of an isolated nucleic acid comprising คาสเซตต์การแสดงออก. In another example, a คาสเซตต์การแสดงออกแบบรีคอมบิแนนต์ may comprise โพลีนิวคลีโอไทด์ combined in such a way that the โพลีนิวคลีโอไทด์ are extremely unlikely to be found in nature.
instance, restriction sites or plasmid vector sequences manipulated by man may flank or
separate the โปรโมเตอร์ from the second โพลีนิวคลีโอไทด์. One of skill will recognize that โพลีนิวคลีโอไทด์ can be manipulated in many ways and are not limited to the examples above.
In case คำว่า "รีคอมบิแนนต์" is used to specify an organism or cell, e.g. a microorganism, it is used to express that the organism or cell comprises at least one "transgene",
" transgenic" or "รีคอมบิแนนต์" โพลีนิวคลีโอไทด์, which is usually specified later on.
A โพลีนิวคลีโอไทด์ "exogenous to" an individual organism is a โพลีนิวคลีโอไทด์ which is in•
troduced into the organism by any means other than by a sexual cross.
คำว่าs " การเชื่อมแบบที่ทำงานได้" or " ที่เชื่อมในรูปแบบที่ทำงานได้" are generally understood as
meaning an arrangement in which a genetic control sequence, e.g. a โปรโมเตอร์, เอนฮานเซอร์ or terminator, is capable of exerting its function with regard to a โพลีนิวคลีโอไทด์ being ที่เชื่อมในรูปแบบที่ทำงานได้กับ it, for example a โพลีนิวคลีโอไทด์ encoding a โพลีเปปไทด์. Function, in this context, may mean for example control of the การแสดงออก, i.e. transcription and/or translation, of the
nucleic acid sequence. Control, in this context, encompasses for example initiating,
increasing, governing or suppressing the การแสดงออก, i.e. transcription and, if appropriate, translation. Controlling, in turn, may be, for example, tissue- and I or time-specific. It may also be inducible, for example by certain chemicals, stress, pathogens and the like. Preferably, operable linkage is understood as meaning for example the sequential
arrangement of a โปรโมเตอร์, of the nucleic acid sequence to be expressed and, if appropriate, further เอลีเมนต์ตัวควบคุมs such as, for example, a terminator, in such a way that each of the เอลีเมนต์ตัวควบคุม can fulfill its function when the nucleic acid sequence is expressed. การเชื่อมในรูปแบบที่ทำงานได้does not necessarily require a direct linkage in the
chemical sense. Genetic control sequences such as, for example, เอนฮานเซอร์ sequences are
also capable of exerting their function on the target sequence from positions located at a distance to the โพลีนิวคลีโอไทด์, which ถุกเชื่อมในรูปแบบที่ทำงานได้. Preferred arrangements are those
in which the nucleic acid sequence to be expressed is positioned after a sequence acting as โปรโมเตอร์ so that the two sequences are linked covalently to one another. The distance between the โปรโมเตอร์ and the amino acid sequence encoding โพลีนิวคลีโอไทด์ in an
คาสเซตต์การแสดงออก, is preferably less than 200 base pairs, especially preferably less than
100 base pairs, very especially preferably less than 50 base pairs. The skilled worker is familiar with a variety of ways in order to obtain such an คาสเซตต์การแสดงออก However, an คาสเซตต์การแสดงออก may also be constructed in such a way that the nucleic acid sequence to be expressed is brought under the control of an endogenous genetic control element, for
example an endogenous โปรโมเตอร์, for example by means of homologous recombination or else by random insertion. Such constructs are likewise understood as being คาสเซตต์การแสดงออกs for the purposes of the invention.
The term " คาสเซตต์การแสดงออก" means those construct in which the nucleic acid sequence encoding an amino acid sequence to be expressed ถุกเชื่อมในรูปแบบที่ทำงานได้กับ
one genetic control element which enables or regulates its การแสดงออก (i.e. transcription and
I or translation). The การแสดงออก may be, for example, stable or transient, constitutive or inducible.
คำว่าs " express," " expressing," " expressed" and " การแสดงออก" refer to การแสดงออก of a gene product (e.g., a biosynthetic enzyme of a gene of a pathway or
reaction defined and described in this application) at a level that the resulting enzyme
activity of this protein encoded for, or the pathway or reaction that it refers to allows metabolic flux through this pathway or reaction in the organism in which this gene/pathway
is expressed in. The การแสดงออก can be done by genetic alteration of the microorganism that is used as a starting organism. 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 or in a comparable microorganism which has not been altered. 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).
In some embodiments, a microorganism can be physically or environmentally altered to
express a gene product at an increased or lower level relative to level of การแสดงออก of the gene product unaltered microorganism. For example, a microorganism can be treated with, or cultured in the presence of an agent known, or suspected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased. Alternatively, a microorganism can be cultured at a
temperature selected to increase transcription of a particular gene and/or translation of a particular gene product such that transcription and/or translation are enhanced or increased.
คำว่าs " deregulate," " deregulated" and " deregulation" refer to alteration or
modification of at least one gene in a microorganism, wherein the alteration or modification results in increasing efficiency of production of a given compound in the microorganism relative to production in absence of the alteration or modification. In some embodiments, a
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gene that is altered or modified
transport protein, such that the
an enzyme in a biosynthetic pathway, or a
or activity of the biosynthetic enzyme in the
microorganism is altered or modified, or that the transport specificity or efficiency is or modified. In some embodiments, at least one gene that encodes an enzyme in a
biosynthetic pathway, i.e. a โพลีเปปไทด์ bringing about a specific activity in the biosynthetic pathway, is altered or modified such that the level or activity of the enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene.
Deregulation also includes altering the coding region of one or more genes to yield, for example, an enzyme that is feedback resistant or has a higher or lower specific activity.
Also, deregulation further encompasses genetic alteration of genes encoding transcriptional factors (e.g., activators, repressors) which regulate การแสดงออก of genes coding for enzymes or transport proteins. คำว่าs" deregulate," " deregulated" and " deregulation" can further be specified in regard to the kind of deregulation present.
In case the particular activity, is altered or modified such that the level or activity of the
enzyme is enhanced or increased relative to the level in presence of the unaltered or wild type gene, คำว่า" up-regulated" is used. In case particular activity, is altered or modified such that the level or activity of the enzyme is lowered or decreased relative to the level in presence of the unaltered or wild type gene, คำว่า " down-regulated" is used.
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 expres