Model for the production of L-tryptophan from L-serine and indole by immobilized cells in a three-phase liquid-impelled loop reactor

Production of L-tryptophan from L-serine and indole catalyzed by Escherichia coli, immobilized in k-carrageenan gel beads, is technically feasible in the liquidimpelled loop reactor (LLR), using an organic solvent, e.g. n-dodecane.With L-serine in large excess intrinsic reaction kinetics is approximately first order with respect to indole, with a reaction constant of 8.5×10^–5 m³ kg _dw ^–1 s^–1.The overall process kinetics is jointly controlled by intrinsic kinetics and by intraparticle mass transfer resistance, which can be quantified using an effectiveness factor.Mass transfer of indole from the organic to the aqueous phase and from the aqueous to the gel phase are relatively fast and thus have negligible influence in the overall process kinetics, under the operational conditions tested. However, they may become important if the process is intensified by increasing the cell concentration in the gel and/or the gel hold-up in the reactor.A simple model which includes indole mass balances over the aqueous and organic phases, mass transfer and reaction kinetics, with parameters experimentally determined in independent experiments, was successful in simulating L-tryptophan production in the LLR.List of Symbols a, b, c coefficients of the equilibrium curve for indole between organic and aqueous phases - A, B, C, D, E, F auxiliary variables used in liquid-liquid mass transfer studies - a _x specific interfacial area referred to the volume of the aqueous phase (m^–1) - A _x interfacial area (m²) - a _Y specific interfacial area referred to the volume of the organic phase (m^–1) - A _Y interfacial area (m²) - C _b substrate concentration in the bulk of the aqueous phase (kg m^–3) - C _e substrate concentration in exit stream (kg m^–3) - C _E biocatalyst concentration referred to the aqueous phase (kg m^–3) - C _{E s} biocatalyst concentration referred to the volume of gel (kg m^–3) - C _s substrate concentration at the gel surface (kgm^–3) - d, e, f coefficients of the equilibrium curve for indole between aqueous and organic phases - dp particle diameter (m) - K ₂ kinetic constant (s^–1) - K ₁ kinetic constant K₂/K_M (kg^–1 m³ s^–1) - K _M Michaälis-Menten constant (kgm^–3) - K _X mass transfer coefficient referred to the aqueous phase (ms^–1) - K _Xa_X volumetric mass transfer coefficient based on the volume of the aqueous phase (s^–1) - k _Y mass transfer coefficient referred to the organic phase (ms^–1) - K _Ya_Y volumetric mass transfer coefficient based on the volume of the organic phase (s^–1) - N _X mass flux of indole from organic to aqueous Phase (kg m^–2s^–1) - N _Y mass flux of indole from aqueous to organic phase (kg m^–2s^–1) - Q _e volumetric flow rate in exit stream (m³s^–1) - Q _f volumetric flow rate in feed stream (m³s^–1) - _obs observed reaction rate (kg s^–1 m^–3) - intrinsic reaction rate (kg s^–1 m^–3) - Re Reynolds number - Sc Schmidt number - Sh Sherwood number - t time (s) - u superficial velocity (m s^–1) - V _max maximum reaction rate (kg s^–1m^–3) - V _S volume of the support (m³) - V _X volume of aqueous phase (m³) - V _Y volume of the organic phase (m³) - X indole concentration in the aqueous phase (kgm^–3) - Y indole concentration in the organic phase (kg m^–3 Greek Letters overall effectiveness factor - _e external effectiveness factor - _i internal effectiveness factor - Thiele module A fellowship awarded to one of us (D.M.R.)by INICT is gratefuly acknowledged.     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

The interaction of the trp repressor from Escherichia coli with L-tryptophan and indole propanoic acid

The binding of the corepressor, L-tryptophan, and an inducer, indole propanoic acid, to the trp repressor from Escherichia coli was studied by absorbance, fluorescence, circular dichroic and proton NMR spectroscopy. The two ligands bind to the same site on the repressor in the same orientation; they are molecular competitors. The binding site is of relatively low polarity and contains at least one methyl group that lies 0.3 nm over the indole moiety near the C5 proton of the bound ligand, and an aromatic residue, probably tyrosine. The dissociation constant was determined as a function of temperature and pH. At 25 degrees C in 0.1 M phosphate buffer, pH 7.6, the dissociation constant is 18 +/- 2 microM for both ligands. In the same buffer system, the van't Hoff enthalpy for dissociation is 35.5 +/- 1 kJ/mol for tryptophan, and 30.5 +/- 2 kJ/mol for indole propanoic acid. The affinity of the repressor for indole propanoic acid is independent of pH in the range 7 less than 10, but decreases four fold for tryptophan in the same range. The amino group of tryptophan makes a significant contribution to its binding affinity. Difference NMR spectra showed that there are few changes of protein resonances on binding ligands. The NMR signals of the bound resonances were assigned by difference and nuclear Overhauser effect spectroscopy. The properties of the bound resonances are consistent with the ligands being largely immobilised within the binding site. The difference spectra, and the known functional differences of the two ligands, suggest that tryptophan induces a slightly different conformational state in the repressor from that induced by indole propanoic acid. There is no evidence for a global transition. The rate of dissociation of ligands is relatively large, being in the range 400-600 s-1.     

