Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with... Ann Microbiol (2013) 63:1219–1224 DOI 10.1007/s13213-012-0579-4 ORIGINAL ARTICLE Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase Jian Wang & Jing Huang & Jianming Shi & Qingyang Xu & Xixian Xie & Ning Chen Received: 29 July 2012 /Accepted: 22 November 2012 /Published online: 10 January 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Acetate is a primary inhibitory metabolite in Introduction Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be L-Tryptophan is an essential aromatic-group amino acid derived from acetyl coenzyme A by the phosphotransacety- with a unique indole side chain that makes it a fundamental lase (Pta)–acetate kinase (AckA) pathway. In this study, the precursor to a number of neurotransmitters in the brain, fermentation characteristics of Pta mutant strain E. coli including serotonin, melatonin, and niacin, which are essen- TRTHΔpta were compared with those of the control strain tial factors for the regulation of appetite, sleep, mood, and E. coli TRTH in a 30-L fermentor. The effects of glucose pain levels (Kocabaş et al. 2006). L-Tryptophan also serves concentration and dissolved oxygen (DO) level were inves- as a nutrient enhancer, a preservative, and a feed additive. tigated, and the results suggest that DO and glucose con- The current production of L-tryptophan by microbiological centration are vital influencing parameters for the methods includes enzymatic catalysis, microbial fermenta- production of L-tryptophan. Based on our experimental tion, and microbial transformation. The large-scale produc- results, we then tested a DO-stat fed-batch fermentation tion of L-tryptophan by microbial fermentation with E. coli strategy. When DO was controlled at about 20 % during and Corynebacterium glutamicum has been reported L-tryptophan fermentation in the DO-stat fed-batch system, (Sprenger 2007). Processes using E. coli remain the produc- the pta mutant was able to maintain a higher growth rate at tion systems of choice because of the susceptibility of this the exponential phase, and the final biomass and L-trypto- microorganism to genetic manipulation and its efficient pro- phan production were increased to 55.3 g/L and 35.2 g/L, duction of recombinant proteins (Pavlou and Reichert 2004). respectively. Concomitantly, as the concentration of acetate Escherichia coli excretes acetate as a major by-product of decreased to 0.7 g/L, the accumulation of pyruvate and its aerobic metabolism (Majewski and Domach 1990), and lactate increased in the mutant strain as compared with acetate is undesirable because it retards growth and inhibits the control strain. This characterization of the recombi- protein formation (Koh et al. 1992; Jensen and Carlsen nant mutant strain provides useful information for the 1990; Mey et al. 2007). Moreover, acetate production rep- rational modification of metabolic fluxes to improve trypto- resents a diversion of carbon that might otherwise have phan production. generated biomass or a specific protein product (March et al. 2002). Two enzymatic pathways for acetate formation in . . . Keyword pta gene L-Tryptophan Escherichia coli E. coli have been identified: (1) acetate can be derived DO-stat fed-batch fermentation Acetate directly from pyruvate by pyruvate oxidase (PoxB), but the activity of this enzyme in E. coli is thought to be too low to account for the amount of acetate produced (Majewski and Domach 1990); (2) acetate can be derived from acetyl coen- J. Wang (*) zyme A (CoA) by thephosphotransacetylase (Pta)–acetate College of Agricultural and Biological Engineering, kinase (AckA) pathway, which is reversible and constitutively Jilin University, Changchun, China e-mail: wangjian99@jlu.edu.cn expressed (Chang et al. 1994). In the work presented here, we examined the effects of : : : : J. Huang J. Shi Q. Xu X. Xie N. Chen pta gene knockout on L-tryptophan fermentation. Based on College of Biological Engineering, Tianjin University of Science our investigation of the effects of glucose and dissolved & Technology, Tianjin, China 1220 Ann Microbiol (2013) 63:1219–1224 oxygen (DO) concentrations on cell growth and L-tryptophan Analytical methods production with E. coli TRTH Δpta, we then designed and tested a DO-stat feeding strategy in which substrate inflow rate The DO, pH, and temperature were measured automatically is controlled by a suitable DO concentration to achieve max- with electrodes attached to the fermenters. Biomass was rep- imal L-tryptophan and to minimize the consumption of nutri- resented by dry cell weight (DCW), and dry cell weight was ent components in the synthesis of metabolic by-products. measured as follows. Aliquots of culture broth (10 ml) were centrifuged at 10,000 rpm for 20 min. The pellets were washed twice with distilled water and dried to constant weight Materials and methods at 80 °C. Glucose concentration was measured by an SBA- 40E Biosensor Analyzer (Biology Institute, Shandong Acad- Strains and cultivation systems emy of Sciences, Jinan, Shandong Province). For analysis of the extracellular metabolites, 1 ml of culture sample was Escherichia coli strain TRTHΔpta used in this study was centrifuged, and the supernatant was then filtered through a constructed from E. coli TRTH (trpEDCBA+tet )by Huang 0.22-μm pore-size syringe filter. The concentration of L-tryp- et al. (2011). E. coli TRTH (trpEDCBA+tet ) strain was used tophan in the broth was determined by high-performance as the control. liquid chromatograph using an Agilent 1200 system (Agilent The seed medium contained (/L) 20 g glucose, 15 g yeast Technologies, Santa Clara, CA) equipped with an Agilent C18 extract, 10 g (NH ) SO , 0.5 g citrate sodium, 5 g (150×4.6 mm, i.d. 3.5 μm) column and refractive index 4 2 4 MgSO ·7H O, 1.5gKH PO ,15 mgFeSO ·7H O, and detectors (RID). A mobile phase (flow rate 1 mL/min) using 4 2 2 4 4 2 100 mg vitamin B1. The initial pH was adjusted to 7.0–7.2. a solvent of (0.03 %) KH PO /methanol (90:10, v/v) was 2 4 The production medium for fed-batch cultures contained applied to the column. The column was operated at 39 °C, (/L) 20 g glucose, 1 g yeast extract, 4 g (NH ) SO ,2g and the detection wavelength was 278 nm. Concentrations of 4 2 4 citrate sodium, 5 g MgSO ·7H