A primer to directed evolution: current methodologies and future directions.

Directed evolution is one of the most powerful tools for protein engineering and functions by harnessing natural evolution, but on a shorter timescale. It enables the rapid selection of variants of biomolecules with properties that make them more suitable for specific applications. Since the first in vitro evolution experiments performed by Sol Spiegelman in 1967, a wide range of techniques have been developed to tackle the main two steps of directed evolution: genetic diversification (library generation), and isolation of the variants of interest. This review covers the main modern methodologies, discussing the advantages and drawbacks of each, and hence the considerations for designing directed evolution experiments. Furthermore, the most recent developments are discussed, showing how advances in the handling of ever larger library sizes are enabling new research questions to be tackled.

Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways.

BACKGROUND: Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock. Taken together, the production of bio commodities from low-cost gas streams containing CO, CO2, and H2, obtained from the gasification of any carbon-containing waste streams or off-gases from heavy industry (steel mills, processing plants, or refineries), embodies an opportunity for affordable and renewable chemical production. To achieve this, by studying non-model autotrophic acetogens, current limitations concerning low growth rates, toxicity by gas streams, and low productivity may be overcome. The Acetobacterium wieringae strain JM is a novel autotrophic acetogen that is capable of producing acetate and ethanol. It exhibits faster growth rates on various gaseous compounds, including carbon monoxide, compared to other Acetobacterium species, making it potentially useful for industrial applications. The species A. wieringae has not been genetically modified, therefore developing a genetic engineering method is important for expanding its product portfolio from gas fermentation and overall improving the characteristics of this acetogen for industrial demands. RESULTS: This work reports the development and optimization of an electrotransformation protocol for A. wieringae strain JM, which can also be used in A. wieringae DSM 1911, and A. woodii DSM 1030. We also show the functionality of the thiamphenicol resistance marker, catP, and the functionality of the origins of replication pBP1, pCB102, pCD6, and pIM13 in all tested Acetobacterium strains, with transformation efficiencies of up to 2.0 × 103 CFU/µgDNA. Key factors affecting electrotransformation efficiency include OD600 of cell harvesting, pH of resuspension buffer, the field strength of the electric pulse, and plasmid amount. Using this method, the acetone production operon from Clostridium acetobutylicum was efficiently introduced in all tested Acetobacterium spp., leading to non-native biochemical acetone production via plasmid-based expression. CONCLUSIONS: A. wieringae can be electrotransformed at high efficiency using different plasmids with different replication origins. The electrotransformation procedure and tools reported here unlock the genetic and metabolic manipulation of the biotechnologically relevant A. wieringae strains. For the first time, non-native acetone production is shown in A. wieringae.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

Versatile selective evolutionary pressure using synthetic defect in universal metabolism.

The non-natural needs of industrial applications often require new or improved enzymes. The structures and properties of enzymes are difficult to predict or design de novo. Instead, semi-rational approaches mimicking evolution entail diversification of parent enzymes followed by evaluation of isolated variants. Artificial selection pressures coupling desired enzyme properties to cell growth could overcome this key bottleneck, but are usually narrow in scope. Here we show diverse enzymes using the ubiquitous cofactors nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) can substitute for defective NAD regeneration, representing a very broadly-applicable artificial selection. Inactivation of Escherichia coli genes required for anaerobic NAD regeneration causes a conditional growth defect. Cells are rescued by foreign enzymes connected to the metabolic network only via NAD or NADP, but only when their substrates are supplied. Using this principle, alcohol dehydrogenase, imine reductase and nitroreductase variants with desired selectivity modifications, and a high-performing isopropanol metabolic pathway, are isolated from libraries of millions of variants in single-round experiments with typical limited information to guide design.

Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria.

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO2 capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO2, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO2, avoiding fossil carbon or competition with food production.

Design and Implementation of Multi-protein Expression Constructs and Combinatorial Libraries using Start-Stop Assembly.

Start-Stop Assembly is a multi-part, modular, Golden Gate-based DNA assembly system with two key features which distinguish it from previous DNA assembly methods. Firstly, coding sequences are assembled with upstream and downstream sequences via overhangs corresponding to start and stop codons, avoiding unwanted 'scars' in assembled constructs at coding sequence boundaries. Scars at these crucial, sensitive locations can affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. Start-Stop Assembly is therefore both functionally scarless (an advantage usually only achieved using bespoke, overlap-based assembly methods) and suitable for efficient, unbiased and combinatorial assembly (a general advantage of Golden Gate-based methods). Secondly, Start-Stop Assembly has a new, streamlined assembly hierarchy, meaning that typically only one new vector is required in order to assemble constructs for any new destination context, such as a new organism or genomic location. This should facilitate more rapid and convenient development of engineered metabolic pathways for diverse nonmodel organisms in order to exploit their applied potential. This chapter explains both design considerations and practical procedures to implement multi-part, hierarchical assembly of multi-protein expression constructs, either individually or as combinatorial libraries, using Start-Stop Assembly.

