The engineering of enzymes with altered activity, specificity, and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. In vitro recombination techniques such as DNA shuffling, staggered extension process (StEP), random chimeragenesis on transient templates (RACHITT), iterative truncation for the creation of hybrid enzymes (ITCHY), recombined extension on truncated templates (RETT), and so on have been developed to mimic and accelerate nature's recombination strategy. This review discusses gradual advances in the techniques and strategies used for the directed evolution of biocatalytic enzymes aimed at improving the quality and potential of enzyme libraries, their advantages, and disadvantages.

Developments in directed evolution for improving enzyme functions.

1. Introduction: From Physiology to DNA and Back.- 2. RNA Chain Initiation and Promoter Escape by RNA Polymerase.- Promoter Function is Regulated at Two Distinct Phases of Transcription: Promoter Binding and RNA Chain Initiation.- The Biochemistry of the RNA Chain Initiation Phase of Transcription.- Parameters That Describe the RNA Chain Initiation Reaction at Different Promoters.- Factors That Affect the Initiation Reaction: Intrinsic Factors.- Factors That Affect the Initiation Reaction: Extrinsic Factors.- Models for the Mechanism of RNA Chain Initiation: Some Simple Models Do Not Account for What Is Known.- Models for the Mechanism of RNA Chain Initiation-Models Based on Recent Models for RNA Chain Elongation.- 3. Transcription Termination and Its Control.- Termination.- Antitermination.- 4. Codon Context, Translational Step-Times and Attenuation.- Overview.- Attenuation.- Codon Context and Translational Efficiency.- Effects of Codon Pair Bias on Translational Step-Times.- Discussion.- 5. Control by Antisense RNA.- Antisense RNAs Control Diverse Biological Functions.- Antisense RNAs Control Gene Expression at Many Different Post-Transcriptional Levels.- Antisense RNAs Pair to Their Target RNAs by Defined Mechanisms.- Overview.- 6. Translational Control of Gene Expression in E. Coli and Bacteriophage.- Translation Initiation.- Translational Operators.- Translational Repressors.- Mechanisms of Control.- Translational Control and mRNA Processing and/or Degradation.- The Role of Translational Control in Growth Rate Regulation.- Conclusions and Perspectives.- 7. Effects of DNA Supercoiling on Gene Expression.- Synopsis.- The Dependence of Transcription on the Cellular Level of DNA Gyrase and DNA Topoisomerase I.- Mechanistic Considerations.- Supercoiling of the DNA Template by Transcription.- Concluding Remarks.- 8. The HU and IHF Proteins: Accessory Factors for Complex Protein-DNA Assemblies.- Perspective.- Structure.- Interaction with Nucleic Acids.- Control of Intracellular Concentration and Activity.- Participation of IHF and HU in Well-Characterized Biochemical Processes.- Unfinished Business.- 9. The lac and gal Operons Today.- The lac and gal Operons Encode Enzymes of a Continuous Biochemical Pathway.- The Regulatory Circuits and Their Components.- Modulation of Promoters by cAMP*CRP.- Control of P2 by UTP in gal.- Natural Polarity.- Negative Control by Repressor-Operator Interactions.- Epilogue.- 10. The Maltose System.- and Scope.- The Positive Transcriptional Activator MalT.- The Maltose/Maltodextrin Transport System.- The Enzymes of the Maltose System.- Nonclassical Regulatory Phenomena.- Perspectives.- 11. The Phosphoenolpyruvate-Dependent Carbohydrate: Phosphotransferase System (PTS) and Control of Carbon Source Utilization.- Regulatory Phenomena Related to Carbon Source Utilization.- Bacterial Transport Systems and Global Regulatory Networks Form a Unit.- The Bacterial PTS Is a Transport and Signal Transduction System.- IIAGlc of the PTS Is Central to Carbon Catabolite Repression.- IIAGlc, the Regulation of Adenylate Cyclase Activity and of Intracellular cAMP Levels.- Not Only cAMP Levels, but Also CRP Levels Are Essential in Catabolite Repression.- IIAGlc and Inducer Exclusion.- Catabolite Repression and Inducer Exclusion Act in Concert.- Carbon Catabolite Repression through PTS-Control Is Part of a Stimulon.- Concluding Remarks.- 12. The Cap Modulon.- The Long History of CAP.- Cyclic AMP and Gene Expression.- CAP as a Global Regulator: The CAP Modulon.- CAP Binding at Target Promoters and Structural Studies.- Activation by CAP at "Simple"CRP.- Control of P2 by UTP in gal.- Natural Polarity.- Negative Control by Repressor-Operator Interactions.- Epilogue.- 10. The Maltose System.- and Scope.- The Positive Transcriptional Activator MalT.- The Maltose/Maltodextrin Transport System.- The Enzymes of the Maltose System.- Nonclassical Regulatory Phenomena.- Perspectives.