Derivation of Aromatic Amino Acid Mutants from a Methanol-Utilizing Yeast, Hansenula polymorpha

Three classes of mutants, deregulated to enhance the flow of aromatic intermediates through the tryptophan biosynthetic branch, were obtained. 5-Fluorotryptophan, an antimetabolite of tryptophan, was employed to obtain one class of deregulated mutants. By sequential resistance development, three resistant mutants were isolated. Hansenula polymorpha strains showed greater sensitivity to 5-fluorotryptophan when growing on methanol than when growing on glucose. Yeast extract stimulated the production of total indole metabolites (indoles) by wild-type and mutant strains, with each 5-fluorotryptophan mutant producing higher amounts of these metabolites than its predecessor. Two other mutant classes were isolated: (i) a mutant resistant to anthranilate (an inhibitory intermediate in the tryptophan biosynthetic branch) and (ii) a phenylalanine-plus-tyrosine bradytroph. Each of these produced a higher extracellular titer of total indoles than its immediate parent. With respect to the overproduction of indoles, resistance to 5-fluorotryptophan was a more useful selection method than were anthranilate resistance and phenylalanine-plus-tyrosine bradytrophy.     

Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.     

Cloning and expression of naphthalene dioxygenase genes from Comamonas sp. MQ for indigoids production

The naphthalene dioxygenase (NDO) genes were cloned from Comamonas sp. MQ and successfully expressed in Escherichia coli BL21 (DE3) (designated as ND_IND). The whole cells of recombinant strain ND_IND possessed relatively high transformation efficiency towards indole and most indole derivatives. According to the UV–vis and HPLC–MS analyses, the major products derived from the indoles could be indigo with different substituent groups. Furthermore, strain ND_IND was able to produce 205 mg/l indigo from 300 mg/l indole with a specific production rate of 8.4 mg/(g dry cell weight h). The effects of phenol, pyridine and quinoline on indigo production were determined, which indicated that phenol and pyridine had little inhibition on indigo production while quinoline would result in a 32% decrease in indigo yield. The present study proposed the potential application of recombinant strain ND_IND in indigoid pigments production, and offered the promise of applying strain ND_IND for the production of indigo using indole-containing wastewater as the raw materials.     

Abiotic stress-related genes governing signal transduction cascades in wild plants with emphasis to those in Hordeum spontaneum

The study aims at discussing abiotic stress-related genes and metabolites in the crosstalking signal transduction pathways with emphasis to those in leaves of wild barley Hordeum spontaneum. Selected pathways involve tryptophan and target of rapamycin (or TOR) complex as core metabolites. In H. spontaneum, route towards tryptophan production seems to be mediated via anthranilate synthase acting as a master switch. Downstream auxin-related factors and proteins can secure plant’s ability to maintain proper growth rates and stress tolerance. Detected TOR in H. spontaneum participates in several signal transduction related pathways with a central role in nutrient starvation, ribosome biogenesis and protein synthesis and in standing oxidative and other abiotic stresses. TOR also participates as a master regulator of lifespan, aging and autophagy in H. spontaneum to regulate growth and maintain plant yield and its attributes under stress conditions. In conclusion, the present study emphasizes some mechanisms by which wild plant species, e.g., H. spontaneum, utilize to cope with abiotic stresses.

     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

Relating genotype and phenotype for tryptophan synthesis in an aphid–bacterial symbiosis

The symbiotic bacteria Buchnera provide their aphid hosts with tryptophan and other essential amino acids. Tryptophan production by Buchnera varied among 12 parthenogenetic clones of the pea aphid Acyrthosiphon pisum (Harris), as determined from both the incorporation of radioactivity from ¹⁴C‐anthranilate into tryptophan and the protein‐tryptophan growth rate of larval aphids on tryptophan‐free diet. The values of tryptophan production obtained for the two methods were correlated significantly with each other but not with the level of amplification of the Buchnera genes trpEG, which code for anthranilate synthase, a key enzyme in tryptophan biosynthetic pathway. This study provides the first direct demonstration of interclonal variation in production of any nutrient in an aphid–Buchnera symbiosis and indicates that a key aspect of Buchnera phenotype (tryptophan production) does not vary in a simple fashion with Buchnera genotype.     

