearing cells grown on either mannitol, lactose, fructose, or maltose as carbon source and casein as well as yeast extract as nitrogen source compared with the control medium. The increase in copper uptake was in the following order: parental cells, then pUC9-bearing cells, then VHb-expressing cells.

Effect of Vitreoscilla hemoglobin gene ( vgb) and metabolic inhibitors on cadmium uptake by the heterologous host Enterobacter aerogenes

The effect of metabolic inhibitor/uncoupler(s) (CCCP and NaN 3) and sulfhydryl reagents (dithiothreitol, 2-mercaptoethanol glutathione) on cadmium uptake was investigated in Enterobacter aerogenes strains. They include a transformed strain bearing the Vitreoscilla hemoglobin gene, vgb as well as control strains that lack this transformed gene. The vgb-harboring strains showed better uptake of cadmium than vgb-lacking strains. Under low aeration, there was 2-fold enhancement of Cd +2 uptake in vgb-harboring strains compared with 1.6-fold enhancement under high aeration. The CCCP caused 36, 40 and 58% inhibition in cadmium uptake of parental, pUC9-harboring and VHb-expressing cells, respectively. Similarly, the sodium azide exerted 44, 38 and 55% inhibition in Cd +2 uptake of parental, pUC9-harboring and VHb-expressing cells, respectively. Less extensive inhibition of Cd +2 uptake in the range of 11–39% was observed with sulfhydryl reagents.

Expression of double Vitreoscilla hemoglobin enhances growth and alters ribosome and tRNA levels in Escherichia coli.

In several organisms, expression of a gene encoding dimeric hemoglobin (VHb) from the obligate aerobic bacterium Vitreoscilla stercoraria has been shown to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism. In an attempt to further improve these effects of VHb, a gene encoding two vhb genes connected by a short linker of six base pairs was constructed and expressed in Escherichia coli(double VHb). Escherichia coli cells expressing double VHb reached a cell density 19% higher than that of cells expressing native VHb. The protein production per cell remained constant since the increase in cell growth was accompanied by an increase in protein content by 16%. Investigation of ribosome and tRNA content revealed that cells expressing double VHb reached their maximal capacity of protein synthesis later during cultivation than cells expressing native VHb, and furthermore they reached considerably higher levels of ribosome and tRNA compared to that of the VHb-expressing cells.

Effect ofVitreoscilla hemoglobin dosage on microaerobicEscherichia coli carbon and energy metabolism

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-beta-D-thiogalactopyranoside- (IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (Vhb(-)) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb(-) control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 mumol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 60%, respectively. Increasing VHb concentration to 3.8 mumol/g DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb(+) cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADH/NADPH transhydrogenase while the VHb(-) cells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb(+) cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb(+) cells. (c) 1996 John Wiley & Sons, Inc.

Effect of Vitreoscilla hemoglobin dosage on microaerobic Escherichia coli carbon and energy metabolism

The amount of Vitreoscilla hemoglobin (VHb) expression was modulated over a broad range with an isopropyl-β-D-thiogalactopyranoside- (IPTG-) inducible plasmid, and the consequences on microaerobic Escherichia coli physiology were examined in glucose fed-batch cultivations. The effect of IPTG induction on growth under oxygen-limited conditions was most visible during late fed-batch phase where the final cell density increased initially linearly with increasing VHb concentrations, ultimately saturating at a 2.7-fold increase over the VHb-negative (Vhb−) control. During the same growth phase, the specific excretions of fermentation by-products, acetate, ethanol, formate, lactate, and succinate from the culture expressing the highest amount of VHb were reduced by 25%, 49%, 68%, 72%, and 50%, respectively, relative to the VHb− control. During the exponential growth phase, VHb exerted a positive but smaller control on growth rate, growth yield, and respiration. Varying the amount of VHb from 0 to 3.8 μmol/g dry cell weight (DCW) increased the specific growth rate, the growth yield, and the oxygen consumption rate by 33%, 35%, and 60%, respectively. Increasing VHb concentration to 3.8 μmol/g DCW suppressed the rate of carbon dioxide evolution in the exponential phase by 30%. A metabolic flux distribution analysis incorporating data from these cultivations discloses that VHb+ cells direct a larger fraction of glucose toward the pentose phosphate pathway and a smaller fraction of carbon through the tricarboxylic acid cycle from acetyl coenzyme A. The overall nicotinamide adenine dinucleotide [NAD(P)H] flux balance indicates that VHb-expressing cells generate a net NADH flux by the NADH/NADPH transhydrogenase while the VHb− cells yield a net NADPH flux under the same growth conditions. Flux distribution analysis also reveals that VHb+ cells have a smaller adenosine triphosphate (ATP) synthesis rate from substrate-level phosphorylation but a larger overall ATP production rate under microaerobic conditions. The thermodynamic efficiency of growth, based on reducing equivalents generated per unit of biomass produced, is greater for VHb+ cells. © 1996 John Wiley & Sons, Inc.

