Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Production of vitamin A and vitamin E: expression of vitreoscilla hemoglobin gene in Erwinia herbicola.

Vitamin A prevents eye problems, blindness and skin problems by strengthening the immune system. Vitamin E is a nutrient that has important roles in many areas such as skin health, eye health and hormonal order. Vitreoscilla hemoglobin (VHb) gives an advantage in later phases of grown conditions to cells. In this study, the intracellular and extracellular production of vitamin A and E in E. herbicola and its recombinant strains (vgb- and vgb+) in the three different M9 mediums with supplemented 0.1% glucose, 0.1% fructose and 0.1% sucrose was investigated. Additionally, the viable cell number and total cell mass (OD600) were measured by the host and the recombinant bacteria in these mediums. The VHb gene expression in E. herbicola enhanced vitamin A under different carbon conditionals. Especially, in the vgb + strain (carrying vgb gene) the production of total vitamin in 0.1% glucose medium was recorded as 0.14 µg/ml, while the production in fructose and sucrose media was recorded as 0.07 µg/ml. The production of intracellular vitamin E in the host strain (0.025 µg/ml) was about 13-fold (0.002 µg/ml) higher than vgb + recombinant strain in 0.1% fructose. The vgb + strain showed about 2-fold higher extracellular vitamin E production than the host strain.

Design of a microaerobically inducible replicon for high-yield plasmid DNA production.

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.

Construction and application of a Xanthomonas campestris CGMCC15155 strain that produces white xanthan gum.

In the industrial production of xanthan gum using Xanthomonas campestris CGMCC15155, large amounts of ethanol are required to extract xanthan gum from the fermentation broth and remove xanthomonadin impurities. To reduce the amount of ethanol and the overall production cost of xanthan gum, a xanthomonadin-deficient strain of CGMCC15155 was constructed by inserting the Vitreoscilla globin (vgb) gene, under the control of the LacZ promoter, into the region of the pigA gene, which is involved in xanthomonadin synthesis. The insertion of vgb inactivated pigA, resulting in the production of white xanthan gum. The lack of xanthomonadins resulted in a decreased yield of xanthan gum. However, the expression product of vgb gene, VHb, could increase the metabolism of X. campestris, which allowed the production of xanthan gum to reach wild-type levels in the engineered strain. The yield, molecular weight, and rheological properties of the xanthan gum synthesized by the engineered and wild-type bacteria were essentially the same. When the same volume of ethanol was used, the whiteness values of the xanthan gum extracted from engineered and wild-type bacteria were 65.20 and 38.17, respectively. To extract xanthan gum with the same whiteness, three and seven times the fermentation volume of ethanol was required for the engineered and wild-type strains, respectively. Thus, the engineered train reduced the requirement for ethanol in xanthan gum production by 133.3%. The results demonstrated that the engineered bacteria used less ethanol, thus reducing the downstream processing cost in xanthan gum production.

Anaerobic production of medium-chain fatty alcohols via a ß-reduction pathway.

In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse ß-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and ß-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse ß-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation.

Oxygen plays a key role during bacterial cellulose (BC) biosynthesis by Gluconacetobacter xylinus. In this study, the Vitreoscilla hemoglobin (VHb)-encoding gene vgb, which has been widely applied to improve cell survival during hypoxia, was heterologously expressed in G. xylinus via the pBla-VHb-122 plasmid. G. xylinus and G. xylinus-vgb + were statically cultured under hypoxic (10 and 15% oxygen tension in the gaseous phase), atmospheric (21%), and oxygen-enriched conditions (40 and 80%) to investigate the effect of oxygen on cell growth and BC production. Irrespective of vgb expression, we found that cell density increased with oxygen tension (10-80%) during the exponential growth phase but plateaued to the same value in the stationary phase. In contrast, BC production was found to significantly increase at lower oxygen tensions. In addition, we found that BC production at oxygen tensions of 10 and 15% was 26.5 and 58.6% higher, respectively, in G. xylinus-vgb + than that in G. xylinus. The maximum BC yield and glucose conversion rate, of 4.3 g/L and 184.7 mg/g, respectively, were observed in G. xylinus-vgb + at an oxygen tension of 15%. Finally, BC characterization suggested that hypoxic conditions enhance BC's mass density, Young's modulus, and thermostability, with G. xylinus-vgb + synthesizing softer BC than G. xylinus under hypoxia as a result of a decreased Young's modulus. These results will facilitate the use of static culture for the production of BC.

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.

Synergic regulation of redox potential and oxygen uptake to enhance production of coenzyme Q10 in Rhodobacter sphaeroides.

