Regulation of EPS production in Lactobacillus casei LC2W through metabolic engineering.

UNLABELLED: Lactobacillus casei LC2W is an exopolysaccharide(EPS)-producing strain with probiotic effects. The low efficiency and unclear regulation mechanism of EPS biosynthesis have become main constraints for its application in food industry. To investigate the major rate-limiting factors of EPS biosynthesis and to improve its yield, metabolic engineering was applied to this strain. Eight relevant genes related to central metabolism, sugar-nucleotides supply, glycosyltransferase and cofactor engineering were cloned and overexpressed. The results suggested that nox, pfk, rfbB and galT genes were the largest contributors to EPS biosynthesis in this study, which elevated EPS yield by 46·0, 20, 17·4 and 19·6% respectively. Notably, under aerobic condition which was not a suitable condition for lactobacilli to grow in, recombinant strain LC-nox achieved the highest EPS yield of 263·7 mg l(-1) , which was increased by 75% compared to that of the starting strain. The oxygen stress was excluded since the phenomenon was not observed in the control strain under the same condition. Therefore, it was probably that higher NADH oxidase activity led to a decreased NADH availability and reduced lactate concentration, which resulted in the elevation of EPS yield. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributed to the understanding of EPS biosynthesis in Lact. casei through metabolic engineering and provided a starting point for introducing cofactor engineering into this strain. Overexpression of NADH oxidase was found to have a most significant effect on the EPS production. It is the first report that EPS could be accumulated to such a high level under aerobic condition in lactobacilli. Our results provided a novel strategy for the improvement of EPS production in lactic acid bacteria.

Functional analysis of the mouse galactose-1-phosphate uridyl transferase (GALT)promoter.

Galactose-1-phosphate uridyltransferase (GALT) is expressed in most tissues, but the near total absence of catalytic activity in humans with the disease galactosemia leads to specific organ dysfunction, the pathophysiology of which remains an enigma. To characterize the transcriptional regulation of the mouse GALT gene, we isolated and sequenced over 3 kb of a 5'-flanking sequence and functionally characterized the region using in vitro transient transfection and in transgenic mice. A minimal promoter of 145 bp was found to function in both HepG2 cells and NS20Y mouse neuroblastoma cells. The minimal promoter contains regions of homology to the corresponding rat and human GALT genes. In transgenic mice expressing a luciferase transgene under control of a 1.9-kb fragment of the mGALT promoter region, reporter activity was found in most tissues, with higher than expected reporter levels in neonatal brain. To determine if high galactose levels in tissues could induce promoter activity, we bred the mGALT:luciferase transgene into a line of mice in which the GALT gene function has been eliminated by homologous recombination. High tissue levels of galactose and metabolites did not induce reporter activity above background. The studies show that GALT transcriptional regulation is complex and not directly induced by substrate levels.

In vivo and in vitro expression of rat galactose-1-phosphate uridyltransferase (GALT) in the developing central and peripheral nervous system.

Galactose-1-phosphate uridyltransferase (GALT) is a key enzyme in the metabolism of galactose. GALT activates the galactose-glucose interconversion and enables the synthesis of glucose-1-phosphate and UDP-galactose (UDP-Gal). UDP-Gal is the galactosyl donor for the incorporation of galactose into complex oligosaccharides, glycoproteins and glycolipids. The expression of GALT was characterized both in vivo and in vitro during late embryonic and postnatal development of the brain and peripheral nerve of the rat. Assays of GALT mRNA and protein showed that it is weakly expressed during late embryonic development with a second peak of expression concomitant with myelinogenesis. GALT was prominently expressed in myelinating Schwann cells in a rat dorsal root ganglia culture system. GALT deficiency in humans results in galactosemia, a disease characterized by long-term intellectual impairment, and probably dysmyelination. The developmentally regulated pattern of GALT expression during maturation of the nervous system may provide a molecular basis for these neurological complications which seriously compromise the outcome of many galactosemic patients.

Developmental and tissue-specific modulation of rat galactose-1-phosphate uridyltransferase steady state messenger RNA and specific activity levels.

To assess the role of genetic regulation as a modulating factor in the variability of rat tissue galactose-1-phosphate uridyltransferase (GALT) sp act, we have determined steady state GALT mRNA and sp act in rat liver during postnatal development. Steady state GALT mRNA levels increase from birth to d 5 and subsequently decrease toward adult levels. GALT sp act mirrors the mRNA pattern. A survey of steady state mRNA and GALT sp act of several adult rat tissues revealed marked tissue differences with a good correlation of the two parameters. Liver had the highest GALT mRNA and sp act; kidney, ovary, and heart had similar but lower mRNA and sp act; skeletal muscle and testes had the least GALT mRNA and enzyme sp act. These findings suggest that genetic regulation is important in the variable expression of GALT tissue sp act.

Rat galactose-1-phosphate uridyltransferase coding sequence, transcription start site and genomic organization.

Galactosemia is an inborn error of metabolism in humans due to deficient activity of the enzyme galactose-1-phosphate uridyl-transferase (GALT). Using a 1.3 kb Bam HI restriction fragment of the human GALT cDNA as a probe, three cDNAs, 786 bp, 265 bp and 1.4 kb were isolated from a rat liver cDNA library. The total rat GALT coding sequence obtained from these three clones was 1051 nt. Repeat cDNA library screening failed to isolate cDNA clones with additional 5'-coding sequence. Using the 786 bp cDNA as a probe, three genomic clones, RGG1, RGG2, and RGG3 were isolated from a rat genomic library. RGG2 and RGG3 contained additional 5'-coding sequence, missing from the cDNA clones, which was identified by comparison with the human cDNA sequence. The rat GALT sequence, from transcription start site to the polyadenylation tail, is 1254 nt and is divided into 11 exons which span over 3.5 kb of genomic sequence. Primer extension identified the transcription start site as 17 nt 5' to the translation start site. Rat GALT is 379 amino acids long, identical to human GALT, with a derived molecular weight of 43,312 Da similar to the 43,360 Da weight for human GALT. The derived rat amino acid sequence is 90% identical to the human sequence. The isolation of rat GALT genomic sequence will facilitate future investigations of the regulation of expression of the rat GALT gene.

A yeast expression system for human galactose-1-phosphate uridylyltransferase.

Galactose-1-phosphate uridylyltransferase (GALT) (UTP: alpha-D-hexose-1-phosphate uridylyltransferase, EC 2.7.7.10) is an essential enzyme of the Leloir pathway of galactose metabolism. Mutations in human GALT are associated with the potentially lethal disorder galactosemia, which affects 1 in 30,000-60,000 live-born infants. Although a number of base substitutions have been identified in the GALT alleles of galactosemia patients, the detailed biochemical impact of these mutations on GALT enzymatic activity remains obscure. Similarly, little is known about the sequence/structure/function relationships for wild-type human GALT. As a first step toward addressing these questions, we have developed a yeast-based expression system for the human enzyme. The wild-type human GALT coding sequence has been introduced into a strain of Saccharomyces cerevisiae that carries a disruption of the GALT-encoding GAL7 gene and, therefore, expresses no endogenous GALT. Transformants were tested for restoration of GALT activity both indirectly, by cell growth on galactose, and directly, by analysis of enzyme activity in cell extracts. The results of both tests were striking; wild-type human GALT functioned in yeast almost as well as the endogenous enzyme. In contrast, cells transformed with either human or yeast GALT sequences engineered to carry a common human GALT mutation, Q188R (changing Gln188 to Arg), exhibited essentially no detectable GALT activity and failed to grow on galactose. Lymphoblasts from patients homozygous for the Q188R mutation similarly exhibited essentially no detectable GALT activity in parallel assays. The results reported here establish the utility of the yeast-based expression system for human GALT and set the stage for more detailed studies of this important enzyme and its role in galactosemia.

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.

[Use of neonatal induction of enzymes for the correction of experimental hereditary enzymopathies]. / Korrektsiia nasledstvennykh fermentopatii v éksperimente putem neonatal'noi induktsii fermentov.