Some tryptophan pathways in the phytopathogen Xanthomonas oryzae pv. oryzae

Xanthomonas oryzae pv.oryzae, the causal or-ganism of bacterial blight of rice which produces leaf blight as well as kresek (wilt) symptoms in plants were tested for indole, auxin production in culture supplemented withl-tryptophan. On the basis of indoleacetic acid (IAA) production the isolates were grouped into IAA-positive and IAA-negative. Out of 17 isolates, 11 were IAA-positive while 6 were IAA-negative. The isolates metabolized tryptophan through two different routes and the isolates vary in the pathway of tryptophan utilization. The IAA-positive isolates converted tryptophan to IAA as the end product, whereas the IAA-negative isolates formed anthranilate as an intermediate metabolite and finally produced pyrocatecholvia the kynurenine pathway. Quantification of tryptophan metabolism revealed that the maximum production of IAA and pyrocatechol in culture occurred during 2-d incubation at 30±2°C.     

不同碳源和生长因子条件下粒毛盘菌代谢产物初步研究

采用GC-MS法对一种粒毛盘菌(Lachnum sp.)在不同碳源、生长因子条件下发酵代谢产物的挥发性成分组成与差异进行分析。结果显示,不同碳源和生长因子条件下产生的代谢产物不同,主要包括有机酸、胺类、烷烃类、酯类、醇类、吡咯等物质。分别以20 g/L的葡萄糖、蔗糖、淀粉为碳源的发酵液中检测到的挥发性代谢产物为7、7、10种;添加1 mg/L的V_C、V_(B1)、甘氨酸、色氨酸作为不同生长因子的发酵液中检测到的挥发性代谢产物分别为6、7、7、12种。结果显示粒毛盘菌YM406发酵代谢产物具有丰富的多样性,并且在不同的培养条件下产生的代谢产物存在一定的差异。     

Anthranilate synthase and chorismate mutase activities in transgenic tobacco plants overexpressing tryptophan decarboxylase fromCatharanthus roseus

TransgenicNicotiana tabacum L. Petit Havana SR1 F₁-plants expressing tryptophan decarboxylase cDNA (tdc) fromCatharanthus roseus (L.) G. Don under the control of the CaMV 35S promoter and terminator exhibited tryptophan decarboxylase (TDC) enzyme activity and accumulated tryptamine. The plants with the highest TDC activity contained 19 pkat per mg of protein. The influence of transgenic expression oftdc on the activities of anthranilate synthase (AS) and chorismate mutase (CM) were examined in 10 transgenic tobacco plants. The specific activities of these two chorismate-utilizing enzymes were not significantly affected by expression oftdc, despite their important functions as branch point enzymes in the shikimate pathway. The results indicate that the normal route of tryptophan biosynthesis in plants is sufficient to supply a considerable amount of this essential amino acid for the biosynthesis of secondary metabolites. Despite their increased tryptamine content, the growth and development of the transgenic tobacco plants expressingtdc appeared normal.     

Oxygen uptake and citric acid production by Candida lipolytica Y 1095

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L . h, and volumetric oxygen uptake rate, 343 mg O(2)/L . h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell . h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell . h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell . h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L . h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions. (c) 1994 John Wiley & Sons, Inc.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Free tryptophan pool and tryptophan biosynthetic enzymes in Saccharomyces cerevisiae

