Content uploaded by Allen E Silverstone
Author content
All content in this area was uploaded by Allen E Silverstone
Content may be subject to copyright.
A preview of the PDF is not available
Catabolite-Insensitive Revertants of Lac Promoter Mutants
Abstract and Figures
The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.
Figures - uploaded by Allen E Silverstone
Author content
All figure content in this area was uploaded by Allen E Silverstone
Content may be subject to copyright.
... With the advent of molecular biology, different parts of the lac operon have been modified to permit optimal expression of recombinant proteins by E. coli . The first adaptation was to the wild type promoter which was mutated so that the derivative, designated lacUV5, was no longer under cAMP regulation (Silverstone et al., 1970 ;Pastan & Adhya, 1976 ). Subsequently, the gene encoding RNA polymerase from phage T7 was cloned into the E. coli chromosome, so that this gene was also under the same regulatory mechanism as the lac operon (lacUV5 promoter/O1 operator) (Studier and Moffatt, 1986 ). ...
Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts.
One-Sentence Summary
A computational model made freely available to optimize recombinant protein expression in Escherichia coli other heterologous hosts.
... Expression of the chromosomally localized gene encoding the T7 RNA polymerase is governed by the IPTG-inducible lacUV5 promoter (Studier and Moffatt, 1986). This promoter is a more powerful variant of the wild-type lac promoter and it is not well titratable (Silverstone et al., 1970;Wanner et al., 1977). The idea behind this setup is that the more mRNA encoding the recombinant protein is synthesized, the more protein can be produced. ...
Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.
... One of the main factors affecting metabolic burden is promoter strength. lac-derived promoters (P T7lac , P trc , and P tac ) are all based on the negative regulation by LacI, and the expression is induced by lactose or non-metabolizable isopropyl β-D-1-thiogalactopyranoside (IPTG), used as analogous molecule of lactose, while pBAD plasmids carry a BAD promoter (P BAD ) positively induced by L-arabinose (Müller-Hill et al., 1968;Silverstone et al., 1970;Terpe, 2006;Brautaset et al., 2009). P trc and P tac promoters are considered as strong promoters and are well characterized. ...
Recombinant protein production for medical, academic, or industrial applications is essential for our current life. Recombinant proteins are obtained mainly through microbial fermentation, with Escherichia coli being the host most used. In spite of that, some problems are associated with the production of recombinant proteins in E. coli, such as the formation of inclusion bodies, the metabolic burden, or the inefficient translocation/transport system of expressed proteins. Optimizing transcription of heterologous genes is essential to avoid these drawbacks and develop competitive biotechnological processes. Here, expression of YFP reporter protein is evaluated under the control of four promoters of different strength (P T7lac , P trc , P tac , and P BAD) and two different replication origins (high copy number pMB1 and low copy number p15A). In addition, the study has been carried out with the E. coli BL21 wt and the ackA mutant strain growing in a rich medium with glucose or glycerol as carbon sources. Results showed that metabolic burden associated with transcription and translation of foreign genes involves a decrease in recombinant protein expression. It is necessary to find a balance between plasmid copy number and promoter strength to maximize soluble recombinant protein expression. The results obtained represent an important advance on the most suitable expression system to improve both the quantity and quality of recombinant proteins in bioproduction engineering.
... Unlike P lac , P lacUV5 works independently of activator proteins or other cis-regulatory elements and has lost responsiveness to catabolite repression. The result is less sensitivity to increased cAMP levels that occur upon transition from the non-glucose-limited state (Batch) to the glucose-limited state (Fed-batch), and consequently lower basal level expression, compared to P araBAD , upon entering glucoselimited conditions [40][41][42]. ...
Background
Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β- d -1-thiogalactopyranoside (IPTG)-inducible P T7-lacO promoter can be induced solely with l -arabinose in a newly constructed Escherichia coli expression host BL21-AI< gp2 >, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production.
Results
Here, we show that BL21-AI< gp2 > is able to precisely regulate protein production rates on a cellular level in an l -arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l -arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting.
Conclusions
In all cases tested, BL21-AI< gp2 > outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.
... Given their frequency, it is remarkable that this class of UV mutations has not previously been reported or analyzed in any systematic way. There have been hints in the literature that UV light can cause AC>TT mutations (e.g., Reis et al., 2000), perhaps the best-known example being the UV-induced lacUV5 promoter mutation (Silverstone et al., 1970). This mutation class is highly relevant to melanoma, because these tandem substitutions are responsible for the oncogenic BRAF V600R and V600K mutations. ...
