Abstract: A method of performing high-throughput screening of various enzymatic activities with high sensitivity using artificial genetic circuits is provided. Particularly, the invention is screening and quantifying the activity of isoprene biosynthesis enzymes using an artificial genetic circuit capable of sensing isoprene. The artificial genetic circuit comprises an isoprene-sensing transcriptional regulator which recognizes isoprene, at least one reporter gene, a isoprene-sensing transcriptional regulator binding region and promoters for genes encoding isoprene-sensing transcriptional regulators and reporter proteins. The artificial genetic circuit detects isoprene liberated from many enzymatic reactions and measures the activity of reporter genes. This system is widely applicable for high throughput and quantitative screening of isoprene biosynthesis enzymes and MEP/MVA pathway enzymes. Therefore, the invention can be advantageously used in the protein engineering technology for enzyme modification.

Abstract: A method of performing high-throughput screening of various enzymatic activities with high sensitivity using artificial genetic circuits is provided. Particularly, the invention is screening and quantifying the activity of isoprene biosynthesis enzymes using an artificial genetic circuit capable of sensing isoprene. The artificial genetic circuit comprises an isoprene-sensing transcriptional regulator which recognizes isoprene, at least one reporter gene, a isoprene-sensing transcriptional regulator binding region and promoters for genes encoding isoprene-sensing transcriptional regulators and reporter proteins. The artificial genetic circuit detects isoprene liberated from many enzymatic reactions and measures the activity of reporter genes. This system is widely applicable for high throughput and quantitative screening of isoprene biosynthesis enzymes and MEP/MVA pathway enzymes. Therefore, the invention can be advantageously used in the protein engineering technology for enzyme modification.

Abstract: Disclosed are Hansenula polymorpha mutants useful as host cells through which various proteins can be produced as being intact at high yield and a process for preparing recombinant proteins using the host cells. Using various vectors, Hansenula polymorpha is made to be a mutant which is deprived of methanol assimilating ability and incapable of utilizing methanol as a carbon source. This Hansenula polymorpha mutant is used as a high yield host to produce recombinant proteins without continuous feeding of methanol, with the aid of an expression cassette carrying a promoter capable of inducing the expression at a low concentration of methanol. Further, the mutant is also lacking in carboxypeptidase Y, protease Y and/or carboxypeptidase a activity, so the recombinant protein of interest is not degraded at its carboxyl terminal when being expressed in the cell. Thus, intact recombinant protein can be obtained.

Abstract: Disclosed are Hansenula polymorpha mutants useful as host cells through which various proteins can be produced as being intact at high yield and a process for preparing recombinant proteins using the host cells. Using various vectors, Hansenula polymorpha is made to be a mutant which is deprived of methanol assimilating ability and incapable of utilizing methanol as a carbon source. This Hansenula polymorpha mutant is used as a high yield host to produce recombinant proteins without continuous feeding of methanol, with the aid of an expression at a cassette carrying a promoter capable of inducing the expression at a low concentration of methanol. Further, the mutant is also lacking in carboxypeptidase Y, protease Y and/or carboxypeptidase &agr; activity, so the recombinant protein of interest is not degraded at its carboxyl terminal when being expressed in the cell. Thus, intact recombinant protein can be obtained.

Abstract: Disclosed are Hansenula polymorpha mutants useful as host cells through which various proteins can be produced as being intact at high yield and a process for preparing recombinant proteins using the host cells. Using various vectors, Hansenula polymorpha is made to be a mutant which is deprived of methanol assimilating ability and incapable of utilizing methanol as a carbon source. This Hansenula polymorpha mutant is used as a high yield host to produce recombinant proteins without continuous feeding of methanol, with the aid of an expression cassette carrying a promoter capable of inducing the expression at a low concentration of methanol. Further, the mutant is also lacking in carboxypeptidase Y, protease Y and/or carboxypeptidase a activity, so the recombinant protein of interest is not degraded at its carboxyl terminal when being expressed in the cell. Thus, intact recombinant protein can be obtained.