The free tryptophan pool and the levels of two enzymes of tryptophan biosynthesis (anthranilate synthase and indoleglycerolphosphate synthase) have been determined in a wild type strain of Saccharomyces cerevisiae and in mutants with altered regulatory properties.The tryptophan pool of wild type cells growing in minimal medium is 0.07 mole per g dry weight. Addition of anthranilate, indole or tryptophan to the medium produces a fifteen- to forty-fold increase in tryptophan pool, but causes no repression of the biosynthetic enzymes. Inclusion of 5-methyltryptophan in the growth medium causes a reduction in growth rate and a derepression of the biosynthetic enzymes, and this is shown here not to be correlated with a decrease in the free tryptophan pool.Mutants with an altered anthranilate synthase showing decreased sensitivity to inhibition by l-tryptophan or by the analogue dl-5-methyltryptophan have a tryptophan pool far higher than the wild type strain, but no repression of indoleglycerolphosphate synthase was observed. Mutants with an anthranilate synthase more sensitive to tryptophan inhibition show a slightly reduced tryptophan pool, but no derepression of indoleglycerolphosphate synthase was found.A mutant with constitutively derepressed levels of the biosynthetic enzymes shows a considerably increased tryptophan pool. Addition of 5-methyltryptophan to the growth medium of non-derepressible mutants causes a decrease in growth rate accompanied by a decrease in the tryptophan pool.Abbreviations CDRP 1-(o-carboxyphenylamino)-1-deoxyribulosephosphate - paba paraaminobenzoic acid - PRA N-(5-phosphoribosyl)-anthranilate - tRNA transfer ribonucleic acid; trp1 to trp5 refer to the structural genes for corresponding tryptophan biosynthetic enzymes     

Enzymatic Basis for Overproduction of Tryptophan and Its Metabolites in Hansenula polymorpha Mutants

3-Deoxy-d-arabinoheptulosonate 7-phosphate (DAHP) synthetase and anthranilate synthetase are key regulatory enzymes in the aromatic amino acid biosynthetic pathway. The DAHP synthetase activity of Hansenula polymorpha was subject to additive feedback inhibition by phenylalanine and tyrosine but not by tryptophan. The synthesis of DAHP synthetase in this yeast was not repressed by exogenous aromatic amino acids, singly or in combinations. The activity of anthranilate synthetase was sensitive to feedback inhibition by tryptophan, but exogenous tryptophan did not repress the synthesis of this enzyme. Nevertheless, internal repression of anthranilate synthetase probably exists, since the content of this enzyme in H. polymorpha strain 3-136 was double that in the wild-type and less sensitive 5-fluorotryptophan-resistant strains. The biochemical mechanism for the overproduction of indoles by the 5-fluorotryptophan-resistant mutants was due primarily to a partial desensitization of the anthranilate synthetase of these strains to feedback inhibition by tryptophan. These results support the concept that inhibition of enzyme activities rather than enzyme repression is more important in the regulation of aromatic amino acid biosynthesis in H. polymorpha.     

Control of Aromatic Amino Acid Biosynthesis in Cultured Plant Tissues: Effect of Intermediates and Aromatic Amino Acids on Free Levels

Shikimate, anthranilate, indole, l -tryptophan, phenylpyruvate, l -p henylalanine, p-hydroxyphenylpyruvate or l -tyrosine were added to suspension-cultured Nicotiana tabacum (tabacco) and Daucus carota (carrot) tissues and incubated for 24 hours. Uptake of each compound was substantial as measured by its decrease in the medium. The levels of free tryptophan, phenylalanine and tyrosine were determined in the tissues after the 24 hours incubation. Shikimate did not change the aromatic animo acid levels in carrot tissue, but did increase all three in tobacco (3-fold or more), indicating a less stringent feedback control in tobacco. Anthranilate and indole increased the tissue tryptophan levels in both species by at least 17-fold, showing that the flow from anthranilate and indole to tryptophan was apparently unhindered by enzymatic control mechanisms. When tryptophan levels were elevated in both carrot and tobaccotissues by anthranilate, indole or tryptophan addition, there was also an increase in free phyenylalanine and tyrosine. This might be due to the reversal of phenylalanine and tyrosine feedback inhibition of chorismate mutase by the high tryptophan in the tissue. Chorismate mutase activity in tobacco crude extracts could be inhibited by 66–90% by 1 mM phenylalanine and /or tyrosine. Tryptophan at 1 mM stimulated the enzyme activity by 1/3 and completely reversed the phenylalanine and/or tyrosine inhibition of enzyme activity. Chorsimate mutase activity amino acids under a variety of conditions. Phenylpyruvate increased the phenylalanine levels and p-hydroxyphenylpyruvate increased the tyrosine levels in carrot and tobacco tissues indicating that there was no feedback control of the last step in phenylalanine and tyrosine biosynthesis.     