Somatic mutations in skin cancers and other ultraviolet (UV)-exposed cells are typified by C>T and CC>TT substitutions at dipyrimidine sequences; however, many oncogenic “driver” mutations in melanoma do not fit this UV signature. Here, we use genome sequencing to characterize mutations in yeast repeatedly irradiated with UV light. Analysis of ~50,000 UV-induced mutations reveals abundant non-canonical mutations, including T>C, T>A, and AC>TT substitutions. These mutations display transcriptional asymmetry that is modulated by nucleotide excision repair (NER), indicating that they are caused by UV photoproducts. Using a sequencing method called UV DNA endonuclease sequencing (UVDE-seq), we confirm the existence of an atypical thymine-adenine photoproduct likely responsible for UV-induced T>A substitutions. Similar non-canonical mutations are present in skin cancers, which also display transcriptional asymmetry and dependence on NER. These include multiple driver mutations, most prominently the recurrent BRAF V600E and V600K substitutions, suggesting that mutations arising from rare, atypical UV photoproducts may play a role in melanomagenesis.
... The evidence for this is indirect. Such a role of cyclic AMP seems to be ruled out by (i) the demonstration that isolated gal or lac mRNA, synthesized in the presence of cyclic AMP and CRP, can be translated efficiently by a S30 protein-synthesizing system without the cyclic nucleotide (92,102); and (ii) the observation that a mutant lac operon (UV5) does not need cyclic AMP and CRP to promote /3-galactosidase synthesis (104). The lac RNA from UV5 is normal in structure and does not require cyclic AMP for its translation. ...
The Pseudomonas aeruginosa virulence factor regulator (Vfr) is a cyclic AMP (cAMP)-responsive transcription factor homologous to the Escherichia coli cAMP receptor protein (CRP). Unlike CRP, which plays a central role in E. coli energy metabolism and catabolite repression, Vfr is primarily involved in the control of P. aeruginosa virulence factor expression. Expression of the Vfr regulon is controlled at the level of vfr transcription, Vfr translation, cAMP synthesis, and cAMP degradation. While investigating mechanisms that regulate Vfr translation, we placed vfr transcription under the control of the rhaBp rhamnose-inducible promoter system (designated PRha) and found that PRha promoter activity was highly dependent upon vfr. Vfr dependence was also observed for the araBp arabinose-inducible promoter (designated PBAD). The observation of Vfr dependence was not entirely unexpected. Both promoters are derived from E. coli, where maximal promoter activity is dependent upon CRP. Like CRP, we found that Vfr directly binds to promoter probes derived from the PRha and PBAD promoters in vitro. Because Vfr-cAMP activity is highly integrated into numerous global regulatory systems, including c-di-GMP signaling, the Gac/Rsm system, MucA/AlgU/AlgZR signaling, and Hfq/sRNAs, the potential exists for significant variability in PRha and PBAD promoter activity in a variety of genetic backgrounds, and use of these promoter systems in P. aeruginosa should be employed with caution.
In a transcription system containing λpgal8 DNA as template and Escherichia coli RNA polymerase, the addition of cyclic adenosine monophosphate receptor protein (CRP) and 3',5'-cyclic AMP causes a 15-fold increase in gal mRNA synthesis. Gal mRNA was measured by hybridizing the [³H]RNA product to the separated strands of λpgal8 DNA after removal of λ mRNA by preliminary hybridization to λ DNA. Competition experiments indicate that a large fraction of this RNA is identical with in vivo gal mRNA. With λpgal8 DNA as template but not with λ DNA, 3',5'-cyclic AMP and CRP cause a 2-fold increase in λ mRNA. Since the increment in λ mRNA hybridizes to the left-hand half of the ι strand of λ DNA and is abolished by the addition of ρ, it is interpreted as read through from the 3',5'-cyclic AMP-CRP sensitive site in the gal region of λpgal8 DNA. The concentrations of CRP and 3',5'-cyclic AMP necessary for half-maximal stimulation of gal mRNA synthesis are 5 x 10⁻⁸ and 5 x 10⁻⁶m, respectively. 2',3'-Cyclic AMP and 5'-AMP do not stimulate gal mRNA synthesis; 3',5'-cyclic GMP is an inhibitor. When 3',5' cyclic AMP, CRP, DNA, and RNA polymerase are incubated together for 10 min, a rifampicin-resistant preinitiation complex is formed, and, upon the addition of nucleoside triphosphates, gal mRNA synthesis occurs. 3',5'-Cyclic GMP prevents the formation of this complex and can dissociate it after it has been formed. These results suggest that 3',5'-cyclic AMP and CRP stimulate gal transcription at a step prior to chain elongation.
The work presented in this thesis describes experiments carried out in order to determine the three-dimensional structure of a DNA repair enzyme, uracil-DNA glycosylase. An open reading frame, UL2, in the herpes simplex virus type 1 genome, is known to encode a uracil-DNA glycosylase. By sequence homology, there are three candidate start codons which might express a functional uracil-DNA glycosylase. Expression from two of these was attempted in Escherichia coli, using plasmids designed for high level production of recombinant proteins. The second candidate start codon produces high levels of a soluble, functional uracil-DNA glycosylase in Escherichia coli both in a native form, and as part of a fusion protein. Both the fusion and the native form of the enzyme have been purified to apparent homogeneity, as has a recombinantly expressed insoluble Escherichia coli uracil-DNA glycosylase. Preliminary attempts were made at deriving structural and functional information from the soluble, native recombinant herpes simplex enzyme with the use of circular dichroism. This form of uracil-DNA glycosylase has subsequently been crystallised in two ways, firstly as the free enzyme, and secondly in a complex with a single stranded DNA oligonucleotide. Extensive optimisation of the crystallisation parameters have been carried out in conjunction with modifications to the original purification protocol, and large, single crystals of both free, and DNA bound forms, suitable for X-ray diffraction studies are now readily reproduced. A systematic search for isomorphous heavy atom derivatives has been carried out for both types of crystal, and preliminary phases have been obtained for the DNA-bound form of the enzyme. This has enabled the calculation of an electron density map in which protein secondary structure features can be located. Improvement of this map will reveal the molecular structure of the enzyme/ DNA complex.
