Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously

Venkat, Sumana; Sturges, Jourdan; Stahman, Alleigh; Gregory, Caroline; Gan, Qinglei; Fan, Chenguang

doi:10.1021/acssynbio.7b00408

Cited by 53 publications

(40 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this context, we also analyzed their dependence on their most direct regulator (glucose) and some direct effectors (deacetylase CobB, peptidyl N(ε)-Lysine acetyl-transferase PatZ). Although this topic belongs to the ambit of molecular physiology, the clarification of these mechanisms could have important consequences in the field of biotechnological applications (Kuczynska-Wiśnik et al, 2016 ; Ahsan et al, 2017 ; Basak et al, 2017 ; Ishigaki et al, 2017 ; Venkat et al, 2018 ), and even for some medical issues (Bernal et al, 2016 ; Ou et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Guebel¹,

Torres

2018

Front. Microbiol.

View full text Add to dashboard Cite

Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, we aimed to progress in the characterization of this regulation by dealing with three convergent aspects: the effector enzymes involved, the master regulator CRP, and the dependence on glucose availability.Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 relevant scenarios. These conditions arise when the wild type and four isogenic mutants (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and R-type mutants of protein CRP) are studied under three levels of glucose availability (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). The Q-type emulates a permanent stage of CRPacetylated, whereas the R-type emulates a permanent stage of CRPdeacetylated. The data were analyzed by an optimized factorial microarray method (Q-GDEMAR).Results: (a) By analyzing one mutant against the other, we were able to unravel the true genes that participate in the interaction between ΔcobB/ΔpatZ mutations and glucose availability; (b) Increasing stages of glucose limitation appear to be associated with the up-regulation of specific sets of target genes rather than with the loss of genes present when glucose is in excess; (c) Both CRPdeacetylated and CRPacetylated produce extensive changes in specific subsets of genes, but their number and identity depend on the glucose availability; (d) In other sub-sets of genes, the transcriptional effect of CRP seems to be independent of its acetylation or deacetylation; (e) Some specific ontology functions can be associated with each of the different sets of genes detected herein.Conclusions: CRP cannot be thought of only as an effector of catabolite repression, because it acts along all the glucose conditions tested (excess, limited, and exhausted), exerting both positive and negative effects through different sets of genes. Acetylation of CRP does not seem to be a binary form of regulation, as there is not a univocal relationship between its activation/inhibitory effect and its acetylation/deacetylation stage. All the combinatorial possibilities are observed. During the exponential growth phase, CRP also exerts a very significant transcriptional effect, mainly on flagellar assembly and chemotaxis (FDR = 7.2 × 10−44).

show abstract

Section: Discussionmentioning

confidence: 99%

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Guebel¹,

Torres

2018

Front. Microbiol.

View full text Add to dashboard Cite

show abstract

“…X is the growth rate in the exponential phase, which was automatically provided by Gen5 software designed for the BioTek microplate reader (Winooski, VT, USA). The monitoring of GFP expression by fluorescence followed previous studies [ 13 , 14 ].…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Chen

Venkat

Wilson

et al. 2018

Genes

Self Cite

View full text Add to dashboard Cite

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

show abstract

“…Recently, our group utilized mutually orthogonal SepRS and AcKRS systems in response to two stop codons to simultaneously incorporate Sep and AcK into target proteins. We also demonstrated mutual orthogonality of PylRS, Mj TyrRS and SepRS systems, implying the possibilities to incorporate three different PTMs into a single protein [ 142 ]. Similarly, Wright et al produced site-specifically acetylated Thioredoxin reductase 1 that also contains selenocysteine (Sec) by simultaneous UAG codon reassignment to AcK and UGA codon recoding to Sec [ 143 ].…”

Section: Summary and Perspectivementioning

confidence: 99%

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

et al. 2018

Self Cite

View full text Add to dashboard Cite

Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field.

show abstract

Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously

Cited by 53 publications

References 58 publications

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

Contact Info

Product

Resources

About