O, 2 g KH PO , and 100 mg acetate, lactate, pyruvate, ammonium (NH ), and potassium 4 2 2 4 4 FeSO ·7H O. The initial pH was adjusted to 7.0–7.2. ion (K ) were measured on a Bioprofile 300A Biochemical 4 2 Analyzer (Nova Biomedical, Waltham, MA). Culture methods Specific growth rate (μ) and specific production rate (π) were calculated according to Cheng et al. (2012). A 500-mL baffled flask containing 30 mL of seed medium was inoculated with a single colony of either E. coli TRTH or E. coli TRTHΔpta strain and cultivated at 32 °C over- Results and discussion night in a shaker (rotation speed 200 rpm). A 30-mL sample of inoculum from this culture was added aseptically to a 5-L Effect of pta knockout on L-tryptophan fermentation seed fermentor (Biotech-2002 Bioprocess Controller; Baox- ing, Shanghai, China) with a working volume of 3 L and Effect of pta knockout on cell growth and L-tryptophan cultivated at 32 °C. This seed culture was then inoculated production into a 30-L reactor (Biotech-2002 Bioprocess Controller; Baoxing) containing 18 L production medium when the It has been reported that acetic acid inhibits the growth of OD reached 10 (10 %v/v). The culture temperature was strain E. coli TRTH as well as the formation of L-trypto- maintained at 30 °C and the pH was adjusted to 7.0 with phan(Chengetal. 2012). The Ack–pta pathway is the 25 % (v/v) ammonia. The inlet airflow used was approxi- major pathway for acetate biosynthesis from acetyl- mately 2.0 L/min. The DO level was maintained at approx- CoA. Aerobic fermentation of E. coli TRTHΔpta was imately 30 % saturation by adjusting the agitation rate if not compared with that of the control strain E. coli TRTH otherwise indicated. When the initial glucose was depleted, to investigate the effect of pta gene knockout on cell 800 g/L glucose solution was fed into the fermenter to growth and L-tryptophan production. The growth kinet- maintain the glucose concentration at about 5 g/L before ics and tryptophan production data are shown in Fig. 1. optimization. In the DO-stat feeding strategy, a concentrated The results indicate that E. coli TRTHΔpta grew slowly glucose solution (800 g/L) was automatically fed with a at the beginning of the exponential phase but that it was peristaltic pump at appropriate speeds to maintain a constant able to maintain a higher growth rate at the mid- DO concentration when the residual glucose dropped, and exponential phase compared with the control strain. The max- −1 the DO concentration sharply changed as described previ- imum specific growth rate of E. coli TRTHΔpta was 0.32 h , ously (Cheng et al. 2012). Each experiment was performed which was lower than that of the E. coli TRTH strain −1 three times, and data were presented as the mean value ± (0.38 h ). Finally, the biomass (43.7 g/L) and L-tryptophan standard deviation (SD). production (26.3 g/L) of E. coli TRTHΔpta were improved by Ann Microbiol (2013) 63:1219–1224 1221 was reduced to one-fifth of that produced by the control strain. Based on these results, pta knockout is an effec- tive way to reduce the accumulation of acetate during the L-tryptophan fermentation process. PoxB represents a possible route for the generation of acetate in the pta mutant (Chang et al. 1994; Nahku et al. 2010). The concentration of pyruvate and lactate was 1.3 and 4.5 g/L, respectively, during the exponential growth of E. coli TRTHΔpta strain, while in the control strain the excretion of pyruvate and lactate was quan- titatively insignificant. The excretion of pyruvate and lactate are characteristics of a mutant with a defective Pta–AckA pathway (Diaz-Ricci et al. 1991;Kakuda et al. 1994). The accumulated acetyl-CoA inhibits the activity of the pyruvate dehydrogenase complex, which in turn causes the accumula- tion of pyruvate. The excretion of lactate may be the result of an accumulation of pyruvate. However, pyruvate and lactate in the broth of E. coli TRTHΔpta strain were all consumed by the end of the fermentation period. Analysis of ammonium and potassium concentration during L-tryptophan fermentation by E. coli TRTHΔpta Luli and Strohl (1990) pointed out that protonated acetate can pass through the cell membrane into the interior of the − + cell and dissociate to CH COO and H . To regulate intra- cellular pH, which drops due to increased levels of H , E. coli consumes ATP to pump H to the extracellular domain, which disrupts cell metabolism and physiological activity. Fig. 1 Effect of phosphotransacetylase (pta) gene knockout on cell In addition, a high-affinity potassium uptake system (Kdp) growth and L-tryptophan production. a Effect of pta gene knockout on cell growth (μ Specific growth rate), b effect of pta gene knockout on capable of transporting ammonium ions into the cell will induce a L-tryptophan production (π Specific production rate). Filled symbols futile cycle, and energy will be wasted due to both the energy Escherichia coli TRTH, open symbols E. coli TRTHΔpta requirement of this ammonium transporting system and the need to extrude the protons in order to maintain pH homeostasis in the + + 23.5 and 17.5 %, respectively, as compared to the control cell. The observed variations in NH and K during L-trypto- strain (Fig. 1). phan fermentation are shown in Fig. 3. The highest concentration of NH of E. coli TRTHΔpta during L-tryptophan fermentation Effect of pta knockout on the accumulation of organic acids was 140.3 mmol/L, which is 25.9 % lower than that of the control during L-tryptophan fermentation (Fig. 3). The lower concentration of NH was probably due to the lower production of acetate in the pta mutant. The significant As the capacity of the tricarboxylic acid (TCA) cycle is lower concentration of acetate and NH will result in lower ATP limited, acetyl-CoA can not be completely oxidized to CO . consumption, which improves energy efficiency. The concentra- Consequently, excessive acetyl-CoA is converted into acetate tion of K was not significantly different in the two experimental through the Pta–Ack pathway (Castaño-Cerezo et al. 2009; strains during the fermentation process of L-tryptophan. Shin et al. 2009). The effects of pta knockout on the accumu- lation of organic acids during L-tryptophan fermentation are Optimization of the fermentation conditions