E ect of Vitreoscilla hemoglobin biosynthesis in Escherichia coli on production of poly(l-hydroxybutyrate) and fermentative parameters

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1 FEMS Microbiology Letters 214 (2002) 223^227 E ect of Vitreoscilla hemoglobin biosynthesis in Escherichia coli on production of poly(l-hydroxybutyrate) and fermentative parameters Huimin Yu a;, Yue Shi a, Yanping Zhang a, Shengli Yang b, Zhongyao Shen a a Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Beijing , PR China b Shanghai Research Center of Biotechnology, Academic Sinica, Shanghai , PR China Received 17 May 2002; received in revised form 3 July 2002; accepted 19 July 2002 First published online 14 August 2002 Abstract In order to attain high cell density and low cost production of poly(l-hydroxybutyrate) (PHB), the Vitreoscilla globin gene (vgb) was introduced into a novel recombinant strain, Escherichia coli VG1 (ptu14). Experiments showed that the expression of vgb was under the regulation of dissolved oxygen (DO) in broth and the introduction of vgb in VG1 (ptu14) induced the parent promotion effect on cell growth and PHB accumulation, especially under low DO conditions. Further experiments indicated that the introduction of vgb in VG1 (ptu14) not only decreased the critical oxygen concentration, but also affected the volumetric oxygen transfer coefficient of the recombinant strain. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Poly(L-hydroxybutyrate); Vitreoscilla globin gene (vgb); Dissolved oxygen; The critical oxygen concentration; The volumetric oxygen transfer coe cient 1. Introduction Vitreoscilla hemoglobin (VHb) is an oxygen-binding protein that allows the bacterium to grow aerobically even under microaerophilic conditions. Although the role of hemoglobins in higher eukaryotes as facilitators of oxygen di usion is well established, the cellular function of the Hb and Hb-like avohemoproteins in prokaryotes is still unknown and addresses an intriguing question yet to be solved. Suggested roles for these proteins are facilitation of oxygen transfer and storage, enhancement of energy status of cells, oxygen and nitrogen compound sensing, modulations of the redox status of the cell, and biological nitrogen xation [1,2]. The Vitreoscilla globin (vgb) gene, responsible for VHb production, has been cloned in Escherichia coli along with its natural promoter, and its expressive regulation has been shown to be at the level of transcription [3^5]. Recently, with the development of biotechnology, the utilization of VHb in microbial * Corresponding author. Tel.: +86 (10) ; Fax: +86 (10) address: yuhm@mail.tsinghua.edu.cn (H. Yu). fermentation has been studied, especially in the process of high cell density culture of aerobic organisms [6]. Poly(L-hydroxybutyrate) (PHB), an intracellular energy and carbon reserve material that can be synthesized by many prokaryotes under unbalanced growth conditions, has been drawing considerable interest in the areas of medicine, food and agriculture, both because of its physical properties which are similar to conventional petrochemical-derived plastics and because of its unique properties such as biodegradability and biocompatibility [7]. Large scale commercial applications of PHB are not yet feasible due to its high production cost [8]. One of the major problems is the low productivity caused by insu cient oxygen supply. Traditional methods, such as enhancement by stirring and aeration, the introduction of pure or rich oxygen, and the a liation of the oxygen vector, are usually characterized by high costs but low e ciency. In our research, a recombinant E. coli, VG1 (ptu14), was constructed to attain high cell density and low cost production of PHB. In this novel strain, three exogenous genes, including vgb, PHB biosynthetic genes (phbcab) and lytic genes of phage V with an S amber mutation (S 3 RRz), were simultaneously contained. The functions / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (02)00887-X

