High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37–63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.     

Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene （cbh1） promoter optimization

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene （cbhl） promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbhl promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbhl promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase （gus） reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.     

Cloning and nucleotide sequence of the gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus

The putative gene coding for a subunit of the respiratory NADH dehydrogenase from Bacillus stearothermophilus was cloned in Escherichia coli and the nucleotide sequence was determined. A large open reading frame (ORF1) was recognized, which was composed of 879 bp corresponding to 293 amino acids and a molecular weight of 33,600. Possible promoter and Shine-Dalgarno sequences were found upstream from the initiation codon. The deduced amino acid sequence of the gene was homologous to the NADH dehydrogenase of Paramecium aurelia.     

Identification of functional DNA variants in the constitutive promoter region of MDM2

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (?1494?G?>?A; indel 40?bp; and ?182?C?>?G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40?bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.     

Analysis of the promoter region of the RuBisCO gene of Arthrospira (Spirulina) platensis

Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), a key enzyme involved with photosynthetic carbon assimilation, catalyzes the carboxylation and oxidization of ribulose-1,5-bisphosphate. Interestingly, the promoter region of this gene from Arthrospira platensis could drive the expression of a downstream gene in Escherichia coli. In this study, using green fluorescent protein as a reporter of gene expression, the structure and function of the promoter region of the RuBisCO gene of A. platensis was analyzed. There are three hypothetical promoter elements predicted in the 200 bp upstream of the open reading frame (ORF) of RuBisCO. Through deletion analysis of the promoter, it was demonstrated that one of these elements was the active promoter, which was located between ?94 and the ORF of RuBisCO. The ?35 box of the RuBisCO promoter (TTGACT) was very similar to that of the rpoD gene (TTGACA) of E. coli, with only the sixth nucleotide divergent. Site-directed mutational analysis showed that when the sixth nucleotide (T) was changed to A, the activity of the promoter remained unchanged. However, when the first and second Ts were mutated, the activities of the promoters decreased drastically. Determining the structure and function of promoters would help elucidate the molecular mechanisms of the gene expression and regulation.