of E. coli shown in Fig. 2. Acetate began to accumulate at 2 h during the TRTHΔpta strain L-tryptophan fermentation process by E. coli TRTH strain. While the specific growth rate increased, acetate accumulated Effect of initial glucose concentration on L-tryptophan rapidly and achieved a maximal level, 12.6 g/L, at 18 h fermentation (Fig. 2). Acetate production in the pta mutant was not com- pletely eliminated during the growth phase. The concentration The high concentration of glucose causes severe osmotic stress problems, which in turn affects carbohydrate transport of acetate produced by E. coli TRTHΔpta on glucose (2.5 g/L) 1222 Ann Microbiol (2013) 63:1219–1224 Fig. 3 Concentration of potassium and ammonium during L-trypto- Fig. 2 Accumulation of organic acids during L-tryptophan fermenta- phan fermentation of E. coli TRTH and its pta mutant. a Concentration tion of E. coli TRTH and its pta mutant. a Accumulation of acetate and of potassium, b concentration of ammonium. Filled symbols E. coli succinate, b accumulation of lactate and pyruvate. Filled symbols E. TRTH, open symbols E. coli TRTHΔpta coli TRTH, open symbols E. coli TRTHΔpta glucose concentration of 40 g/L. These results clearly show (Roth et al. 1985) and the distribution of carbon flux (Nanchen that a low initial glucose concentration (10 g/L) improved the et al. 2006). Therefore, we also investigated the effects of glucose to L-tryptophan. different initial glucose concentrations on L-tryptophan pro- duction by E. coli TRTHΔpta strain. When the initial glucose Effect of glucose concentration maintenance was depleted, an 800 g/L glucose stock solution was fed into on L-tryptophan fermentation the fermenter to maintain the glucose concentration at about 5 g/L. The initial glucose concentration was found to have a The rate at which acetate forms is directly related to the rate at profound effect on the production of L-tyrptophan. An inverse which the cells grow or the rate at which they consume glucose relationship between biomass and initial glucose concentra- (Eiteman and Altman 2006). In the common operational mode tion was observed (Table 1). After 38 h of fermentation, the of a fed-batch process, the growth rate of the culture depends on biomass was 47.3, 43.7, 37.6, and 33.6 g/L in cultures having the feeding rate of the glucose. It was therefore necessary to an initial glucose concentration of 10, 20, 30, and 40 g/L, determine the optimal concentration of glucose by maintaining respectively. At the same time, the concentration of L-trypto- the glucose concentration approximately at 2, 5, 10, or 15 g/L, phan attained 28.4, 26.3, 25.4, and 19.8 g/L at initial glucose respectively, during L-tryptophan fermentation when the initial concentrations of 10, 20, 30, and 40 g/L, respectively. At the glucose concentration was 10 g/L. lowest initial glucose concentration of 10 g/L, biomass and By maintaining the glucose concentration at 2 g/L, biomass L-tryptophan production were consistently the highest among and tryptophan concentration, which reached 50.3 and 32.2 g/ all of the initial glucose concentrations examined, being re- L, respectively, were consistently the highest of all concen- spectively 28.9 and 30.3 % higher than that at an initial trations examined (Table 1). In contrast, at 15 g/L, as a result Ann Microbiol (2013) 63:1219–1224 1223 Table 1 Effect of phosphotransacetylase gene (pta) knockout, initial glucose concentration, maintenance of glucose concentration, dissolved oxygen (DO) and DO-stat fed-batch process on L-tryptophan fermentation Measures Escherichia Escherichia coli TRTH Δpta coli TRTH Initial glucose concentration Maintaining glucose DO DO-stat (g/L) concentration (g/L) fed-batch system 10 20 30 40 2 5 10 15 10 % 20 % 30 % 40 % Biomass (g/L) 35.4 47.3 43.7 37.6 33.6 50.3 47.3 44.8 37.9 34.6 52.4 50.3 47.3 55.3 Tryptophan (g/L) 22.4 28.4 26.3 25.4 19.8 32.2 28.4 24.4 21.3 21.7 33.5 32.2 24.8 35.2 The same conditions as described in Effect of pta knockout on L-tryptophan fermentation of an imbalance between the fast carbon influx into the central avoid substrate over-feeding and O limitation. Based on metabolism and the limited capacity of the TCA cycle, acetate previous results, we designed and tested a DO-stat fed-batch excretion was observed and the biomass (37.9 g/L) and con- fermentation strategy in which glucose would be fed to the centration of L-tryptophan (21.3 g/L) clearly decreased. reactor using a peristaltic pump via the DO-stat control mode and operated based on the measured DO level preva- Effect of DO on L-tryptophan fermentation lent in the reaction mixture (Cheng et al. 2012). In this system, the DO level can be maintained at approximately Dissolved oxygen is an important factor for microorganism growth and product accumulation. It was therefore neces- sary to determine the optimal DO level by adjusting the agitation speed to maintain DO at 10 %, 20 %, 30 %, or 40 %, respectively. The results show that the DO level had important impacts on L-tryptophan fermentation. When the DO level was main- tained at 20 %, the biomass and concentration of L-tryptophan attained 52.4 and 33.5 g/L, respectively, which were consis- tently the highest values among all of the DO levels tested (Table 1). When the DO level was maintained at 10 %, the biomass and concentration of L-tryptophan reached 34.6 and 21.7 g/L, respectively. This decrease is due to insufficient levels of oxygen limiting the function of the TCA cycle, resulting in the accumulation of acetate, which in turn inhibits cell growth, causing cell autolysis (Huang et al. 2008)and seriously affecting tryptophan production. High DO levels (40 %) led to rapid growth and excessive recession, resulting in low biomass (47.3 g/L) and L-tryptophan production (24.8 g/L). These results clearly indicate that a 20 % DO level should be maintained to achieve maximal biomass and max- imal production of L-tryptophan. The application of the DO-stat fed-batch process on L-tryptophan fermentation The results of our independent studies suggest that DO and glucose concentration are the vital influencing parameters in the fermentation of L-tryptophan. Excessive glucose and limited capacity of the TCA cycle would cause the “Crab- Fig. 4 Application of dissolved oxygen (DO)-stat