- 11. The Phosphoenolpyruvate-Dependent Carbohydrate: Phosphotransferase System (PTS) and Control of Carbon Source Utilization.- Regulatory Phenomena Related to Carbon Source Utilization.- Bacterial Transport Systems and Global Regulatory Networks Form a Unit.- The Bacterial PTS Is a Transport and Signal Transduction System.- IIAGlc of the PTS Is Central to Carbon Catabolite Repression.- IIAGlc, the Regulation of Adenylate Cyclase Activity and of Intracellular cAMP Levels.- Not Only cAMP Levels, but Also CRP Levels Are Essential in Catabolite Repression.- IIAGlc and Inducer Exclusion.- Catabolite Repression and Inducer Exclusion Act in Concert.- Carbon Catabolite Repression through PTS-Control Is Part of a Stimulon.- Concluding Remarks.- 12. The Cap Modulon.- The Long History of CAP.- Cyclic AMP and Gene Expression.- CAP as a Global Regulator: The CAP Modulon.- CAP Binding at Target Promoters and Structural Studies.- Activation by CAP at "Simple" Promoters.- Activation by CAP at Complex Promoters.- CAP as a Repressor and a Co-Repressor.- CAP: Paradigm or Artifact?.- 13. Regulation of Nitrogen Assimilation.- The glnALG(glnA ntrBC) Operon.- The ?54-Dependent Promoter.- Transcriptional Enhancers.- Phosphorylation of NRI.- NRI/NRII as Two-Component Paradigm.- Activation of Transcription.- Response to Nitrogen Availability.- 14. History of the Pho System.- 15. Are the Multiple Signal Transduction Pathways of the Pho Regulon Due to Cross Talk or Cross Regulation?.- Genes for Pi Control of the Pho Regulon.- Transmembrane Signaling by Environmental Pi.- Genes for Pi Independent Controls of the Pho Regulon.- Activation by CreC and Acetyl Phosphate.- Cross Talk, Cross Regulation and a Hypothesis.- Is There Evidence for Cross Regulation?.- Overview and Prospects for Future Studies.- 16. The FNR Modulon and FNR-Regulated Gene Expression.- The Metabolic Arena.- The FNR Modulon.- The FNR Protein and Relationships with CAP.- The DNA-Binding Specificity of FNR.- In Vitro Transcription Activation and Repression.- Transcriptional Organization of Representative Promoters.- Potential FNR Contacts with RNA Polymerase and DNA-Bending.- The Mystery of Redox-Sensing.- Structural and Functional Homologs of FNR.- Concluding Remarks.- 17. The NAR Modulon Systems: Nitrate and Nitrite Regulation of Anaerobic Gene Expression.- Anaerobic Respiration.- The Characterization of the Nar Regulatory System.- Dual Two-Component Regulatory Systems.- The Role of Nitrite in the Nar Regulatory System.- The Sensor Proteins.- The Response Regulators.- Indirect Nitrate Regulation of Gene Expression.- Concluding Remarks.- 18. Regulation of Aerobic and Anaerobic Metabolism by the Arc System.- Identification of the arc Genes.- In Vivo Studies of arc Mutants.- In Vitro Phosphorylation Studies.- The Arc Modulon.- ArcA DNA Binding.- The Arc Stimulus.- Future Studies.- 19. The Porin Regulon: A Paradigm for the Two-Component Regulatory Systems.- Background.- The History of Porin Regulation.- The Structure of the ompB Locus.- The Structure of the ompF and ompC Genes.- The Roles of OmpR and EnvZ.- Phosphorylation and Signal Transduction.- Summary and Conclusions.- 20. The Leucine\Lrp Regulon.- The Leucine-Responsive Regulatory Protein.- Regulation of Lrp Synthesis.- Target Operons of Lrp and Mutant Phenotypes.- Lrp as a Chromosome Organizer.- Molecular Aspects of Lrp Interactions at Individual Promoters.- 21. Adaptive responses to Oxidative Stress: The soxRS and oxyR Regulons.- Reactive Oxygen Species.- Antioxidant Defenses.- Oxidative Stress.- Global Responses to Oxidative Stress.- The soxRS Regulon.- The oxyR Regulon.- Control of Antibiotic Resistance Genes.- 22. The SOS Regulatory System.- and Current Regulatory Model.- Development of the SOS Model.- Recent Developments.- Behavior of the SOS Gene Regulatory Circuitry.- Future Prospects.- 23. Heat Shock Regulation.- Properties of Important Heat Shock Proteins.- Regulation of the ?32-Promoted Heat Shock Response.- A Second Heat Shock Regulon.- The ?54-Promoted Stress Response.- Heat Shock or Stress Responses in Other Eubacteria.- 24. Roles for Energy-Dependent Proteases in Regulatory Cascades.- The Proteases and Their Targets.- Summary and General Conclusions.- 25. Control of rRNA and Ribosome Synthesis.- rRNA Gene Organization.- High Activity of rRNA Synthesis Rates.- Stringent Control.- Growth Rate Dependent Control.- Additional Considerations.- Conclusion and Future Prospects.- 26. Cell Division.- The Cell Division Process.- Essential Cell Division Genes.- Transcriptional Regulation of Cell Division Genes.- Translational Control.- Division Inhibitors.- Cell Division Inhibition by Mutations in Genes That Do Not Code for Cell Division Proteins.- Past, Present and Future.- 27. Regulation of Gene Expression in Stationary Phase.- The ?s Regulon.- RpoS Regulation.