Administration of enzyme-inducing agents into newborn animals resulted in stable changes of the activities of the relevant inducible enzymes for long periods of their life in adulthood. The phenomenon designated as enzymic imprinting was used for correction of inherited enzymopathies in animals. Neonatal administration of galactose into the W/ssm rats with inherited galactosemia stably decreased galactose transport into the erythrocytes, increased activities of hexose oxidizing enzymes and prevented development of cataracts and other galactosemia symptoms. Neonatal administration of the inducer of mixed function oxidases into the SWR/J mice with inherited hypercholesterolemia stably increased the activities of these cholesterol oxidizing enzymes and abolished the hypercholesterolemia symptoms in adulthood. It is suggested that enzymic imprinting is due to amplification of genes coding the inducible enzymes on account of preferential copying of the induced mRNA's by reverse transcription. It is shown that the enzymic induction was accompanied by activation of RNA dependent DNA synthesis and the activity of this enzymatic system was distinctly higher in newborn animals.

[Correction of the symptoms of hereditary galactosemia in W/SSM strain rats by enzymatic imprinting]. / Korrektsiia simptomov nasledstvennoi galaktozemii u krys linii W/SSM putem fermentativnogo imprintinga.

It was recently shown in this laboratory that treatment of newborn animals with certain enzymic inducers causes stable changes in the activities of the inducible enzymes at a later adult stage. Cataracts, hepato-splenomegaly and other galactosemia symptoms in galactosemic W/SSM rats develop spontaneously. The increased uptake of galactose by erythrocytes, but not the decreased level of galactose-1-phosphate uridyl transferase (Gal-1-PUT) activity was assumed to be the major cause of the disease. The administration of galactose to the newborn W/SSM rats (2 mg/g of body weight for 14 days) resulted in a sustained decline in the uptake of 14C-galactose by erythrocytes at least for five months, in an increase of glucoso-6-phosphate dehydrogenase activity and in a continuous fall of Gal-1-PUT activity. The neonatal treatment of the galactosemic rats with galactose abolished the main symptoms of galactosemia (cararacts, hepato-splenomegally) in adult animals, perhaps ar a consequence of the stable changes in the galactose metabolism.

Escherichia coli gal operon proteins made after prophage lambda induction.

Expression of the EScherichia coli gal operon under the control of the prophage lambda promoter pL leads to gross discoordinacy of gal expression. Expression of the most promoter-distal cistron galK is much greater than expression of the promoter-proximal cistron galE. We had previously shown that transcription of the gal operon is coordinate after prophage induction. A survey of protein synthesized after prophage induction indicated that lack of expression of galE is due to a failure of translation of the galE sequence in the pL-gal transcript. This failure of translation of the galE sequence may be due to extensive dyad symmetry present in the vicinity of the gal promoter region of the pL-gal transcript. This symmetry could result in a ribonucleic acid stem-loop structure, blocking the attachment of ribosomes at the Shine-Dalgarno sequence of galE. To test this model, strains bearing the IS1 or IS2 insertion, deletion, or new promoter mutation within the symmetrical region were constructed. The restoration of some galE expression after such disruptions of the symmetrical region indicated that the ribonucleic acid stem-loop structure did play a role in the discoordinate expression of gal from pL. However, failure to obtain galE expression coordinated with high levels of galK expression suggested that other components were involved, perhaps other symmetries between galE and the pL transcript.

Post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA in Saccharomyces cerevisiae.

Thermal elution poly(U)-Sepharose chromatography was utilized to fractionate yeast mRNA based on poly(A) size. Analysis of the in vitro translation products of the fractionated RNAs in a wheat-embryo cell-free protein synthesis system shows a heterogeneous but equal distribution of these abundant translatable mRNAs in the different poly(A) size classes. By comparing the translational activity of inducible galactose-1-phosphate uridyl transferase mRNA, which can be monitored as a function of age, to contitutive mRNAs, we demonstrate that initially galactose-1-phosphate uridyl transferase mRNA has a uniformly large poly(A) tail which becomes heterogeneous and shorter with age in the cytoplasm. These observations are consistent with the previously observed cytoplasmic poly(A) catabolism in yeast and with cytoplasmic post-transcriptional modification of the poly(A) length of galactose-1-phosphate uridyl transferase mRNA.

Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway.

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.

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.

CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli.

Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so.