2 224 H. Yu et al. / FEMS Microbiology Letters 214(2002) 223^227 of VHb in VG1 (ptu14) were examined and discussed from an engineering point of view. 2. Materials and methods 2.1. Bacterial strains, plasmid and culture conditions The hosts used were E. coli JM105 and its derivative, E. coli VG1, in which the gene vgb was integrated into the chromosomal DNA [9]. Plasmid ptu14 (Ap r ) was constructed as previously described [9]. Except where noted, seed and ask cultures of each strain were completed under the conditions previously described [9]. The semi-de ned medium: glucose (20 g l 31 ), (NH 4 ) 2 SO 4 (2.0 g l 31 ), yeast extract (6.0 g l 31 ), Na 2 HPO 4 W12H 2 O (3.0 g l 31 ), KH 2 PO 4 (0.8 g l 31 ), NaCl (3.0 g l 31 ), CaCl 2 W2H 2 O(5mgl 31 ), MgSO 4 W7H 2 O (150 mg l 31 ), ph 7.0 was optimized for a scale-up culture in the fermenter. The feed solution used for the fed-batch culture contained, per liter: 800 g glucose, 400 g yeast extract and 400 g (NH 4 ) 2 SO 4. When cultivated in a 10-l fermenter, the temperature was maintained at 37 C and the ph was controlled at 6.8 with 6 mol l 31 NaOH solution. Agitation and aeration were set to 750 rev min 31 and 5.0 l min 31.In another 5-l fermenter (B. Braun Company), 1000 rev min 31 agitation was maintained throughout the process of fermentation Analytical methods Cell growth, cell morphology, PHB concentration, PHB content and VHb activity detection were all performed as described previously [9]. A quantitative assay of dissolved oxygen (DO) in a shaking ask was completed using a DO electrode connected with the control system of a fermenter. The stabilization and calibration of the zero point of the DO probe were performed using the oxygen-free solution: extra-saturated Na 2 SO 3 solution (280 ml) plus Na 2 B 4 O 7 W10H 2 O (5 g). The cell-free medium maintained in the same culture conditions with other asks was used to calibrate the solution at the point of 100% oxygen saturation. The volumetric oxygen transfer coe cient (k L a,s 31 )at a certain cell mass concentration in fermentation systems was measured using the dynamic method stated by Taguchi and Humphrey [10,11], according to the following procedures: the oxygen uptake rate of cells (QX, where Q is the speci c oxygen uptake rate and X is the cell concentration in culture) is determined rst by measuring the decreasing rate of DO concentration (3dC/dt) while the oxygen supply is temporarily stopped, then the increasing rate of the DO concentration (dc/dt) is estimated after the oxygen supply is resumed, and nally the k L a is estimated using the rearranged equation: C =(31/k L a)(dc/dt+qx)+ C*, where C is the actual DO concentration in the bulk liquid phase, and C* is the DO concentration at the gas^ liquid interface in equilibrium with the existing oxygen partial pressure in the gas phase. In this study, the relative DO concentration (%) was used to substitute the actual concentration (C) in the process of k L a estimation. The DO probe was stabilized and calibrated as accurately as possible with the oxygen-free solution and cell-free medium. During the process of the fed-batch culture of the recombinant strains with or without the vgb gene, the k L a was assayed at various cell mass concentrations. All the analyses were carried out at least in duplicate. 3. Results and discussion 3.1. DO in broth determining the expression of vgb As described previously, vgb could be successfully coexpressed with the other two exogenous genes, S 3 RRz and phbcab, in recombinant E. coli VG1 (ptu14) [9,12]. Using the method of carbon monoxide (CO) di erence spectra, the time pro les of VHb amounts under di erent DO levels were assayed (Fig. 1). Fig. 1 showed that the DO level in broth decreased continuously with the growth of VG1 (ptu14) cells; at the same time, the amount of VHb expressed per gram of wet cell increased, especially under hypoxic DO conditions such as 5%: more than 10-fold of VHb could be detected in contrast to 80%. This result was similar to the earlier studies which found that the expression of vgb was under the regulation of DO in broth and was merely induced abundantly under oxygen-limited conditions [4^6]. The amount of VHb in VG1 (ptu14) was quickly enhanced when DO was maintained between 5 and 50%. This phenomenon may not be very consistent with the other studies, because VHb was already in great expression at less Fig. 1. Time pro les of VHb amounts under di erent DO levels in broth. Quantitative assay of DO (F) in shaking ask was completed as described in Section 2; quantitative analysis of VHb activity (b) was performed by the method of CO di erence spectra of whole cells of VG1 (ptu14).