fed-batch process tree Effect,” and lead to the generation of acetate. We on L-tryptophan fermentation. Glucose concentration and fermentation therefore postulated that under oxygen-limited conditions, control parameter, biomass, tryptophan level, and by-product concen- tration are presented cell respiration is repressed. DO-stat feeding can be used to 1224 Ann Microbiol (2013) 63:1219–1224 Eiteman MA, Altman E (2006) Overcoming acetate in Escherichia coli 20 % saturation by adjusting the agitation rate. The feed recombinant protein fermentations. Trends Biotechnol 24:530– pump is switched on only when the DO level is above the 536. doi:10.1016/j.tibtech.2006.09.001 set point, and feeding stops when DO decreases below the Huang J, Xu QY, Wen TY, Chen N (2008) Metabolic flux analysis of set point. In this manner, minimal glucose concentration L-Threonine biosynthesis strain under diverse dissolved oxygen conditions. Acta Microbiol Sin 48:1056–1060 levels would be maintained in the reaction medium. Huang J, Shi JM, Liu Q, Xu QY, Xie XX, Wen TY, Chen N (2011) Using the DO-stat control strategy described, we were able Effects of gene pta disruption on L-tryptophan fermentation. Acta to fully maintain glucose-limiting conditions for the entire Microbiol Sin 51:480–487 operation (<1.5 g/L). This resulted in the concomitant de- Jensen EB, Carlsen S (1990) Production of recombinant human growth hormone in Escherichia coli: expression of different precursors crease in DO level until the set point was reached, during and physiological effects of glucose, acetate, and salts. Biotechnol which feeding was stopped. Under pump “off ” conditions, Bioeng 36:1–11 DO consumption continued as residual glucose was still avail- Kakuda H, Shiroishi K, Hosono K, Ichihara S (1994) Construction of able for synthesis to L-tryptophan by active cells. Upon com- Pta–Ack pathway deletion mutant of Escherichia coli and char- acteristic growth profiles of the mutants in a rich medium. Biosci plete conversion of glucose to L-tryptophan, DO started to Biotechnol Biochem 58:2232–2235 increase again above the set point. In this way, substrate Kocabaş P, Çalık P, Özdamar TH (2006) Fermentation characteristics feeding was successfully regulated based on the activity of of L-tryptophan production by thermoacidophilic Bacillus acid- the whole cell biosynthesis. The DO profile in Fig. 4 shows ocaldarius in a defined medium. Enzyme Microb Technol 39:1077–1088. doi:10.1016/j.enzmictec.2006.02.012 that this control strategy was effectively implemented, with Koh BT, Nakashimada U, Pfeiffer M, Yap MGS (1992) Comparison of DO levels tightly fluctuating at about 20 % during the fermen- acetate inhibition on growth of host and recombinant Escherichia tation process. The biomass increased to 55.3 g/L and the coli K12 strains. Biotechnol Lett 14:1115–1118. doi:10.1007/ BF01027012 L-tryptophan concentration reached 35.2 g/L. There was no Luli GW, Strohl WR (1990) Comparison of growth, acetate produc- significant residual glucose detected in the reactor throughout tion, and acetate inhibition of Escherichia coli strains in batch and the operation. The concentration of acetic acid, lactate, and fed-batch fermentations. Appl Environ Microbiol 56:1004–1011 pyruvate decreased to 0.7, 2.5, and 0.5 g/L respectively. Majewski RA, Domach MM (1990) Simple constrained-optimization view of acetate overflow in E. coli. Biotechnol Bioeng 35:732– 738. doi:10.1002/bit.260350711 Acknowledgments This work was supported by the National Sci- March JC, Eiteman MA, Altman E (2002) Expression of an anaplerotic ence and Technology Major Project of China for “Significant New enzyme, pyruvate carboxylase, improves recombinant protein Drugs Creation” (2008ZX09401-05) and Key Projects in the National production in Escherichia coli. Appl Environ Microbiol Science & Technology Support Program during the Eleventh Five-year 68:5620–5624. doi:10.1128/AEM.68.11.5620-5624.2002 Plan Period of China (2008BAI63B01). Mey DM, Leoueux GJ, Beauprez JJ, Maertens J, Van Horen E, Soetaert WK, Vanrolleghem PA, Vandamme EJ (2007) Comparison of dif- ferent strategies to reduce acetate formation in Escherichia coli. Biotechnol Prog 23:1053–1063. doi:10.1021/bp070170g References Nahku R, Valgenpea K, Lahtvee PJ, Erm S, Abner K, Adamberg K, Vilu R (2010) Specific growth rate dependent transcriptome pro- Castaño-Cerezo S, Pastor JM, Renilla S, Bernal V, Iborra JL, filing of Escherichia coli K12 MG1655 in accelerostat cultures. J Cánovas M (2009) An insight into the role of phosphotran- Biotechnol 145:60–65. doi:10.1016/j.jbiotec.2009.10.007 sacetylase (pta) and the acetate/acetyl-CoA node in Escher- Nanchen A, Schicker A, Sauer U (2006) Nonlinear dependency of ichia coli. Microb Cell Factories 8:54. doi:10.1186/1475- intracellular fluxes on growth rate in miniaturized continuous cul- 2859-8-54 tures of Escherichia coli. Appl Environ Microbiol 72:1164–1172 Chang YY, Wang AY, Cronan JE Jr (1994) Expression of Escherichia Pavlou AK, Reichert JM (2004) Recombinant protein therapeutics- coli pyruvate oxidase (PoxB) depends on the sigma factor success rates, market trends and values to 2010. Nat Biotechnol encoded by the rpoS (katF) gene. Mol Microbiol 11:1019–1028. 22:1513–1519. doi:10.1038/nbt1204-1513 doi:10.1111/j.1365-2958.1994.tb00380.x Roth WG, Leckie MP, Dietzler DN (1985) Osmotic stress drastically Cheng LK, Wang J, Xu QY, Xie XX, Zhang YJ, Zhao CG, Chen N inhibits active transport of carbohydrates by Escherichia coli. (2012) Effect of feeding strategy on L-tryptophan production by Biochem Biophys Res Commun 126:434–441. doi:10.1016/ recombinant Escherichia coli. Ann Microbiol 62:1625–1634. 0006-291X(85)90624-2 doi:10.1007/s13213-012-0419-6 Shin S, Chang DE, Pan JG (2009) Acetate consumption activity directly Diaz-Ricci JC, Regan L, Bailey JE (1991) Effect of alteration of the determines the level of acetate accumulation during Escherichia coli acetic acid synthesis pathway on the fermentation pattern of W3110 growth. J Microbiol Biotechnol 19:1127–1134 Escherichia coli. Biotechnol Bioeng 38:1318–1324. Sprenger GA (2007) Aromatic amino acids. Microbiol Monogr 5:94– doi:10.1002/bit.260381109 116. doi:10.1007/7171_2006_067 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase

Loading next page...