The physiological role of Coenzyme Q10 (CoQ10) as an electron carrier suggests its association with redox potential. Overexpression of glyceraldehyde-3-phosphate dehydrogenase type I (gapA-1) in Rhodobacter sphaeroides elevated the NADH/NAD+ ratio and meanwhile enhanced the CoQ10 content by 58%, but at the sacrifice of biomass. On the other hand, Vitreoscilla hemoglobin was heterologously expressed to enhance the oxygen uptake ability of the cells, leading to 127% improvement of biomass. Subsequent coexpression of gapA-1 and vgb resulted in a CoQ10 titer of 83.24mg/L, representing 71% improvement as compared to the control strain RspMCS. When gapA-1 and vgb genes were co-expressed in a previously created strain RspMQd [1], 163.5mg/L of CoQ10 was produced. Finally, 600mg/L of CoQ10 production was achieved in fed-batch fermentation. These results demonstrated the synergic effect of redox potential regulation and oxygen uptake improvement on enhancing CoQ10 production in R. sphaeroides.

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 production of trans-4-hydroxy-l-proline by chromosomal integration of the Vitreoscilla hemoglobin gene into recombinant Escherichia coli with expression of proline-4-hydroxylase.

trans-4-Hydroxy-l-proline (Hyp) is a chiral amino acid conventionally produced by acid hydrolysis of animal collagen, a process which involves the bottleneck problems of low efficiency and heavy environmental pollution. Biotransformation of l-proline into Hyp using recombinant whole-cell biocatalysis with proline-4-hydroxylase (P4H) is an environmentally-friendly alternative method. Since biohydroxylation of proline by whole cells is a high-oxygen-demand process, oxygen transfer needs to be improved. To solve this problem, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosome of recombinant Escherichia coli expressing the P4H gene originally from Dactylosporangium sp. RH1. Expression of Vitreoscilla hemoglobin (VHb) resulted in a 94.4% increase of Hyp production in a 100-mL shaking flask culture compared to the same strain without VHb expression. Meanwhile in a fed-batch fermentation with a 1.4 L bioreactor, the expression of VHb led to an increase in Hyp production by 73.2% and biomass improved by 106%. We also found that acetic acid concentration was decreased by the expression of VHb during the fermentation. This work demonstrates that vgb chromosomal integration is an efficient way to improve Hyp production by enhancing oxygen transfer in recombinant E. coli.

Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli.

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of PadhE, Ppfl and Pvgb. The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for PadhE, Ppfl and PnarK. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

Overexpression of Vitreoscilla hemoglobin increases waterlogging tolerance in Arabidopsis and maize.

BACKGROUND: Vitreoscilla hemoglobin (VHb) is a type of hemoglobin found in the Gram-negative aerobic bacterium Vitreoscilla that has been shown to contribute to the tolerance of anaerobic stress in multiple plant species. Maize (Zea mays L.) is susceptible to waterlogging, causing significant yield loss. In this study, we approached this problem with the introduction of an exogenous VHb gene. RESULTS: We overexpressed the VHb gene in Arabidopsis and maize under the control of the CaMV35S promoter. After 14 days of waterlogging treatment, the transgenic VHb Arabidopsis plants remained green, while the controls died. Under waterlogging, important plant growth traits of VHb plants, including seedling height, primary root length, lateral root number, and shoot dry weight were significantly improved relative to those of the controls. The VHb gene was also introduced into a maize line through particle bombardment and was then transferred to two elite maize inbred lines through marker-assisted backcrossing. The introduction of VHb significantly enhanced plant growth under waterlogging stress on traits, including seedling height, primary root length, lateral root number, root dry weight, and shoot dry weight, in both Zheng58 and CML50 maize backgrounds. Under the waterlogging condition, transgenic VHb maize seedlings exhibited elevated expression of alcohol dehydrogenase (ADH1) and higher peroxidase (POD) enzyme activity. The two VHb-containing lines, Zheng58 (VHb) and CML50 (VHb), exhibited higher tolerance to waterlogging than their negative control lines (Zheng58 and CML50). CONCLUSIONS: These results demonstrate that the exogenous VHb gene confers waterlogging tolerance to the transgenic maize line. In Maize in the place of to the transgenic maize line, the VHb gene is a useful molecular tool for the improvement of waterlogging and submergence-tolerance.

A new approach for improving epothilone B yield in Sorangium cellulosum by the introduction of vgb epoF genes.

Epothilone B has drawn great attention due to its much stronger anticancer activity and weaker side effects compared with taxol. The relative low yield of epothilone B limited its application. In this study, we report the successful introduction of the vgb gene and the epoF gene into Sorangium cellulosum So ce M4 by electroporation for the first time, which was demonstrated by Southern blot analysis. Results of qRT-PCR, SDS-PAGE and western blot analysis confirmed the transcription and expression of the vgb and epoF genes. LC-MS results showed that the epothilones B, A yields were improved and epothilones D, C yields were decreased. The yields of epothilone B were improved by 57.9 ± 0.3, 62.7 ± 0.8 and 122.4 ± 0.7 % through the introduction of vgb gene, epoF gene and both genes into strain So ce M4, respectively. Our study provides a new approach for improving epothilone B yield in S. cellulosum.

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis.