3 H. Yu et al. / FEMS Microbiology Letters 214(2002) 223^ Fig. 2. Cell growth and PHB accumulation of VG1 (ptu14) and JM105 (ptu14) with DO above 50%. Under the same conditions, VG1 (ptu14) and the control strain, JM105 (ptu14), were comparatively batch-cultured in a 10-l fermenter. Cell concentrations of VG1 (ptu14) (F) and JM105 (ptu14) (b), PHB concentrations of VG1 (ptu14) (O) and JM105 (ptu14) (P) were measured and plotted. Fig. 4. Time pro les of DO changes of VG1 (ptu14) and JM105 (ptu14). Culture experiments were performed in a 10-l fermenter under the same conditions. The oxygen supply was stopped when DO was decreased to 80% with unchanged agitation. Then the DO changes of VG1 (ptu14) (a) and JM05 (ptu14) (P) were recorded and plotted. than 50% DO, although not in oxygen-de cient conditions E ect of the cloned vgb gene on cell growth and PHB accumulation in VG1 (ptu14) Fig. 3. Cell growth and PHB accumulation of VG1 (ptu14) and JM105 (ptu14) with DO between 50 and 5%. Keeping the other parameters unchanged, DO was controlled by bubbling N 2 into broth. Cell concentrations of VG1 (ptu14) (F) and JM105 (ptu14) (b), PHB concentrations of VG1 (ptu14) (O) and JM105 (ptu14) (P) were detected and plotted. In order to detect the e ect of the cloned vgb gene on cell growth and PHB accumulation in VG1 (ptu14), a control strain, E. coli JM105 (ptu14) without the vgb gene was also constructed [9], and VG1 (ptu14) and JM105 (ptu14) were simultaneously cultured under different conditions. Fig. 2 plotted the culture results of these two strains when DO was maintained above 50% throughout the whole process. The cell growth and PHB accumulation of VG1 (ptu14) were and 1.84-fold higher than those of JM105 (ptu14), respectively, due to the introduction of vgb. The superiority of VG1 (ptu14) increased to and 4.2-fold, respectively, when DO was controlled between 50 and 5% (Fig. 3). Thus it could be pointed out that the introduction of vgb in VG1 (ptu14) induced the parent promotion e ect on cell growth and PHB accumulation, especially when DO was lower than 50% and VHb was in great expression E ect of the cloned vgb gene on critical oxygen concentration (COC) of cells The COC of cells is an important characteristic for an obligate aerobe. Cells can maintain the same high speci c oxygen uptake rate and metabolic activity only when cultured in broth with higher DO than COC. Thus, the lower the COC is, the better. Does the cloning of the vgb a ect the COC of the recombinant strain? To answer this question, VG1 (ptu14) and JM05 (ptu14) were simultaneously cultured and the oxygen supply in the process was stopped. The time pro les of two strains of DO were recorded in Fig. 4. Fig. 4 showed that the DO in VG1 (ptu14) broth decreased at an identical rate dramatically until 5%, and this turning point of DO might be considered as the COC of VG1 (ptu14). While for JM105 (ptu14), the phenomenon was di erent in that the identical rate drop of DO ceased earlier, with the turning point at about 20%. So it was clear that just because of the introduction of the vgb gene, the COC of the recombinant VG1 (ptu14) was markedly reduced. By decreasing the COC, the cell activity of VG1 (ptu14) under low DO was maintained for a long time and the cell growth and PHB accumulation were thus promoted.

