The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform.
An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved. This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.
Wartmann
T
T
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, 06466 Gatersleben, Germany.
Bellebna
C
C
Böer
E
E
Bartelsen
O
O
Gellissen
G
G
Kunze
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D004274:DNA, Recombinant; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D023061:Gene Order; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006657:Histones / Q000737:chemistry / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D020161:Physical Chromosome Mapping; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012709:Serum Albumin / Q000378:metabolism
D004271:DNA, Fungal; D004274:DNA, Recombinant; D005656:Fungal Proteins; D006657:Histones; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D049452:Green Fluorescent Proteins