Two recombinants of alkaliphilic Bacillus subtilis LOCK 1086, constructed via different strategies such as cloning the gene encoding bacterial hemoglobin from Vitreoscilla stercoraria (vhb) and overexpression of the gene encoding acetoin reductase/2,3-butanediol dehydrogenase (bdhA) from B. subtilis LOCK 1086, did not produce more 2,3-butanediol (2,3-BD) than the parental strain. In batch fermentations, this strain synthesized 9.46 g/L in 24 h and 12.80 g/L 2,3-BD in 46 h from sugar beet molasses and an apple pomace hydrolysate, respectively. 2,3-BD production by B. subtilis LOCK 1086 was significantly enhanced in fed-batch fermentations. The highest 2,3-BD concentration (75.73 g/L in 114 h, productivity of 0.66 g/L × h) was obtained in the sugar beet molasses-based medium with four feedings with glucose. In a medium based on the apple pomace hydrolysate with three feedings with sucrose, B. subtilis LOCK 1086 produced up to 51.53 g/L 2,3-BD (in 120 h, productivity of 0.43 g/L × h).

Heterologous coexpression of Vitreoscilla hemoglobin and Bacillus megaterium glucanase in Streptomyces lydicus A02 enhanced its production of antifungal metabolites.

Streptomyces lydicus A02 is a novel producer of commercially important polyene macrocyclic antibiotic natamycin and a potential biocontrol agent to several plant fungal diseases, including wilt caused by Fusarium oxysporum f. spp. To improve the natamycin production and the antifungal activity of S. lydicus A02, we coexpressed gene vgb encoding Vitreoscilla hemoglobin (VHb) and bglC encoding Bacillus megaterium L103 glucanase, both under the control of the strong constitutive ermE* promoter, in S. lydicus A02. Our results showed that coexpressing VHb and glucanase improved cell growth, and the engineered strain produced 26.90% more biomass than the wild-type strain after 72h fermentation in YSG medium. In addition, coexpressing genes encoding VHb and glucanase led to increased natamycin production, higher endogenous chitinase activity and exogenous glucanase activity, as well as enhanced antifungal activity in the engineered S. lydicus AVG02 and AGV02, regardless of the position of the two genes on the plasmids. Compared with model strains, few reports have successfully coexpressed VHb and other foreign proteins in industrial strains. Our results illustrated an effective approach for improving antifungal activity in an industrial strain by the rational engineering of combined favorable factors.

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.

Enhanced production of fatty acids and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla hemoglobin.

Dissolved oxygen is a critical factor for heterotrophic cell growth and metabolite production. The aim of this study was to investigate the effects of an oxygen-involved protein on cell growth and fatty acid and astaxanthin production in the biologically important thraustochytrid Aurantiochytrium sp. The hemoglobin of the Vitreoscilla stercoraria (VHb) gene was fused upstream with a zeocin resistance gene (ble) and driven by the Aurantiochytrium tubulin promoter. The expression construct was introduced into two strains of Aurantiochytrium sp. by electroporation. Transgenic Aurantiochytrium sp. strains MP4 and SK4 expressing the heterologous VHb achieved significantly higher maximum biomass than their corresponding controls in microaerobic conditions. Furthermore, the transformants of Aurantiochytrium sp. SK4 produced 44% higher total fatty acid and 9-fold higher astaxanthin contents than the wild type control in aerobic conditions. The present study highlights the biotechnological application of VHb in high-cell density fermentation for enhanced biomass production as well as high-value metabolites.

Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically.

Micro-aeration is a situation that will be encountered in bacterial cell growth especially when the saturated dissolved oxygen level cannot match the demand from cells grown to a high density. Therefore, it is desirable to separate aerobic growth and micro-aerobic product formation into two stages using methods including anaerobic or micro-aerobic promoters that are inducible under low aeration intensity. Eleven potential low aeration-inducible promoters were cloned and studied for their induction strengths under micro-aerobic conditions. Of them, Vitreoscilla hemoglobin promoter (P vgb ) was found to be the strongest among all 11 promoters. At the same time, six E. coli hosts harboring poly(R-3-hydroxybutyrate) (PHB) synthesis operon phaCAB were compared for their ability to accumulate poly(hydroxyalkanoates) (PHA). E. coli S17-1 was demonstrated to be the best host achieving a 70 % (mass fraction) PHB in the cell dry weigh (CDW) after 48 h under micro-aerobic growth. Cascaded P vgb repeats (P nvgb ) were investigated for enhanced expression level under micro-aerobic growth. The highest PHA production was obtained when a promoter containing eight cascaded P vgb repeats (P 8vgb ) was used, 5.37 g/l CDW containing 90 % PHB was obtained from recombinant in E. coli S17-1. Cells grew further to 6.30 g/l CDW containing 91 % PHB when oxygen-responsive transcription factor ArcA (arcA) was deleted in the same recombinant E. coli S17-1. This study revealed that vgb promoter containing cascaded P vgb repeats (P 8vgb ) is useful for product formation under low aeration intensity.

Effect of vitreoscilla hemoglobin and culture conditions on production of bacterial L-asparaginase, an oncolytic enzyme.

L-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the L-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher L-asparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for L-asparaginase synthesis, it supported the highest biomass production. In glucose medium, L-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both L-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of L-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes.