D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC

d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."     

Galactose transport in Saccharomyces cerevisiae. I. Nonmetabolized sugars as substrates and inducers of the galactose transport system

The inducible galactose transport system in bakers' yeast carries out the facilitated diffusion of the nonmetabolized galactose analogues d-fucose and l-arabinose. This capacity depends on the activity of the Ga 2 gene. In some strains, d-fucose and l-arabinose are also gratuitous inducers. Mutants in which the inducibility of the galactose pathway enzymes is altered show a parallel alteration of the inducibility of the galactose transport system.     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Effect of Galactose on β-Galactosidase Synthesis in Escherichia coli K-12

In wild-type strains of Escherichia coli K-12, the rate of thiomethylgalactoside (TMG)-induced beta-galactosidase synthesis is decreased in the presence of galactose or glucose. A spontaneous mutant of a K-12 strain, 58-161, which synthesizes beta-galactosidase at a low rate was isolated. In this mutant, galactose, after a lag of about one generation time, evoked the same final differential rate of enzyme synthesis as did the gratuitous inducer TMG. However, constitutive, TMG-induced and galactose-induced synthesis in the mutant were subject to inhibition by exogenous glucose. It is concluded that repression of beta-galactosidase synthesis derived from glucose is distinct from the inhibition derived from galactose.     

Galactose transport in Saccharomyces cerevisiae. II. Characteristics of galactose uptake and exchange in galactokinaseless cells

The characteristics of the inducible galactose system in Saccharomyces cerevisiae were studied by using the nonmetabolized galactose analogues, l-arabinose and d-fucose, and galactokinaseless and transportless mutants. Induced wild-type cells transport l-arabinose by facilitated diffusion. Transportless cells transport neither galactose nor l-arabinose above the noninduced rate, whereas galactokinaseless cells transport galactose l-arabinose and d-fucose by facilitated diffusion. Determination of unidirectional rate of (14)C-labeled galactose uptake by preloaded galactokinaseless cells, containing a large unlabeled free-galactose pool, showed that the rate of galactose uptake by facilitated diffusion is greater than the rate of galactose metabolism at similar external galactose concentrations.     

L-Arabinose is not a gratuitous inducer of α-galactosidase from Saccharomyces carlsbergensis

L-Arabinose has been described as a gratuitous inducer of the yeast -galactosidase. This has been found to be an artefact resulting from galactose contamination of commercial samples of L-arabinose. The inactivation produced on UDP-glucose 4-epimerase by the pentose does not amplify the inducer activity of contaminating D-galactose.     

Catabolite inhibition: a general phenomenon in the control of carbohydrate utilization

When Escherichia coli is grown in synthetic medium with radioactive galactose or lactose as the carbon source, the addition of glucose rapidly inhibited utilization of the radioactive substrate, whether the formation of (14)CO(2) or acid-insoluble products was measured. The inhibition was reversed after the removal of glucose. Experiments with mutants blocked in subsequent steps of galactose and lactose metabolism demonstrated that the inhibition occurs prior to the formation of the first metabolic product. The utilization of a variety of sugars, including maltose, lactose, mannose, galactose, l-arabinose, xylose, and glycerol was inhibited by glucose. Of a number of carbohydrates tested as potential inhibitors, only glucose and, to a lesser extent, glucose-6-phosphate (G-6-P) were capable of inhibiting the utilization of all of the substrates. Glucose did not inhibit G-6-P utilization but G-6-P inhibited glucose utilization. With all substrates, except glycerol, there was a delay before the onset of inhibition by G-6-P. We conclude that E. coli has a general regulatory mechanism, termed catabolite inhibition, which controls the activity of early reactions in carbohydrate metabolism, allowing certain substrates to be utilized preferentially.     

