The term " deregulated" includes การแสดงออก of a gene product at a level lower or higher than that expressed prior to manipulation of the microorganism or in a comparable microorganism which has not been manipulated. In one embodiment, the microorganism can be genetically manipulated (e.g., genetically engineered) to express a level of gene product at a lesser or higher level than that expressed prior to manipulation of the
microorganism or in a comparable microorganism which has not been manipulated. Genetic manipulation can include, but is not limited to, altering or modifying regulatory sequences or sites associated with การแสดงออก of a particular gene (e.g., by removing strong โปรโมเตอร์, inducible โปรโมเตอร์ or multiple โปรโมเตอร์), modifying the chromosomal location of a particular gene, altering nucleic acid sequences adjacent to a particular gene such as a
ribosome binding site or transcription terminator, decreasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, เอนฮานเซอร์s, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating การแสดงออก of a particular gene routine in the art (including but not limited to use of antisense nucleic acid
molecules, or other methods to knock-out or block การแสดงออก of the target protein).
คำว่า " deregulated gene activity" also means that a gene activity is introduced into a microorganism where the respective gene activity, has not been observed before, e.g. by introducing a รีคอมบิแนนต์ gene, e.g. a เฮเทอโรโลกัส gene, in one or more copies into the
microorganism preferably by means of genetic engineering.
The phrase " deregulated pathway or reaction" refers to a biosynthetic pathway or reaction in which at least one gene that encodes an enzyme in a biosynthetic pathway or reaction is altered or modified such that the level or activity of at least one biosynthetic enzyme is altered or modified. The phrase" deregulated pathway" includes a biosynthetic pathway in which more than one gene has been altered or modified, thereby altering level and/or activity of the corresponding gene products/enzymes. In some cases
the ability to" deregulate" a pathway (e.g., to simultaneously deregulate more than one gene in a given biosynthetic pathway) in a microorganism arises from the particular phenomenon of microorganisms in which more than one enzyme (e.g., two or three biosynthetic enzymes) are encoded by genes occurring adjacent to one another on a contiguous piece of genetic material termed a " cluster" or" gene cluster" In other
cases, in order to deregulate a pathway, a number of genes must be deregulated in a series of sequential engineering steps.
To express the deregulated genes according to the invention, the DNA sequence encoding the โพลีเปปไทด์ must be ที่เชื่อมในรูปแบบที่ทำงานได้กับ regulatory sequences that control transcriptional
การแสดงออก in an การแสดงออก vector and then, introduced into either microorganism. In addition to transcriptional regulatory sequences, such as โปรโมเตอร์ and เอนฮานเซอร์s, การแสดงออก vectors can include translational regulatory sequences and a marker gene which is suitable for selection of cells that carry the การแสดงออก vector.
คำว่าs u overexpress" , " overexpressing" , " overexpressed" and
" overการแสดงออก" refer to การแสดงออก of a gene product, in particular to enhancing the การแสดงออก of a gene product at a level greater than that present prior to a genetic alteration of the starting microorganism. In some embodiments, a microorganism can be genetically altered (e.g., genetically engineered) to express a gene product at an increased
level relative to that produced by the starting microorganism. Genetic alteration includes, but is not limited to, altering or modifying regulatory sequences or sites associated with การแสดงออก of a particular gene (e.g., by adding strong โปรโมเตอร์, inducible โปรโมเตอร์ or multiple โปรโมเตอร์ or by removing regulatory sequences such that การแสดงออก is constitutive), modifying the chromosomal location of a particular gene, altering nucleic acid
sequences adjacent to a particular gene such as a ribosome binding site or transcription terminator, increasing the copy number of a particular gene, modifying proteins (e.g., regulatory proteins, suppressors, เอนฮานเซอร์s, transcriptional activators and the like) involved in transcription of a particular gene and/or translation of a particular gene product, or any other conventional means of deregulating การแสดงออก of a particular gene using routine in
the art (including but not limited to use of antisense nucleic acid molecules, for example, to block การแสดงออก of repressor proteins). Another way to overexpress a gene product is to enhance the stability of the gene product to increase its life time.