 
/lp/springer-journals/fermentation-characterization-of-an-l-tryptophan-producing-escherichia-GLIe0eD3qa

References (21)

Publisher
Springer Journals
Copyright
Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-012-0579-4
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2013) 63:1219–1224 DOI 10.1007/s13213-012-0579-4 ORIGINAL ARTICLE Fermentation characterization of an L-tryptophan producing Escherichia coli strain with inactivated phosphotransacetylase Jian Wang & Jing Huang & Jianming Shi & Qingyang Xu & Xixian Xie & Ning Chen Received: 29 July 2012 /Accepted: 22 November 2012 /Published online: 10 January 2013 # Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract Acetate is a primary inhibitory metabolite in Introduction Escherichia coli cultivation which is detrimental to bacterial growth and the formation of desired products. It can be L-Tryptophan is an essential aromatic-group amino acid derived from acetyl coenzyme A by the phosphotransacety- with a unique indole side chain that makes it a fundamental lase (Pta)–acetate kinase (AckA) pathway. In this study, the precursor to a number of neurotransmitters in the brain, fermentation characteristics of Pta mutant strain E. coli including serotonin, melatonin, and niacin, which are essen- TRTHΔpta were compared with those of the control strain tial factors for the regulation of appetite, sleep, mood, and E. coli TRTH in a 30-L fermentor. The effects of glucose pain levels (Kocabaş et al. 2006). L-Tryptophan also serves concentration and dissolved oxygen (DO) level were inves- as a nutrient enhancer, a preservative, and a feed additive. tigated, and the results suggest that DO and glucose con- The current production of L-tryptophan by microbiological centration are vital influencing parameters for the methods includes enzymatic catalysis, microbial fermenta- production of L-tryptophan. Based on our experimental tion, and microbial transformation. The large-scale produc- results, we then tested a DO-stat fed-batch fermentation tion of L-tryptophan by microbial fermentation with E. coli strategy. When DO was controlled at about 20 % during and Corynebacterium glutamicum has been reported L-tryptophan fermentation in the DO-stat fed-batch system, (Sprenger 2007). Processes using E. coli remain the produc- the pta mutant was able to maintain a higher growth rate at tion systems of choice because of the susceptibility of this the exponential phase, and the final biomass and L-trypto- microorganism to genetic manipulation and its efficient pro- phan production were increased to 55.3 g/L and 35.2 g/L, duction of recombinant proteins (Pavlou and Reichert 2004). respectively. Concomitantly, as the concentration of acetate Escherichia coli excretes acetate as a major by-product of decreased to 0.7 g/L, the accumulation of pyruvate and its aerobic metabolism (Majewski and Domach 1990), and lactate increased in the mutant strain as compared with acetate is undesirable because it retards growth and inhibits the control strain. This characterization of the recombi- protein formation (Koh et al. 1992; Jensen and Carlsen nant mutant strain provides useful information for the 1990; Mey et al. 2007). Moreover, acetate production rep- rational modification of metabolic fluxes to improve trypto- resents a diversion of carbon that might otherwise have phan production. generated biomass or a specific protein product (March et al. 2002). Two enzymatic pathways for acetate formation in . . . Keyword pta gene L-Tryptophan Escherichia coli E. coli have been identified: (1) acetate can be derived DO-stat fed-batch fermentation Acetate directly from pyruvate by pyruvate oxidase (PoxB), but the activity of this enzyme in E. coli is thought to be too low to account for the amount of acetate produced (Majewski and Domach 1990); (2) acetate can be derived from acetyl coen- J. Wang (*) zyme A (CoA) by thephosphotransacetylase (Pta)–acetate College of Agricultural and Biological Engineering, kinase (AckA) pathway, which is reversible and constitutively Jilin University, Changchun, China e-mail: wangjian99@jlu.edu.cn expressed (Chang et al. 1994). In the work presented here, we examined the effects of : : : : J. Huang J. Shi Q. Xu X. Xie N. Chen pta gene knockout on L-tryptophan fermentation. Based on College of Biological Engineering, Tianjin University of Science our investigation of the effects of glucose and dissolved & Technology, Tianjin, China 1220 Ann Microbiol (2013) 63:1219–1224 oxygen (DO) concentrations on cell growth and L-tryptophan Analytical methods production with E. coli TRTH Δpta, we then designed and tested a DO-stat feeding strategy in which substrate inflow rate The DO, pH, and temperature were measured automatically is controlled by a suitable DO concentration to achieve max- with electrodes attached to the fermenters. Biomass was rep- imal L-tryptophan and to minimize the consumption of nutri- resented by dry cell weight (DCW), and dry cell weight was ent components in the synthesis of metabolic by-products. measured as follows. Aliquots of culture broth (10 ml) were centrifuged at 10,000 rpm for 20 min. The pellets were washed twice with distilled water and dried to constant weight Materials and methods at 80 °C. Glucose concentration was measured by an SBA- 40E Biosensor Analyzer (Biology Institute, Shandong Acad- Strains and cultivation systems emy of Sciences, Jinan, Shandong Province). For analysis of the extracellular metabolites, 1 ml of culture sample was Escherichia coli strain TRTHΔpta used in this study was centrifuged, and the supernatant was then filtered through a constructed from E. coli TRTH (trpEDCBA+tet )by Huang 0.22-μm pore-size syringe filter. The concentration of L-tryp- et al. (2011). E. coli TRTH (trpEDCBA+tet ) strain was used tophan in the broth was determined by high-performance as the control. liquid chromatograph using an Agilent 1200 system (Agilent The seed medium contained (/L) 20 g glucose, 15 g yeast Technologies, Santa Clara, CA) equipped with an Agilent C18 extract, 10 g (NH ) SO , 0.5 g citrate sodium, 5 g (150×4.6 mm, i.d. 3.5 μm) column and refractive index 4 2 4 MgSO ·7H O, 1.5gKH PO ,15 mgFeSO ·7H O, and detectors (RID). A mobile phase (flow rate 1 mL/min) using 4 2 2 4 4 2 100 mg vitamin B1. The initial pH was adjusted to 7.0–7.2. a solvent of (0.03 %) KH PO /methanol (90:10, v/v) was 2 4 The production medium for fed-batch cultures contained applied to the column. The column was operated at 39 °C, (/L) 20 g glucose, 1 g yeast extract, 4 g (NH ) SO ,2g and the detection wavelength was 278 nm. Concentrations of 4 2 4 citrate sodium, 5 g MgSO ·7H O, 2 g KH PO , and 100 mg acetate, lactate, pyruvate, ammonium (NH ), and potassium 4 2 2 4 4 FeSO ·7H O. The initial pH was adjusted to 7.0–7.2. ion (K ) were measured on a Bioprofile 300A Biochemical 4 2 Analyzer (Nova Biomedical, Waltham, MA). Culture methods Specific growth rate (μ) and specific production rate (π) were calculated according to Cheng et al. (2012). A 500-mL baffled flask containing 30 mL of seed medium was inoculated with a single colony of either E. coli TRTH or E. coli TRTHΔpta strain and cultivated at 32 °C over- Results and discussion night in a shaker (rotation speed 200 rpm). A 30-mL sample of inoculum from this culture was added aseptically to a 5-L Effect of pta knockout on L-tryptophan fermentation seed fermentor (Biotech-2002 Bioprocess Controller; Baox- ing, Shanghai, China) with a working volume of 3 L and Effect of pta knockout on cell growth and L-tryptophan cultivated at 32 °C. This seed culture was then inoculated production into a 30-L reactor (Biotech-2002 Bioprocess Controller; Baoxing) containing 18 L production medium when the It has been reported that acetic acid inhibits the growth of OD reached 10 (10 %v/v). The culture temperature was strain E. coli TRTH as well as the formation of L-trypto- maintained at 30 °C and the pH was adjusted to 7.0 with phan(Chengetal. 2012). The Ack–pta pathway is the 25 % (v/v) ammonia. The inlet airflow used was approxi- major pathway for acetate biosynthesis from acetyl- mately 2.0 L/min. The DO level was maintained at approx- CoA. Aerobic fermentation of E. coli TRTHΔpta was imately 30 % saturation by adjusting the agitation rate if not compared with that of the control strain E. coli TRTH otherwise indicated. When the initial glucose was depleted, to investigate the effect of pta gene knockout on cell 800 g/L glucose solution was fed into the fermenter to growth and L-tryptophan production. The growth kinet- maintain the glucose concentration at about 5 g/L before ics and tryptophan production data are shown in Fig. 1. optimization. In the DO-stat feeding strategy, a concentrated The results indicate that E. coli TRTHΔpta grew slowly glucose solution (800 g/L) was automatically fed with a at the beginning of the exponential phase but that it was peristaltic pump at appropriate speeds to maintain a constant able to maintain a higher growth rate at the mid- DO concentration when the residual glucose dropped, and exponential phase compared with the control strain. The max- −1 the DO concentration sharply changed as described previ- imum specific growth rate of E. coli TRTHΔpta was 0.32 h , ously (Cheng et al. 2012). Each experiment was performed which was lower than that of the E. coli TRTH strain −1 three times, and data were presented as the mean value ± (0.38 h ). Finally, the biomass (43.7 g/L) and L-tryptophan standard deviation (SD). production (26.3 g/L) of E. coli TRTHΔpta were improved by Ann Microbiol (2013) 63:1219–1224 1221 was reduced to one-fifth of that produced by the control strain. Based on these results, pta knockout is an effec- tive way to reduce the accumulation of acetate during the L-tryptophan fermentation process. PoxB represents a possible route for the generation of acetate in the pta mutant (Chang et al. 1994; Nahku et al. 2010). The concentration of pyruvate and lactate was 1.3 and 4.5 g/L, respectively, during the exponential growth of E. coli TRTHΔpta strain, while in the control strain the excretion of pyruvate and lactate was quan- titatively insignificant. The excretion of pyruvate and lactate are characteristics of a mutant with a defective Pta–AckA pathway (Diaz-Ricci et al. 1991;Kakuda et al. 1994). The accumulated acetyl-CoA inhibits the activity of the pyruvate dehydrogenase complex, which in turn causes the accumula- tion of pyruvate. The excretion of lactate may be the result of an accumulation of pyruvate. However, pyruvate and lactate in the broth of E. coli TRTHΔpta strain were all consumed by the end of the fermentation period. Analysis of ammonium and potassium concentration during L-tryptophan fermentation by E. coli TRTHΔpta Luli and Strohl (1990) pointed out that protonated acetate can pass through the cell membrane into the interior of the − + cell and dissociate to CH COO and H . To regulate intra- cellular pH, which drops due to increased levels of H , E. coli consumes ATP to pump H to the extracellular domain, which disrupts cell metabolism and physiological activity. Fig. 1 Effect of phosphotransacetylase (pta) gene knockout on cell In addition, a high-affinity potassium uptake system (Kdp) growth and L-tryptophan production. a Effect of pta gene knockout on cell growth (μ Specific growth rate), b effect of pta gene knockout on capable of transporting ammonium ions into the cell will induce a L-tryptophan production (π Specific production rate). Filled symbols futile cycle, and energy will be wasted due to both the energy Escherichia coli TRTH, open symbols E. coli TRTHΔpta requirement of this ammonium transporting system and the need to extrude the protons in order to maintain pH homeostasis in the + + 23.5 and 17.5 %, respectively, as compared to the control cell. The observed variations in NH and K during L-trypto- strain (Fig. 1). phan fermentation are shown in Fig. 3. The highest concentration of NH of E. coli TRTHΔpta during L-tryptophan fermentation Effect of pta knockout on the accumulation of organic acids was 140.3 mmol/L, which is 25.9 % lower than that of the control during L-tryptophan fermentation (Fig. 3). The lower concentration of NH was probably due to the lower production of acetate in the pta mutant. The significant As the capacity of the tricarboxylic acid (TCA) cycle is lower concentration of acetate and NH will result in lower ATP limited, acetyl-CoA can not be completely oxidized to CO . consumption, which improves energy efficiency. The concentra- Consequently, excessive acetyl-CoA is converted into acetate tion of K was not significantly different in the two experimental through the Pta–Ack pathway (Castaño-Cerezo et al. 2009; strains during the fermentation process of L-tryptophan. Shin et al. 2009). The effects of pta knockout on the accumu- lation of organic acids during L-tryptophan fermentation are Optimization of the fermentation conditions of E. coli shown in Fig. 2. Acetate began to accumulate at 2 h during the TRTHΔpta strain L-tryptophan fermentation process by E. coli TRTH strain. While the specific growth rate increased, acetate accumulated Effect of initial glucose concentration on L-tryptophan rapidly and achieved a maximal level, 12.6 g/L, at 18 h fermentation (Fig. 2). Acetate production in the pta mutant was not com- pletely eliminated during the growth phase. The concentration The high concentration of glucose causes severe osmotic stress problems, which in turn affects carbohydrate transport of acetate produced by E. coli TRTHΔpta on glucose (2.5 g/L) 1222 Ann Microbiol (2013) 63:1219–1224 Fig. 3 Concentration of potassium and ammonium during L-trypto- Fig. 2 Accumulation of organic acids during L-tryptophan fermenta- phan fermentation of E. coli TRTH and its pta mutant. a Concentration tion of E. coli TRTH and its pta mutant. a Accumulation of acetate and of potassium, b concentration of ammonium. Filled symbols E. coli succinate, b accumulation of lactate and pyruvate. Filled symbols E. TRTH, open symbols E. coli TRTHΔpta coli TRTH, open symbols E. coli TRTHΔpta glucose concentration of 40 g/L. These results clearly show (Roth et al. 1985) and the distribution of carbon flux (Nanchen that a low initial glucose concentration (10 g/L) improved the et al. 2006). Therefore, we also investigated the effects of glucose to L-tryptophan. different initial glucose concentrations on L-tryptophan pro- duction by E. coli TRTHΔpta strain. When the initial glucose Effect of glucose concentration maintenance was depleted, an 800 g/L glucose stock solution was fed into on L-tryptophan fermentation the fermenter to maintain the glucose concentration at about 5 g/L. The initial glucose concentration was found to have a The rate at which acetate forms is directly related to the rate at profound effect on the production of L-tyrptophan. An inverse which the cells grow or the rate at which they consume glucose relationship between biomass and initial glucose concentra- (Eiteman and Altman 2006). In the common operational mode tion was observed (Table 1). After 38 h of fermentation, the of a fed-batch process, the growth rate of the culture depends on biomass was 47.3, 43.7, 37.6, and 33.6 g/L in cultures having the feeding rate of the glucose. It was therefore necessary to an initial glucose concentration of 10, 20, 30, and 40 g/L, determine the optimal concentration of glucose by maintaining respectively. At the same time, the concentration of L-trypto- the glucose concentration approximately at 2, 5, 10, or 15 g/L, phan attained 28.4, 26.3, 25.4, and 19.8 g/L at initial glucose respectively, during L-tryptophan fermentation when the initial concentrations of 10, 20, 30, and 40 g/L, respectively. At the glucose concentration was 10 g/L. lowest initial glucose concentration of 10 g/L, biomass and By maintaining the glucose concentration at 2 g/L, biomass L-tryptophan production were consistently the highest among and tryptophan concentration, which reached 50.3 and 32.2 g/ all of the initial glucose concentrations examined, being re- L, respectively, were consistently the highest of all concen- spectively 28.9 and 30.3 % higher than that at an initial trations examined (Table 1). In contrast, at 15 g/L, as a result Ann Microbiol (2013) 63:1219–1224 1223 Table 1 Effect of phosphotransacetylase gene (pta) knockout, initial glucose concentration, maintenance of glucose concentration, dissolved oxygen (DO) and DO-stat fed-batch process on L-tryptophan fermentation Measures Escherichia Escherichia coli TRTH Δpta coli TRTH Initial glucose concentration Maintaining glucose DO DO-stat (g/L) concentration (g/L) fed-batch system 10 20 30 40 2 5 10 15 10 % 20 % 30 % 40 % Biomass (g/L) 35.4 47.3 43.7 37.6 33.6 50.3 47.3 44.8 37.9 34.6 52.4 50.3 47.3 55.3 Tryptophan (g/L) 22.4 28.4 26.3 25.4 19.8 32.2 28.4 24.4 21.3 21.7 33.5 32.2 24.8 35.2 The same conditions as described in Effect of pta knockout on L-tryptophan fermentation of an imbalance between the fast carbon influx into the central avoid substrate over-feeding and O limitation. Based on metabolism and the limited capacity of the TCA cycle, acetate previous results, we designed and tested a DO-stat fed-batch excretion was observed and the biomass (37.9 g/L) and con- fermentation strategy in which glucose would be fed to the centration of L-tryptophan (21.3 g/L) clearly decreased. reactor using a peristaltic pump via the DO-stat control mode and operated based on the measured DO level preva- Effect of DO on L-tryptophan fermentation lent in the reaction mixture (Cheng et al. 2012). In this system, the DO level can be maintained at approximately Dissolved oxygen is an important factor for microorganism growth and product accumulation. It was therefore neces- sary to determine the optimal DO level by adjusting the agitation speed to maintain DO at 10 %, 20 %, 30 %, or 40 %, respectively. The results show that the DO level had important impacts on L-tryptophan fermentation. When the DO level was main- tained at 20 %, the biomass and concentration of L-tryptophan attained 52.4 and 33.5 g/L, respectively, which were consis- tently the highest values among all of the DO levels tested (Table 1). When the DO level was maintained at 10 %, the biomass and concentration of L-tryptophan reached 34.6 and 21.7 g/L, respectively. This decrease is due to insufficient levels of oxygen limiting the function of the TCA cycle, resulting in the accumulation of acetate, which in turn inhibits cell growth, causing cell autolysis (Huang et al. 2008)and seriously affecting tryptophan production. High DO levels (40 %) led to rapid growth and excessive recession, resulting in low biomass (47.3 g/L) and L-tryptophan production (24.8 g/L). These results clearly indicate that a 20 % DO level should be maintained to achieve maximal biomass and max- imal production of L-tryptophan. The application of the DO-stat fed-batch process on L-tryptophan fermentation The results of our independent studies suggest that DO and glucose concentration are the vital influencing parameters in the fermentation of L-tryptophan. Excessive glucose and limited capacity of the TCA cycle would cause