Regulation of the L-arabinose catabolic pathway in Aerobacter aerogenes

Three enzymes of the l-arabinose catabolic pathway in Aerobacter aerogenes, l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, are specifically induced in the presence of l-arabinose. Mutants constitutive for kinase activity are also constitutive for the isomerase and 4-epimerase activities, suggesting that these three enzymes are coordinately controlled in A. aerogenes. l-Ribulokinase activity can still be induced in the presence of l-arabinose in an isomerase-deficient strain of A. aerogenes. Since l-arabinose is not converted to l-ribulose in such a strain, it appears that l-arabinose must be the inducer of l-ribulokinase, as well as the coordinately controlled isomerase and 4-epimerase. As in the metabolism of l-arabinose, growth of A. aerogenes on l-arabitol also requires a 4-epimerase for the conversion of l-ribulose-5-phosphate to d-xylulose-5-phosphate. However, loss of ability to metabolize l-arabinose, due to a deficiency in 4-epimerase synthesis in the presence of l-arabinose, does not affect growth on l-arabitol. In addition, synthesis of the 4-epimerase associated with l-arabitol metabolism is not accompanied by l-arabinose isomerase or l-ribulokinase synthesis. These results suggest either the existence of two different l-ribulose-5-phosphate 4-epimerases in A. aerogenes, or of two different regulatory mechanisms for the control of the same epimerase.     

Genetic co-regulation of galactose and melibiose utilization in Saccharomyces.

The gal3 mutation of Saccharomyces, which is associated with an impairment in the utilization of galactose, has been shown to be pleiotropic, causing similar impairments in the utilization of melibiose and maltose. Milibiose utilization and alpha-galactosidase production are directly controlled by the galactose regulatory elements i, c, and GAL4. The fermentation of maltose and the induction of alpha-glucosidase are regulated independently of the i, c, GAL4 system. The production of alpha-galactosidase and galactose-1-phosphate uridyl transferase is coordinate in galactokinaseless strains. Galactose serves as a nonmetabolized, gratuitous inducer of alpha-galactosidase in strains lacking the genes for one or more of the Leloir pathway enzymes.     

Application of a gratuitous induction system in Kluyveromyces lactis for the expression of intracellular and secreted proteins during fed-batch culture

A gratuitous induction system in the yeast Kluyveromyces lactis was evaluated for the expression of intracellular and extracellular products during fed-batch culture. The Escherichia coli lacZ gene (beta-galactosidase; intracellular) and MFalpha1 leader-BPTI cassette (bovine pancreatic trypsin inhibitor; extracellular) were placed under the control of the inducible K. lactis LAC4 promotor, inserted into partial-pKD1 plasmids, and transformed into a ga1-209 K. lactis strain. To obtain a high level of production, culture conditions for growth and expression were initially evaluated in tube cultures. A selective medium containing 5 g/L glucose (as carbon source) and 0.5 g/L galactose (as inducer) demonstrated the maximum activity of both beta-galactosidase and secreted BPTI. This level of expression had no significant effect on the growth of the recombinant cells; growth rate dropped by approximately 11%, whereas final biomass concentrations remained the same. In shake-flask culture, biomass concentration, beta-galactosidase activity, and BPTI secreted activity were 4 g/L, 7664 U/g dry cell, and 0.32 mg/L, respectively. Fed-batch culture (with a high glucose concentration and a low galactose [inducer] concentration feed) resulted in a 6.5-fold increase in biomass, a 23-fold increase in beta-galactosidase activity, and a 3-fold increase in BPTI secreted activity. The results demonstrate the success of gratuitous induction during high-cell-density fed-batch culture of K. lactis. A very low concentration of galactose feed was sufficient for a high production level.     

The DeLey-Doudoroff Pathway of Galactose Metabolism in Azotobacter vinelandii

Azotobacter vinelandii cell extracts reduced NAD and oxidized d-galactose to galactonate that subsequently was converted to 2-keto-3-deoxy-galactonate. Further metabolism of 2-keto-3-deoxy-galactonate required the presence of ATP and resulted in the formation of pyruvate and glyceraldehyde 3-P. Radiorespirometry indicated a preferential release of CO(2) at the first carbon position of the d-galactose molecule. This suggested that Azotobacter vinelandii metabolizes d-galactose via the DeLey-Doudoroff pathway. The first enzyme of this pathway, d-galactose dehydrogenase, was partially characterized. It has a molecular weight of about 74,000 Da and an isoelectric point of 6.15. The pH optimum of the galactose dehydrogenase was about 9. The apparent K(m)s for NAD and d-galactose were 0.125 and 0.56 mM, respectively. Besides d-galactose, the active fraction of this galactose dehydrogenase also oxidized l-arabinose effectively. The electron acceptor for d-galactose or l-arabinose oxidation, NAD, could not be replaced by NADP. These substrate specificities were different from those reported in Pseudomonas saccharophila, Pseudomonas fluorescens, and Rhizobium meliloti.     

