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.     

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.     

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic alpha-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable alpha-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of alpha-amylase was observed at 95 degrees C. The half-life of the enzyme was 5 h at 90 degrees C. Over 40% and 30% of the enzyme activity remained after incubation at 100 degrees C for 2 and 3 h, respectively. The enzyme did not require Ca(2+) for thermostability. This alpha-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 alpha-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Parametric analysis of metabolic fluxes of α-amylase and protease-producing Bacillus subtilis

Parametric analysis was applied for a metabolic flux model for the fed-batch culture of Bacillus subtilis producing recombinant α-amylase and protease. The metabolic flux model was formulated as a linear programming problem consisting of 49 reactions (decision variables) and 50 metabolites (equality constraints). This study was aimed to determine the response of the metabolic fluxes and objective function value of minimizing the difference between ATP consumption and ATP production (ATP balance). With regard to intracellular metabolite accumulation, the objective function value was least sensitive to variation in succinate and most sensitive to variation in malate. Amongst the variations in the accumulation rates of extracellular metabolites, the objective function value was least sensitive to variation in glutamate and most sensitive to variation in starch hydrolysis and triglyceride synthesis. A 10% variation in metabolite accumulation rates caused a maximum of 13.8% variation (standard error = 3.8%) in the objective function value.     

Construction and characterization of new corynebacterial plasmids carrying the α-amylase gene

Novel corynebacterial plasmids carrying α-amylase gene fromBacillus have been constructed. The level of α-amylase expression depends on the size of the vector. The highest expression levels were measured in brevibacteria harboring pA61 plasmid.     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

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.     

5′ Regulatory region of ubiquitin 2 gene from Porteresia coarctata makes efficient promoters for transgene expression in monocots and dicots

Key message

Porteresia ubiquitin 5′ regulatory region drives transgene expression in monocots and dicots.

Abstract

Ubiquitin promoters are promising candidates for constitutive transgene expression in plants. In this study, we isolated and characterized a novel 5′ regulatory sequence of a ubiquitin gene from Porteresia coarctata, a stress-tolerant wild grass species. Through functional analysis in heterologous plant systems, we have demonstrated that full length (Port Ubi2.3) or truncated sequence (PD2) of the isolated regulatory fragment can drive constitutive expression of GUS in monocots and/or dicots. In silico analysis of Port Ubi2.3 has revealed the presence of a 640 bp core promoter region followed by two exons and two introns with numerous putative cis-acting sites scattered throughout the regulatory region. Transformation and expression studies of six different deletion constructs in rice, tobacco and sugarcane revealed that the proximal intron has an enhancing effect on the activity of the core promoter in both monocots and dicots, whereas, Port Ubi2.3 was able to render strong expression only in monocots. This regulatory sequence is quite distinct from the other reported ubiquitin promoters in structure and performs better in monocots compared to other commonly used promoters—maize Ubi1 and Cauliflower Mosaic Virus 35S.     

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

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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.     

Comparative and evolutionary analysis of the rhesus macaque extended MHC class II region

The sequence-based map of a part of the rhesus macaque major histocompatibility complex (MHC) extended class II region is presented. The sequenced region encompasses 67,401 bp and contains the SACM2L, RING1, FABGL and KE4 genes, as well as the HTATSF1-like and ZNF-like pseudogenes. Similar to human, but different from rat and mouse, no class I genes are found in the SACM2L- RING1 interval. The rhesus macaque extended MHC class II region shows a high degree of conservation of exonic as well as intronic and intergenic sequences compared with the respective human region. It is concluded that this particular genomic organization of the extended class II region-i.e., the absence of class I genes and the presence of the HTATSF1-like and ZNF-like pseudogenes-can be traced back to a common ancestor of humans and rhesus macaques about 23 million years ago.     

Sugar sensing and α-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) α-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the α-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the α-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. Received: 22 April 1997 / Accepted: 9 September 1997     

Isolation and properties of sorghum α-amylase

α-Amylase was purified and prepared in good yield from sorghum malt by procedures based on ultracentrifugation, salt fractionation, and adsorption on starch granules. The purified enzyme had a specific activity comparable to crystalline barley α-amylase. The enzyme had a pH optimum of 4.6 and was inactivated at pH's below 3.5. It exhibited a minimum solubility in ammonium sulfate solutions at pH 5.8 and required Ca⁺⁺ ions for stabilization.     

Expression of mouse α-amylase gene in methylotrophic yeastPichia pastoris

The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9, yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS ₄ locus (38 of Mut⁺ clone) or into theAOX1 locus (45 of Mut^s clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity, 290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of the recombinantS. cerevisiae.     

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.