Cloning and expression of the α-amylase gene and oxytetracycline production in Streptomyces rimosus

An 8.4 kb Sau3AI DNA fragment containing the Streptomyces rimosus TM-55 -amylase gene (amy) was ligated to a vector pIJ702, named pCYL01, and cloned into amylase deficient mutant S. lividans M2 (amy ^–). Subcloning study showed that the amy gene was localized in 3.3 kbKpnI-PstI fragment. The molecular weight of the purified -amylases of S. lividans M2/pCYL01 and S. rimosus TM-55 were estimated to be 65.7 kDa. Different sizes of recombinant plasmids carrying the amy gene had been retransferred into the parental strain of S. rimosus TM-55. Among these S. rimosus transformants, TM-55/pCYL01, TM-55/pCYL12 and TM-55/pCYL36 showed amylase activity 1.36- to 2.05-fold at the seventh day (1.61 to 2.42 units vs 1.18 units), and oxytetracycline (OTC) production 2.00- to 2.50-fold at the ninth day (approximate 140 to 170 g ml^–1 vs 72 g ml^–1), higher than that of S. rimosus TM-55 alone, respectively. These results showed that industrial microorganisms could be improved by genetic and metabolic engineering.     

Cloning and expression of a chicken α-amylase gene

We have isolated and sequenced a genomic clone for a pancreatic α-amylase gene (amy) of the chicken (Gallus gallus). The gene is interrupted by nine introns, spans over 4 kb, and encodes a protein (AMY) of 512 aa that is 83% identical to the human pancreatic α-amylase enzyme. Southern blot analysis of chicken DNA revealed two distinct pancreatic amy loci. In addition, we have generated a cDNA from chicken pancreatic RNA corresponding to the coding sequence of the genomic clone. The cDNA was inserted into a yeast expression vector, and the resulting construct used to transform Saccharomyces cerevisiae cells. Transformed yeast cells synthesized and secreted active AMY enzyme, and the gel migration pattern of the α-amylase produced by the yeast cells was identical to that of the native chicken enzyme.     

Gibberellic-acid-regulated expression of α-amylase and six other genes in wheat aleurone layers

The effect of gibberellic acid (GA₃) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA₃-treated tissue. At least one mRNA species declines in relative level in response to GA₃. There is also a GA₃-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA₃-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA₃-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA₃-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA₃-treated tissue has also been cloned.     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

Cloning of α-amylase gene from Schwanniomyces occidentalis and expression in Saccharomyces cerevisiae

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Integration,amplification and expression of the Bacillus licheniformis α-amylase gene in Bacillus subtilis chromosome

We have introduced the α-amylase gene from Bacillus licheniformis (amy gene) in a non-replicative plasmid which can be conveniently integrated and amplified at a specific site of the B. subtilis chromosome. Although we were able to select spontaneous and stable gene amplification of about 20 integrated copies, the amylase secretion remained very low. A DNA fragment presenting a high promoter activity in B. subtilis was therefore inserted upstream from the amy gene coding sequence, leading to a significant increase of amylase production. However, the amplified structures obtained with this construction were found to contain no more than 12 copies of the amy gene and to be rather unstable when cells were grown under non-selective conditions.     

Cloning of α-amylase gene fromSchwanniomyces occidentalis and expression inSaccharomyces cerevisiae

The cloning of α-amylase gene ofS. occidentalis and the construction of starch digestible strain of yeast,S. cerevisiae AS. 2. 1364 with ethanol-tolerance and without auxotrophic markers used in fermentation industry were studied. The yeast/E.coli shuttle plasmid YCEp1 partial library ofS. occidentalis DNA was constructed and α-amylase gene was screened in S.cerevisiae by amylolytic activity. Several transformants with amylolysis were obtained and one of the fusion plasmids had an about 5.0 kb inserted DNA fragment, containing the upstream and downstream sequences of α-amylase gene fromS. occidentalis. It was further confirmed by PCR and sequence determination that this 5.0 kb DNA fragment contains the whole coding sequence of α-amylase. The amylolytic test showed that when this transformant was incubated on plate of YPDS medium containing 1 % glum and 1 % starch at 30°C for 48 h starch degradation zones could be visualized by staining with iodine vapour. α-amylase activity of the culture filtratate is 740–780 mU/mL and PAGE shows that the yeast harboring fusion plasmids efficiently secreted α-amylase into the medium, and the amount of the recombinant α-amylase is more than 12% of the total proteins in the culture filtrate. These results showed that α-amylase gene can be highly expressed and efficiently secreted inS. cerevisiae AS. 2.1364, and the promotor and the terminator of α-amylase gene fromS. occidentalis work well inS. cercvisiac AS. 2.1364.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Evolution of the response patterns to dietary carbohydrates and the developmental differentiation of gene expression of α-amylase in Drosophila

Intraspecific variation of -amylase activity in D. melanogaster and D. immigrans, which is distantly related to D. melanogaster, and interspecific variation of -amylase activity in 18 Drosophila species were examined. The amount of intraspecific variation of -amylase activities measured in terms of coefficient of variation in D. melanogaster and D. immigrans was one-half and one-tenth or less, respectively, of the interspecific variation in 18 Drosophila species. We also surveyed the response patterns of -amylase activity to dietary carbohydrates at the larval and adult stages. The levels of -amylase activity depended on both repression by dietary glucose (glucose repression) and induction by dietary starch (starch induction). In general, our data suggest that glucose repression was conserved among species at both stages while starch induction was mainly observed in larvae, although the degree of the response depended on species. In D. lebanonensis lebanonensis and D. serrata, larvae expressed electrophoretically different -amylase variants (isozymes) from those of adult flies. These results may suggest that the regulatory systems responsible both for the response to environment and developmental expression are different among species in Drosophila. Correspondence to: T. Yamazaki     

Sequence heterogeneity and differential expression of the α-Amy2 gene family in wheat

Summary Within plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec⁺ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an M_r of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.     

Origin and evolution of the Amyrel gene in the α-amylase multigene family of Diptera

Alpha-amylase genes often form multigene families in living organisms. In Diptera, a remote paralog, Amyrel, had been discovered in Drosophila, where this gene is currently used as a population and phylogenetic marker. The putative encoded protein has about 40% divergence with the classical amylases. We have searched the presence of the paralog in other families of Diptera to track its origin and understand its evolution. Amyrel was detected in a number of families of Muscomorpha (Brachycera-Cyclorrapha), suggesting an origin much older than previously thought. It has not been found elsewhere to date, and it is absent from the Anopheles gambiae genome. The intron–exon structures of the genes found so far suggest that the ancestral gene (before the duplication which gave rise to Amyrel) had two introns, and that subsequent, repeated and independent loss of one or both introns occurred in some Muscomorpha families. It seems that the Amyrel protein has experienced specific amino acid substitutions in regions generally well conserved in amylases, raising the possibility of peculiar, functional adaptations of this protein.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

The contribution of different α-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values

The participation of various isoenzymes of α-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes α-AMY1 and α-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of α-AMY1 and α-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 ≥ 7.3) were found these isoenzymes belong to α-amylase or late maturing or α-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of α-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of α-amylase is reduced in grain with a low falling number.     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.