Recombinant protein production in high cell density cultures of Escherichia coli with galactose as a gratuitous inducer

A new expression system was developed by introducing two major modifications into the genome of Escherichia coli: a deletion in the gal operon (DeltagalEKT) to allow the use of the inexpensive compound galactose as a gratuitous inducer and the introduction of the gal P2 promoter driving the expression of the T7 RNA polymerase. The novel JRR10 strain containing these two features gives high-level expression of a reporter gene cloned under the T7 phi10 promoter in high cell density cultures. The cost of the induction of this novel system is more than 30 times lower than that of the IPTG-induced system of the widely used BL21 strain.     

Differential selectivity of the Escherichia coli cell membrane shifts the equilibrium for the enzyme-catalyzed isomerization of galactose to tagatose

An Escherichia coli galactose kinase gene knockout (DeltagalK) strain, which contains the l-arabinose isomerase gene (araA) to isomerize d-galactose to d-tagatose, showed a high conversion yield of tagatose compared with the original galK strain because galactose was not metabolized by endogenous galactose kinase. In whole cells of the DeltagalK strain, the isomerase-catalyzed reaction exhibited an equilibrium shift toward tagatose, producing a tagatose fraction of 68% at 37 degrees C, whereas the purified l-arabinose isomerase gave a tagatose equilibrium fraction of 36%. These equilibrium fractions are close to those predicted from the measured equilibrium constants of the isomerization reaction catalyzed in whole cells and by the purified enzyme. The equilibrium shift in these cells resulted from the higher uptake and lower release rates for galactose, which is a common sugar substrate, than for tagatose, which is a rare sugar product. A DeltamglB mutant had decreased uptake rates for galactose and tagatose, indicating that a methylgalactoside transport system, MglABC, is the primary contributing transporter for the sugars. In the present study, whole-cell conversion using differential selectivity of the cell membrane was proposed as a method for shifting the equilibrium in sugar isomerization reactions.     

Production of recombinant hirudin in galactokinase-deficientSaccharomyces cerevisiae by fed-batch fermentation with continuous glucose feeding

The artificial gene coding for anticoagulant hirudin was placed under the control of theGAL10 promoter and expressed in the galactokinase-deficient strain (Δgal1) ofSaccharomyces cereivisiae, which uses galactose only as a gratuitous inducer in order to avoid its consumption. For efficient production of recombinant hirudin, a carbon source other than galactose should be provided in the medium to support growth of the Δgal1 strain. Here we demonstrate the successful use of glucose in the fed-batch fermentation of the Δgal1 strain to achieve efficient production of recombinant hirudin, with a yield of up to 400 mg hirudin/L.     

Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport

Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols. Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92:601-608. 1966.-The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization ("glucose effect") was studied in Escherichia coli. Experiments on the uptake into the cell of labeled inducers (d-galactose-C(14) and d-fucose-H(3)) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport inhibition model was provided by the finding that glucose did not substantially inhibit induction of the gal enzymes when glucose and galactose were produced intracellularly by beta-galactosidase hydrolysis of lactose, even if excess glucose was added. The inducer uptake experiments also showed that d-galactose and d-fucose probably enter the cell via different transport systems, although uptake of both compounds was inhibited by glucose.     

Induction of Nonpigmented Variants of Erwinia herbicola by Incubation at Supraoptimal Temperatures

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.     

Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate.

The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp. ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes. The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate. 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms. In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism. Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist.     

Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects

Although the physiology and metabolism of the growth of yeast strains has been extensively studied, many questions remain unanswered where the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. The strain is able to metabolise the inducer, galactose, which is a much more expensive carbon source than glucose. Both the transport of galactose into the cell-required for the induction of cutinase production-and galactose metabolism are highly repressed by glucose. Different fermentation strategies were tested and the culture behaviour was interpreted in view of the strain metabolism and physiology. A fed-batch fermentation with a mixed feed of glucose and galactose was carried out, during which simultaneous consumption of both hexoses was achieved, as long as the glucose concentration in the medium did not exceed 0.20 g/l. The costs, in terms of hexoses, incurred with this fermentation strategy were reduced to 23% of those resulting from a fermentation carried out using a more conventional strategy, namely a fed-batch fermentation with a feed of galactose.     

Urea transport in Saccharomyces cerevisiae.

Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.