Improvement of Escherichia coli microaerobic oxygen metabolism by Vitreoscilla hemoglobin: New insights from NAD(P)H fluorescence and culture redox potential

On-line NAD(P)H fluorescence and culture redox potential (CRP) measurements were utilized to investigate the role of Vitreoscilla hemoglobin (VHb) in perturbing oxygen metabolism of microaerobic Escherichia coli Batch cultures of a VHb-synthesizing E. coli strain and the iso-genic control under fully aerated conditions were subject to several high/low oxygen transitions, and the NAD(P)H fluorescence and CRP were monitored during these passages. The presence of VHb decreased the rate of net NAD(P)H generation by 2.4-fold under diminishing oxygen tension. In the absence of aeration, the strain producing VHb maintained a steady NAD(P)H level 1.8-fold less than that of the control, indicating that the presence of VHb keeps E. coli in a more oxidized state under oxygen-limited conditions. Estimated from CRP, the oxygen uptake rates near anoxia were 25% higher for cells with VHb than those without. These results suggest that VHb-expressing cells have a higher microaerobic electron transport chain turnover rate. To examine how NAD(P)H utilization of VHb-expressing cells responds to rapidly changing oxygen tension, which is common in large-scale fermentations, we pulsed air intermittently into a cell suspension and recorded the fluorescence response to the imposed dissolved oxygen (DO) fluctuation. Relative to the control, cells containing VHb had a sluggish fluorescence response to sudden changes of oxygen tension, suggesting that VHb buffers intracellular redox perturbations caused by extracellular DO fluctuations.(c) John Wiley & Sons, Inc.

Intracellular expression of Vitreoscilla hemoglobin alters the aerobic metabolism of Saccharomyces cerevisiae.

Vitreoscilla hemoglobin (VHb) has been expressed in Saccharomyces cerevisiae, and its influence on yeast aerobic metabolism has been investigated. New expression vectors were constructed to express VHb constitutively under the control of the ADH-1 promoter. The presence of VHb was shown by Western blot analysis. VHb has been shown to localize predominantly in the cytoplasm. Batch fermentation results indicated that the wild-type strain expressing VHb exhibited a shift in the carbon flux toward ethanol production, with no significant alteration in the specific growth rate. This effect was not observed if cells were grown under respiration inhibition, indicating that the metabolic effect of VHb is likely linked to respiration. Expression of VHb in the adh degrees strain MC65-2A, which produces ethanol only via a respiration-coupled pathway, revealed that ethanol production was decreased and cells reached a higher final cell density in a culture of the VHb-expressing strain. Growth enhancement due to expression of VHb was observed only during the final stage of culture growth when the acetaldehyde produced during the first growth phase was used as a substrate. This metabolic effect of intracellular VHb was seen more clearly in an acetaldehyde fed-batch fermentation in which VHb-expressing cells grew to at least 3-fold higher final cell density. These results suggest that the action of VHb is likely linked to electron transfer.