Continuous production of L-malic acid by immobilized cells

l-Malic acid is used extensively in the pharmaceutical industry and as a food additive. It is now produced on an industrial scale by the enzymatic conversion of fumaric acid using immobilized cells of Brevibacterium flavum. Recent improvements to this system, especially the use of x-carrageenan supports, have resulted in a continuous process capable of yielding 30 tonnes of l-malic acid per month.     

L-serine deaminase of Escherichia coli

The native l-serine deaminase (l-serine hydrolyase, deaminating, EC 4.2.1.13) of Escherichia coli K-12, which seems to be a very labile protein, is rather stable in concentrated solution. Dilution rapidly inactivates it, but in the presence of a saturating concentration of l-serine the molecule is protected from inactivation. It is a very specific enzyme; l-serine is the sole substrate with a K(m) value of 6.60 x 10(-3)m. d-Serine and l-cysteine are competitive inhibitors. Substrate saturation curves of the native enzyme show sigmoid shape, whereas the enzyme liberated from the bacteria in the presence of l-serine exhibits normal Michaelis-Menten kinetics.     

大肠杆菌色氨酸转运系统多基因敲除对色氨酸生产的影响

大肠杆菌中色氨酸向胞内的转运主要是由mtr、tnaB和aroP 3个基因编码的通透酶进行调控.利用Red重组技术,在mtr单基因敲除菌的基础上,成功构建了mtr.tnaB和mtr.aroP双基因敲除菌以及mtr.tnaB.aroP三基因敲除菌,并通过发酵实验首次考察了色氨酸转运系统多基因缺失对大肠杆菌合成色氨酸的影响.发酵结果表明,mtr.tnaB和mtr.aroP双基因缺失后,色氨酸产量分别达到1.38 g/L和1.27 g/L,与出发菌株相比分别提高了17％和9％,而mtr.tnaB.aroP三基因缺失后,菌体生长受到了明显抑制,发酵后色氨酸产量仅为0.63 g/L.在补料分批发酵实验中,mtr.tnaB双基因敲除菌的色氨酸产量进一步提高至12.2 g/L,与出发菌株相比色氨酸产量提高了27％.     

Recombinant Escherichia coli cells immobilized in Ca-alginate beads for metabolite production

Milligram amounts of metabolites of drug candidates are required to identify toxic products. Human drug metabolites are currently produced selectively in a time- and cost-efficient manner in bioreactor systems containing recombinant Escherichia coli co-expressing a human cytochrome P450 isoenzyme/NADPH cytochrome P450 reductase (hCYP/HR) complex. For further optimization, immobilization of the catalytic system in Ca-alginate microbeads was considered. This new concept was designed for CYP3A4 with testosterone as substrate. Immobilized E. coli cells had a high maximal and homogeneously distributed biomass. Viability was stable over at least 1 week of culture and even longer during storage. Gene expression was ideally initiated 6 h after immobilization. Although immobilized E. coli cells expressed a highly functional enzyme system after 2 days, they did not metabolize testosterone, probably due to cell permeability problems resulting from immobilization. Therefore, immobilized cell membranes displaying testosterone bioconversion activity, even after long-term storage, will be used in bioreactors with high organic solvent content.     

大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统改造对产L-色氨酸的影响

L-色氨酸是芳香族氨基酸的一种,被广泛应用于医药、食品和饲料等领域。大肠杆菌磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PTS系统)在葡萄糖转运和磷酸化过程中起重要作用,是糖代谢基因表达调控的核心。利用Red同源重组系统,构建包含两类典型PTS系统突变(ptsHIcrr~-glf-glk~+和ptsG~-)的L-色氨酸生产菌,并对相关菌株进行补料分批发酵研究。结果表明,不同类型PTS系统突变对菌体生长、L-色氨酸产量、糖酸转化率及副产物生成均有较大影响。与出发菌相比,ptsHIcrr~-glf-glk~+突变株最高OD_(600)达到125,提高47.0%,产酸38.5 g/L,提高25.9%,糖酸转化率16.7%,提高26.5%,乙酸生成略有增加;ptsG~-突变株最高OD_(600)达到100,提高17.6%,产酸33.4 g/L,提高9.4%,糖酸转化率15.5%,提高17.4%,乙酸生成略有减少。对葡萄糖转运系统的进一步研究将为大肠杆菌合成L-色氨酸效率的提升提供帮助。     

