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Escherichia coli
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
Hisashi Yasueda, Hiroshi Matsui
One of the historically important promoters is that from the lac operon. In 1979, Goeddel et al. [5] succeeded in the direct expression of human growth hormone (hGH) using the lac promoter. Transcription of the lac operon is negatively regulated by the lac repressor, the lad gene product, and is also positively regulated by the catabolite activator protein (CAP) complexed with cAMP. The typical inducer of lacP is isopropyl-β-d-thiogalactoside (IPTG). A more popular promoter for expression vectors is the lacUV5 promoter, which has an up-promoter mutation. However, in this system it is often necessary to incorporate the laclq mutation, which expresses higher levels of the repressor, into the expression system, since the overproduced repressor can prevent its titration by operators on multicopy plasmids and can tightly regulate transcription from the promoter [6].
Molecular Machines
Published in Thomas M. Nordlund, Peter M. Hoffmann, Quantitative Understanding of Biosystems, 2019
Thomas M. Nordlund, Peter M. Hoffmann
Before attacking the CAP–DNA bending issue, let’s pause to consider the overall cellular task that is being addressed by CAP. When E. coli encounters increased or decreased levels of nutrient (e.g., glucose), rapid adjustment of enzyme concentrations are made. Within a few minutes of a glucose increase, E. coli will have eliminated production of enzymes used to cleave larger carbohydrates into smaller and increased production of other enzymes, including those needed to repress production of the aforementioned enzymes. Eukaryotic cells typically require hours to days to make similar adjustments, but eukaryotic cells enjoy a much more stable environment. A specific 26-base pair DNA sequence is recognized by the lac repressor protein. If lac repressor binds to this sequence, transcription of the LacZ-coding sequence will be inhibited. Lac repressor thus has a task search illustrated in Figure 16.14. Does lac repressor act as a linear track search agent or as a diffusion searcher, binding more strongly to the target site than to others? Direct molecular motor measurements have not answered this question, but classic chemical binding experiments strongly suggest the answer.
Transport of Nutrients and Carbon Catabolite Repression for the Selective Carbon Sources
Published in Kazuyuki Shimizu, Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production, 2017
The E. coli lac operon is only expressed if allolactose (a lactose isomer formed by ^-galactosidase) binds and inactivates the lac repressor. Lactose cannot be transported into the cell in the presence of glucose, because the lactose permease, LacY is inactive in the presence of glucose (Winler and Wilson 1967). As shown in Fig. 9, phosphorylated EIIAGlc is dominant when glucose is absent and does not interact with LacY, whereas unphosphorylated EIIAGlc can bind and inactivates LacY when glucose is present (Hogema et al. 1999, Nelson et al. 1983). This only occurs if lactose is present (Smirnova et al. 2007). The same mechanism may be seen for the transport of other secondary carbon sources such as maltose, melibiose, raffinose, and galactose (Titgemeyer et al. 1994, Misko et al. 1987).
Optimization of the 503 antigen induction strategy of Leishmania infantum chagasi expressed in Escherichia coli M15
Published in Preparative Biochemistry and Biotechnology, 2018
Luan Tales Costa de Paiva Vasconcelos, Marcos Antônio Oliveira Filho, Vitor Troccoli Ribeiro, Jaciara Silva de Araújo, Francisco Canindé de Sousa Junior, Daniella Regina Arantes Martins, Everaldo Silvino dos Santos
The mechanism of transport of IPTG through the cell membrane is also a considerable factor in the expression of heterologous genes, however, it is still not sufficiently characterized.[34] Despite that, it has been reported for intracellular IPTG control that it can pass through the cell membrane independently of lactose permease[35] and that it is actively transported by lacY.[36] In turn, lactose presents as a competitive inducer and it can be used for both expression and growth. By the dynamics of this compound, the real inducer in this system is the allolactose, which is an intermediate product produced by β-galactosidase (lacY of lac operon) during the conversion of lactose to glucose and galactose. In the case of repression, allolactose is not sufficiently produced and does not bind to the lac operon repressor significantly to provide a good expression.[37] However, since experiments using lactose show differences in biomass levels when compared to assays using IPTG, the efficiency of using lactose as a source of carbon rather than for the production of the antigen is evident. Such behavior suggests the role of cyclic adenosine monophosphate (cAMP) on the regulation of the E. coli’s growth rate and the lac operon expression trade-off. As the transcription of the lac operon is adjusted by the lac repressor and the cAMP receptor protein (CRP).[38] Therefore, in a medium with glucose, this sugar drains phosphate from the IIA component of glucose-specific phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) (EAII[Glc]) that remains desphosphorylated.[39] In this way, the EAII[Glc] is not able to activate membrane-bound enzyme adenylate cyclase that generates the signal metabolite cAMP. It is known that cAMP binds to the CRP forming the cAMP-CRP complex, which in turn binds near the lac promoter and then improves its transcription.[40] Hence, the use of lactose as inducer can keep the EAII[Glc] non-phosphorylated,[41] but while glucose lowers cAMP level inhibiting the lactose permease, lactose lowers the signal molecule concentration without inhibiting its own entry to the cell.[42] Therefore, this control limits the lac operon expression and it can be used for favoring cell growth instead.