Regulation of Gene Expression in Escherichia Coli

Control of the Escherichia coli rrnB P1 promoter strength by ppGpp

Over the past decade, synthetic biology has emerged as an engineering discipline for biological systems. Compared with other substrates, biology poses a unique set of engineering challenges resulting from an incomplete understanding of natural biological systems and tools for manipulating them. To address these challenges, synthetic biology is advancing from developing proof-of-concept designs to focusing on core platforms for rational and high-throughput biological engineering. These platforms span the entire biological design cycle, including DNA construction, parts libraries, computational design tools, and interfaces for manipulating and probing synthetic circuits. The development of these enabling technologies requires an engineering mindset to be applied to biology, with an emphasis on generalizable techniques in addition to application-specific designs. This review aims to discuss the progress and challenges in synthetic biology and to illustrate areas where synthetic biology may impact biomedical engineering and human health.

https://www.annualreviews.org/doi/pdf/10.1146/annurev-bioeng-071811-150118

Synthetic Biology: An Emerging Engineering Discipline

This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiological states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.

AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action

The mode of action of regulated promoters is largely determined by kinetic parameters which govern the interaction between promoters and proteins involved in induction and repression of transcription. To gain insight into the interplay between positively and negatively acting transcriptional regulators, in this case AraC and LacR, we have generated a panel of promoter sequences derived from P(lac), the promoter of the Escherichia coli lac operon. The function of these promoters is limited at different steps and to various extents within the pathway of RNA polymerase (RNAP)/promoter interaction. Moreover, in all promoters the cAMP receptor protein binding site was replaced by the binding motif of AraC to prevent pleiotropic effects in vivo upon activation. Analyzing the activation of these promoters by AraC in vivo under conditions of repression by LacR and derepression yielded a three step model of transcription initiation which reveals mechanisms of AraC and LacR action. Our data show three distinct rate limiting steps at which AraC can exert its function. In general, the activator accelerates the formation of the first stable complex between RNAP and promoter. At most promoter sequences, however, its main impact is on the conversion of the closed to the open complex. However, AraC is also capable of eliminating limitations at steps following open complex formation.

Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator.

Stalling of Escherichia coli RNA polymerase in the +6 to +12 region in vivo is associated with tight binding to consensus promoter elements

On the Streptococcus equisimilis H46A chromosome, the divergent coding sequences of the genes for the plasminogen activator streptokinase (skc) and a leucine-rich protein (lrp), the function of which is unknown, are separated by a 328 by intrinsically bent DNA region rich in AT tracts. To begin to understand the expression control of these two genes, we mapped their transcriptional initiation sites by S1 nuclease analysis and studied the influence of the bent intergenic region on promoter strength, using promoter-reporter gene fusions of skc′ and lrp′ to ′lacZ from Escherichia coli. The major transcriptional start sites, in both S. equisimilis and E. coli, mapped 22 bases upstream of the ATG start site of lrp (G), and 24 and 32 bases upstream of the translational initiation codon of skc (A and G, respectively), indicating the existence of two overlapping canonical skc promoters arranged in tandem on opposite faces of the helix. The reporter gene fusions were cloned in E. coli on a vector containing a 1.1 kb fragment of the S. equisimilis dexB gene, thus allowing promoter strength to be measured in multiple plasmid-form copies in the heterologous host and in single-copy genomic form following integration into the skc region of the homologous host. In S. equisimilis, skc′-′lacZ was expressed about 200-fold more strongly than the corresponding lrp′-′lacZ fusion. In contrast, in E. coli, the corresponding levels of expression differed by only about 11-fold. Deletion of the 202 by bent region upstream of the skc and lrp core promoters caused a 13-fold decrease in skc promoter activity in S. equisimilis but did not alter lrp promoter strength in this host. In contrast, when studied in E. coli, this deletion did not alter the strength of the skc double promoter and even increased by 2.4- to 3-fold the activity of the lrp promoter. This comparative promoter analysis shows that skc has a complex promoter structure, the activity of which in the homologous genomic environment specifically depends on sequences upstream of the two core promoters. Thus, the skc promoter structure resembles that of an array of promoters involved in a transcriptional switch; however, the nature of the potential switch factor(s) remains unknown.

Complex transcriptional control of the streptokinase gene of Streptococcus equisimilis H46A

The overlapping tandem promoters of the streptokinase gene, P 1 and P 2, identified previously by S 1 nuclease transcript mapping were functionally dissected by mutagenesis of their –10 regions and fused transcriptionally with or without the 202-bp upstream region (USR) to the luciferase reporter gene (luc) from Photinus pyralis to analyze the contribution of the different sequence elements to promoter activity in Escherichia coli and the homologous Streptococcus equisimilis strain H46A. In E. coli, virtually the entire promoter activity derived from the upstream promoter P 1. In S. equisimilis, luc expression increased in the following order of the involved sequence elements: P 2≌P 2+USR < P 1 < P 1+P 2 < P 1+USR < P 1+ P 2+USR. This shows that (1) in the homologous system, P 1 and P 2 alone are extremely weak, (2) in the USR-less arrangement, only the combined core promoters have substantial activity, and (3) the USR stimulates only P 1 and the combination of P 1+P 2. Thus, the tandem promoters presumably function by mutual contributary action and their full activity strongly depends on the AT-rich and statically bent upstream region. The distinctive feature determining the strength of P 1 in both hosts appears to be its extended –10 region which matches the consensus TRTGN established for strong S. pneumoniae and Bacillus subtilis promoters.

Structural dissection and functional analysis of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A.

Transcriptional start sites of the staphylokinase 42D promoter in E.coli and B.subtilis

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