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

L-serine is a promising building block biochemical with a high theoretical production yield from glucose. Toxicity of L-serine is however prohibitive for high-titer production in E. coli. Here, E. coli lacking L-serine degradation pathways was evolved for improved tolerance by gradually increasing L-serine concentration from 3 to 100 g/L using adaptive laboratory evolution (ALE). Genome sequencing of isolated clones revealed multiplication of genetic regions, as well as mutations in thrA, thereby showing a potential mechanism of serine inhibition. Additional mutations were evaluated by MAGE combined with amplicon sequencing, revealing role of rho, lrp, pykF, eno, and rpoB on tolerance and fitness in minimal medium. Production using the tolerant strains resulted in 37 g/L of L-serine with a 24% mass yield. The resulting titer is similar to the highest production reported for any organism thereby highlighting the potential of ALE for industrial biotechnology.     

Construction of Escherichia coli strains producing L-serine from glucose

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.     

Continuous production of 6-aminopenicillanic acid from penicillin by immobilized microbial cells

Summary For continuous production of 6-aminopenicillanic acid (6-APA) the microbial cells ofEscherichia coli ATCC 9637 having high penicillin amidase (penicillin amidohydrolase, E.C. 3. 5. 1. 11) activity were immobilized by entrapment in a polyacrylamide gel lattice.Enzymatic properties of penicillin amidase of the immobilizedE. coli cells were investigated and compared with those of the intact cells. With regard to optimal pH and temperature, no marked difference was observed. The heat stability was somewhat increased by immobilization of the cells.The enzyme activity of the immobilized cell column was stable, and its half-life was 17 days at 40°C and 42 days at 30°C. From the effluent of the column, 6-APA was easily obtained in a good yield.Abbreviations 6-APA 6-aminopenicillanic acid - BIS N,N-methylenebisacrylamide - DMAPN -dimethylaminopropionitrile - SV space velocity     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Continuous production of glutathione by immobilized Saccharomyces cerevisiae cells

Summary Glutathione was continuously produced by an immobilized Saccharomyces cerevisiae IFO 2044 cell column. The production of glutathione was strongly influenced by the level of activity of the glycolytic pathway. This activity was maintained constant by the addition of NAD.Abbreviations ADP adenosine-5-diphosphate - ATP adenosine-5-triphosphate - NAD nicothinamide adenine dinucleotide     

Continuous production of steroid glycoalkaloids by immobilized plant cells

Summary Non-permeablilized cells ofSolanum aviculare were immobilized by their adsorption and covalent linkage on activated polymeric adsorbent. The retention of extracellular production of steroid glycoalkaloids was studied in a packed-column recycle reactor.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

Characterization of the reaction of L-serine and indole with Escherichia coli tryptophan synthase via rapid-scanning ultraviolet-visible spectroscopy

The pre-steady-state reaction of indole and L-serine with the alpha 2 beta 2 complex of Escherichia coli tryptophan synthase has been investigated under different premixing conditions with rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy for the spectral range 300-550 nm. When alpha 2 beta 2 was mixed with indole and L-serine, the reaction of alpha 2 beta 2 was found to occur in three detectable relaxations (1/tau 1 greater than 1/tau 2 greater than 1/tau 3) with rate constants identical with the three relaxations seen in the partial reaction with L-serine [Drewe, W.F., Jr., & Dunn, M.F. (1985) Biochemistry 24, 3977-3987]. Kinetic isotope effects due to substitution of 2H for the alpha-1H of serine were found to be similar to the effects observed in the reaction with serine only. The observed spectral changes and isotope effects indicate that the aldimine of L-serine and PLP and the first quinoid derived from this external aldimine are transient species that accumulate during tau 1. Conversion of these intermediates to the alpha-aminoacrylate Schiff base during tau 2 and tau 3 limits the rate of formation of the second quinoidal species (lambda max 476 nm) generated via C-C bond formation between indole and the alpha-aminoacrylate intermediate. The pre-steady-state reaction of the alpha 2 beta 2-serine mixture with indole is comprised of four relaxations (1/tau 1* greater than 1/tau 2* greater than 1/tau 3* greater than 1/tau 4*).(ABSTRACT TRUNCATED AT 250 WORDS)