4 226 H. Yu et al. / FEMS Microbiology Letters 214(2002) 223^ E ect of the cloned vgb gene on the change of volumetric oxygen transfer coe cient (k L a) Fig. 6. Changes of k L a and DO with cell growth of VG1 (ptu14). E. coli VG1 (ptu14) was cultured under the same conditions as in Fig. 5. In the process of cell growth, the k L a (b) and corresponding DO (a) in broth were also measured and plotted. In addition to the conventional operating parameters of fermentation such as the aeration rate, agitation speed and power input, other important factors a ecting k L a are viscosity, ionic strength of the medium, and surface-active agents. During the high cell density culture of bacteria, DO will drop rapidly with the quick growth of cells, and k L a will also decrease with the increasing of the culture viscosity caused by the increasing of cell concentration and cell morphologies. Because one of the suggested roles for VHb is facilitation of oxygen transfer and storage in the cell, it is possible that the apparent k L a of the recombinant strain containing the cloned vgb gene will be maintained and even increased under some DO levels in the process of cell growth. In order to validate this hypothesis, E. coli JM105 (ptu14) and VG1 (ptu14) were simultaneously cultured in a 5-l fermenter, and the results were plotted in Figs. 5 and 6. Fig. 5 showed that the DO and k L a in broth both decreased continuously with the cell growth of JM105 (ptu14), with no vgb gene during the fed-batch culture. When DO was down to 20% from 90%, the k L a decreased to 1.3U10 32 (s 31 ) from the original 3.2U10 32 (s 31 ). But for recombinant VG1 (ptu14) with the vgb gene, di erent results were detected (Fig. 6). Firstly, the original k L a was as high as 7.4U10 32 (s 31 ), which was more than 2.3-fold of JM105 (ptu14). Secondly, k L a decreased quickly with the growth of VG1 (ptu14) cells at rst; while this decrease was ceased and kept unchanged for a very long time when DO was lower than about 50%, a slight increase appeared when DO was down to about 10%. As discussed earlier, Fig. 1 showed that the activities of VHb quickly increased when DO was lower than 50%, especially lower than 10%. So through a simple deduction, an apparent conclusion could be drawn that the introduction of vgb in recombinant VG1 (ptu14) maintained or promoted the engineered parameter k L a under low or poor oxygen conditions. To sum up, it can be concluded that the successful introduction of vgb in recombinant E. coli VG1 (ptu14) played an important role in its physiological metabolisms, consequently inducing the great increase of cell growth and PHB accumulation, the decrease of COC in broth and the maintaining or increase of a signi cant engineering parameter, k L a, especially under oxygen-de cient conditions. Thus the endurance ability of recombinant cells for poor oxygen was greatly strengthened. Further study showed that the cell density and PHB content of VG1 (ptu14) attained g l 31 and 89.7%, respectively, and did not need a rich or pure oxygen supply in the whole process of fermentation. According to the economic evaluation for PHB production by Choi and Lee [8], the price of PHB could be reduced at least 30% when using this vgb-integrated novel strain, VG1 (ptu14), due to the combined e ects of high PHB productivity and high PHB content. This result veri ed the great potential to decrease the production cost of PHB and even some other important bio-products. Acknowledgements This work was supported by National Natural Science Foundation of China (No , ). References Fig. 5. Changes of k L a and DO with cell growth of JM105 (ptu14). E. coli JM105 (ptu14) was cultured in a 5-l fermenter (B. Braun) with uniform agitation throughout (1000 rpm). Accompanying the cell growth, DO (E) in broth was recorded by the probe, and the corresponding k L a (F) was calculated as stated in Section 2. [1] Joshi, M., Mande, S. and Dikshit, K.L. (1998) Hemoglobin biosynthesis in Vitreoscilla stercoraria DW: cloning, expression, and characterization of a new homolog of a bacterial globin gene. Appl. Environ. Microbiol. 64, 2220^2228. [2] Kallio, P.T., Kim, D.J., Tsai, P.S. and Bailey, J.E. (1994) Intracellular expression of Vitreoscilla haemoglobin activates E. coli energy metabolism under oxygen-limited conditions. Eur. J. Biochem. 219, 201^208.

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