the “Crab- Fig. 4 Application of dissolved oxygen (DO)-stat fed-batch process tree Effect,” and lead to the generation of acetate. We on L-tryptophan fermentation. Glucose concentration and fermentation therefore postulated that under oxygen-limited conditions, control parameter, biomass, tryptophan level, and by-product concen- tration are presented cell respiration is repressed. DO-stat feeding can be used to 1224 Ann Microbiol (2013) 63:1219–1224 Eiteman MA, Altman E (2006) Overcoming acetate in Escherichia coli 20 % saturation by adjusting the agitation rate. The feed recombinant protein fermentations. Trends Biotechnol 24:530– pump is switched on only when the DO level is above the 536. doi:10.1016/j.tibtech.2006.09.001 set point, and feeding stops when DO decreases below the Huang J, Xu QY, Wen TY, Chen N (2008) Metabolic flux analysis of set point. In this manner, minimal glucose concentration L-Threonine biosynthesis strain under diverse dissolved oxygen conditions. Acta Microbiol Sin 48:1056–1060 levels would be maintained in the reaction medium. Huang J, Shi JM, Liu Q, Xu QY, Xie XX, Wen TY, Chen N (2011) Using the DO-stat control strategy described, we were able Effects of gene pta disruption on L-tryptophan fermentation. Acta to fully maintain glucose-limiting conditions for the entire Microbiol Sin 51:480–487 operation (<1.5 g/L). This resulted in the concomitant de- Jensen EB, Carlsen S (1990) Production of recombinant human growth hormone in Escherichia coli: expression of different precursors crease in DO level until the set point was reached, during and physiological effects of glucose, acetate, and salts. Biotechnol which feeding was stopped. Under pump “off ” conditions, Bioeng 36:1–11 DO consumption continued as residual glucose was still avail- Kakuda H, Shiroishi K, Hosono K, Ichihara S (1994) Construction of able for synthesis to L-tryptophan by active cells. Upon com- Pta–Ack pathway deletion mutant of Escherichia coli and char- acteristic growth profiles of the mutants in a rich medium. Biosci plete conversion of glucose to L-tryptophan, DO started to Biotechnol Biochem 58:2232–2235 increase again above the set point. In this way, substrate Kocabaş P, Çalık P, Özdamar TH (2006) Fermentation characteristics feeding was successfully regulated based on the activity of of L-tryptophan production by thermoacidophilic Bacillus acid- the whole cell biosynthesis. The DO profile in Fig. 4 shows ocaldarius in a defined medium. Enzyme Microb Technol 39:1077–1088. doi:10.1016/j.enzmictec.2006.02.012 that this control strategy was effectively implemented, with Koh BT, Nakashimada U, Pfeiffer M, Yap MGS (1992) Comparison of DO levels tightly fluctuating at about 20 % during the fermen- acetate inhibition on growth of host and recombinant Escherichia tation process. The biomass increased to 55.3 g/L and the coli K12 strains. Biotechnol Lett 14:1115–1118. doi:10.1007/ BF01027012 L-tryptophan concentration reached 35.2 g/L. There was no Luli GW, Strohl WR (1990) Comparison of growth, acetate produc- significant residual glucose detected in the reactor throughout tion, and acetate inhibition of Escherichia coli strains in batch and the operation. The concentration of acetic acid, lactate, and fed-batch fermentations. Appl Environ Microbiol 56:1004–1011 pyruvate decreased to 0.7, 2.5, and 0.5 g/L respectively. Majewski RA, Domach MM (1990) Simple constrained-optimization view of acetate overflow in E. coli. Biotechnol Bioeng 35:732– 738. doi:10.1002/bit.260350711 Acknowledgments This work was supported by the National Sci- March JC, Eiteman MA, Altman E (2002) Expression of an anaplerotic ence and Technology Major Project of China for “Significant New enzyme, pyruvate carboxylase, improves recombinant protein Drugs Creation” (2008ZX09401-05) and Key Projects in the National production in Escherichia coli. Appl Environ Microbiol Science & Technology Support Program during the Eleventh Five-year 68:5620–5624. doi:10.1128/AEM.68.11.5620-5624.2002 Plan Period of China (2008BAI63B01). Mey DM, Leoueux GJ, Beauprez JJ, Maertens J, Van Horen E, Soetaert WK, Vanrolleghem PA, Vandamme EJ (2007) Comparison of dif- ferent strategies to reduce acetate formation in Escherichia coli. Biotechnol Prog 23:1053–1063. doi:10.1021/bp070170g References Nahku R, Valgenpea K, Lahtvee PJ, Erm S, Abner K, Adamberg K, Vilu R (2010) Specific growth rate dependent transcriptome pro- Castaño-Cerezo S, Pastor JM, Renilla S, Bernal V, Iborra JL, filing of Escherichia coli K12 MG1655 in accelerostat cultures. J Cánovas M (2009) An insight into the role of phosphotran- Biotechnol 145:60–65. doi:10.1016/j.jbiotec.2009.10.007 sacetylase (pta) and the acetate/acetyl-CoA node in Escher- Nanchen A, Schicker A, Sauer U (2006) Nonlinear dependency of ichia coli. Microb Cell Factories 8:54. doi:10.1186/1475- intracellular fluxes on growth rate in miniaturized continuous cul- 2859-8-54 tures of Escherichia coli. Appl Environ Microbiol 72:1164–1172 Chang YY, Wang AY, Cronan JE Jr (1994) Expression of Escherichia Pavlou AK, Reichert JM (2004) Recombinant protein therapeutics- coli pyruvate oxidase (PoxB) depends on the sigma factor success rates, market trends and values to 2010. Nat Biotechnol encoded by the rpoS (katF) gene. Mol Microbiol 11:1019–1028. 22:1513–1519. doi:10.1038/nbt1204-1513 doi:10.1111/j.1365-2958.1994.tb00380.x Roth WG, Leckie MP, Dietzler DN (1985) Osmotic stress drastically Cheng LK, Wang J, Xu QY, Xie XX, Zhang YJ, Zhao CG, Chen N inhibits active transport of carbohydrates by Escherichia coli. (2012) Effect of feeding strategy on L-tryptophan production by Biochem Biophys Res Commun 126:434–441. doi:10.1016/ recombinant Escherichia coli. Ann Microbiol 62:1625–1634. 0006-291X(85)90624-2 doi:10.1007/s13213-012-0419-6 Shin S, Chang DE, Pan JG (2009) Acetate consumption activity directly Diaz-Ricci JC, Regan L, Bailey JE (1991) Effect of alteration of the determines the level of acetate accumulation during Escherichia coli acetic acid synthesis pathway on the fermentation pattern of W3110 growth. J Microbiol Biotechnol 19:1127–1134 Escherichia coli. Biotechnol Bioeng 38:1318–1324. Sprenger GA (2007) Aromatic amino acids. Microbiol Monogr 5:94– doi:10.1002/bit.260381109 116. doi:10.1007/7171_2006_067

Journal

Annals of MicrobiologySpringer Journals

Published: Jan 10, 2013

There are no references for this article.