Studies on the root nodules of leguminous plants: The production of indole acetic acid in culture by a Rhizobium sp. from a leguminous climbing shrub Derris scandens benth

The Rhizobium sp. When isolated form the root nodules of a leguminous climbing shrub Derris scandens produced a high amount of indole acetic acid (IAA) (135.2 μg/ml) from the tryptophan-supple-mented basal medium. Growth and IAA production started simultaneously, and the maximum amount of IAA was produced as a secondary metabolite in the stationary phase of growth. The IAA production by the Rhizobium sp. was increased by 503% when the medium was supplemented with mannitol (2%), KNO₃ (0.2%), nicotinic acid (0.1 μg/ml) and MnSO₄ (1 μg/ml) in addition to tryptophan (4 mg/ml)/ The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is also discussed.     

Production of L-Tryptophan by Sulfonamide-resistant Mutants

Sulfaguanidine-resistant mutant S-225, derived from a tryptophan-producing 5-fluoro-tryptophan-resistant mutant of Brevibacterium flavum, accumulated 19 g/liter of L-tryptophan at maximum when cultured for 72 hr in a medium containing 13% glucose as carbon source, 1.8-fold higher than did the parent. Strain S-225 accumulated 17 g/liter of L-tryptophan in a medium containing 10% sucrose as carbon source (17% yield based on the sugar). It also accumulated 450 mg/liter of chorismate, an intermediate common to the biosyntheses of tryptophan and p-aminobenzoate. The accumulation was 1.7-fold higher than that by the parent, suggesting that the intracellular concentration of chorismate was increased through acquisition of the sulfaguanidine resistance. Sulfaguanidine-resistant mutants were also derived from a tryptophan-producing mutant of Corynebacterium glutamicum. The mutants showed 2.2-fold higher maximum tryptophan production than did the parent.     

Metabolic engineering for improving anthranilate synthesis from glucose in Escherichia coli

Background  

Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies.     

A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.     

A hybrid bioreactor for high density cultivation of plant cell suspensions

Summary A hybrid bioreactor was developed for the production of secondary metabolites from high density cultivation of plant cell suspensions. Some of the advantages of both air-lift and cell-lift by agitation were combined. The addition of a decanting column also made it possble to run a perfusion system for high density culture or to run a two-stage culture efficiently. Cell growth and the production of berberine from Thalictrum rugosum in the hybrid bioreactor are reported in this paper. A cell density up to 31 g/l was obtained by perfusion without any problems in mixing or loss of cell viability and the specific berberine productivity was comparable to that in shake flasks. The maximum berberine concentration was 88 mg/l at 3 weeks operation and declined thereafter.Offprint requests to: D.-I. Kim     

Regulation of the synthesis of enzymes of tryptophan dissimilation in Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus dissimilates tryptophan via the -ketoadipate pathway. The first enzyme, tryptophan oxygenase (l-tryptophan: oxygen oxidoreductase; EC 1.13.1.12), is substrate-induced by tryptophan. The second two enzymes, formamidase (aryl-formylamine amidohydrolase; EC 3.5.1.9) and kynureninase (l-kynurenine hydrolase; EC 3.7.1.3), are induced by the next intermediate, kynurenine. The last enzyme specific to tryptophan dissimilation, anthranilate oxidase, is substrate induced. This inductive pattern is in marked contrast to the extensive coordinacy of enzyme synthesis characteristic of the remainder of the -ketoadipate pathway.