A cell-free system allowing for synthesis of beta-galactosidase enzymatic activity has been developed. This system requires DNA containing the beta-galactosidase gene, a cell-free extract of Escherichia coli bacteria, and the low-molecular-weight components necessary for transcription of the DNA and translation of the resulting messenger RNA. Such a system is useful for studying enzyme synthesis, as well as its regulation. The gene for beta-galactosidase is part of the lac operon whose expression is under the control of the lac repressor. In whole cells the lac repressor inhibits almost all of the gene expression for beta-galactosidase. In the cell-free system, we had previously been able to repress about half the gene expression. Adding cyclic adenosine 3'5'-monophosphate to the cell-free system improved the yield of beta-galactosidase enzymatic activity by 8 to 30 times and the efficiency of repression from 50 to 95 per cent.
The mechanism of the induction of β-galactosidase in Escherichia coli at 30°C was investigated by separating the phase of enzyme induction from the phase of enzyme production. This separation was accomplished by removing the inducer by filtration after 3 to 4 min of contact with the cells, that is before enzyme had been formed; the cells then produced the enzyme in inducer-free medium at an exponentially declining rate for approximately 10 min. It was found that interference with the synthesis of normal RNA by 5-fluorouracil during the phase of enzyme induction largely prevents the subsequent production of normal enzyme and causes the formation of an altered protein serologically related to β-galactosidase; on the other hand, such an interference with RNA synthesis during the phase of enzyme production does not affect the formation of the enzyme by previously induced cells. Conversely, interference with protein synthesis by starvation for an amino acid or by chloramphenicol during the phase of induction does not prevent the subsequent production of normal enzyme; but such an interference with protein synthesis during the phase of enzyme production inhibits the formation of the enzyme by previously induced cells to the same extent as that of other protein. These results indicate that during the induction phase a messenger RNA specific for β-galactosidase is produced which directs the subsequent synthesis of the enzyme in the inducer-free medium. No additional β-galactosidase messenger RNA is formed after removal of the inducer and that present in the cell decays exponentially with a half-life of approximately 2·5 min. The rate of this decay is independent of the rate of protein synthesis or the rate of energy metabolism. An excess of intracellular catabolites, produced either by the rapid catabolism of glucose or by the restriction of protein synthesis in the presence of a utilizable energy source, inhibits the induction of the enzyme but does not affect the production of the enzyme by previously induced cells. Apparently, the catabolite repressor inhibits and the inducer stimulates the synthesis of the unstable messenger RNA specific for β-galactosidase.
Transient and catabolite repression of the synthesis of beta-galactosidase in E. coli are related phenomena, the result of a decreased intracellular concentration of cyclic AMP. The lac operon promoter is the site of action of the cyclic AMP.
Cyclic 3′,5′-AMP increases the synthesis of lac mRNA in a cell-free extract of E. coli using λh80dlac DNA as template. Lac mRNA was measured by DNA-RNA hybridization to F'lac DNA and shown to be specific by competition of the 3H-lac mRNA with unlabeled lac mRNA prepared from whole cells.
Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression.
The lac promoter maps between the represser i gene and the operator o gene, so that the operator gene is probably transcribed.
A mutation, GD-1, in the leucine operon imposed unusual growth characteristics upon a leucine auxotrophic strain bearing the leucine operator mutation, leu-500. The strain with the GD-1 mutation was able to grow on a minimal salts medium when citrate was the sole carbon source, but required leucine when glucose was present. Tests with a large number of carbohydrates suggest that in the strain bearing the GD-1 mutation the leucine biosynthetic enzymes are under catabolite repressor control. Recombination studies indicate that the GD-1 mutation is a secondary alteration of the leucine operator at or very close to the site of the leu-500 mutation. Mutations at the supX locus (previously termed su leu 500 and located on the chromosome between the cysteine B and tryptophan gene clusters) result in elimination of the catabolite repression effect. The data are interpreted as an indication that the GD-1 and leu-500 mutations alter the leucine operator with respect to its specificity of response to repressors.
The molecular basis of transient repression of beta-galactosidase by glucose was examined. This repression acted only at the level of transcription. Apparently, it was not mediated by the I-gene product. Analysis of single cells in a culture subjected to transient repression showed that essentially all cells initially experienced repression and later became gradually resistant to repression.