Type IV Pili-Independent Photocurrent Production by the Cyanobacterium Synechocystis sp. PCC 6803.

Biophotovoltaic devices utilize photosynthetic organisms such as the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) to generate current for power or hydrogen production from light. These devices have been improved by both architecture engineering and genetic engineering of the phototrophic organism. However, genetic approaches are limited by lack of understanding of cellular mechanisms of electron transfer from internal metabolism to the cell exterior. Type IV pili have been implicated in extracellular electron transfer (EET) in some species of heterotrophic bacteria. Furthermore, conductive cell surface filaments have been reported for cyanobacteria, including Synechocystis. However, it remains unclear whether these filaments are type IV pili and whether they are involved in EET. Herein, a mediatorless electrochemical setup is used to compare the electrogenic output of wild-type Synechocystis to that of a ΔpilD mutant that cannot produce type IV pili. No differences in photocurrent, i.e., current in response to illumination, are detectable. Furthermore, measurements of individual pili using conductive atomic force microscopy indicate these structures are not conductive. These results suggest that pili are not required for EET by Synechocystis, supporting a role for shuttling of electrons via soluble redox mediators or direct interactions between the cell surface and extracellular substrates.

VAPPER: High-throughput variant antigen profiling in African trypanosomes of livestock.

BACKGROUND: Analysing variant antigen gene families on a population scale is a difficult challenge for conventional methods of read mapping and variant calling due to the great variability in sequence, copy number, and genomic loci. In African trypanosomes, hemoparasites of humans and animals, this is complicated by variant antigen repertoires containing hundreds of genes subject to various degrees of sequence recombination. FINDINGS: We introduce Variant Antigen Profiler (VAPPER), a tool that allows automated analysis of the variant surface glycoprotein repertoires of the most prevalent livestock African trypanosomes. VAPPER produces variant antigen profiles for any isolate of the veterinary pathogens Trypanosoma congolense and Trypanosoma vivax from genomic and transcriptomic sequencing data and delivers publication-ready figures that show how the queried isolate compares with a database of existing strains. VAPPER is implemented in Python. It can be installed to a local Galaxy instance from the ToolShed (https://toolshed.g2.bx.psu.edu/) or locally on a Linux platform via the command line (https://github.com/PGB-LIV/VAPPER). The documentation, requirements, examples, and test data are provided in the Github repository. CONCLUSION: By establishing two different, yet comparable methodologies, our approach is the first to allow large-scale analysis of African trypanosome variant antigens, large multi-copy gene families that are otherwise refractory to high-throughput analysis.

Transcriptional Terminators Allow Leak-Free Chromosomal Integration of Genetic Constructs in Cyanobacteria.

Cyanobacteria are promising candidates for sustainable bioproduction of chemicals from sunlight and carbon dioxide. However, the genetics and metabolism of cyanobacteria are less well understood than those of model heterotrophic organisms, and the suite of well-characterised cyanobacterial genetic tools and parts is less mature and complete. Transcriptional terminators use specific RNA structures to halt transcription and are routinely used in both natural and recombinant contexts to achieve independent control of gene expression and to 'insulate' genes and operons from one another. Insulating gene expression can be particularly important when heterologous or synthetic genetic constructs are inserted at genomic locations where transcriptional read-through from chromosomal promoters occurs, resulting in poor control of expression of the introduced genes. To date, few terminators have been described and characterised in cyanobacteria. In this work, nineteen heterologous, synthetic or putative native Rho-independent (intrinsic) terminators were tested in the model freshwater cyanobacterium, Synechocystis sp. PCC 6803, from which eleven strong terminators were identified. A subset of these strong terminators was then used to successfully insulate a chromosomally-integrated, rhamnose-inducible rhaBAD expression system from hypothesised 'read-through' from a neighbouring chromosomal promoter, resulting in greatly improved inducible control. The addition of validated strong terminators to the cyanobacterial toolkit will allow improved independent control of introduced genes.

Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable 'scar' sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

Synthetic negative feedback circuits using engineered small RNAs.

Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised tet-based autorepressor, incorporating an externally-inducible sRNA to tune the effective feedback strength. This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input-output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.

A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria.

Cyanobacteria are important for fundamental studies of photosynthesis and have great biotechnological potential. In order to better study and fully exploit these organisms, the limited repertoire of genetic tools and parts must be expanded. A small number of inducible promoters have been used in cyanobacteria, allowing dynamic external control of gene expression through the addition of specific inducer molecules. However, the inducible promoters used to date suffer from various drawbacks including toxicity of inducers, leaky expression in the absence of inducer and inducer photolability, the latter being particularly relevant to cyanobacteria, which, as photoautotrophs, are grown under light. Here we introduce the rhamnose-inducible rhaBAD promoter of Escherichia coli into the model freshwater cyanobacterium Synechocystis sp. PCC 6803 and demonstrate it has superior properties to previously reported cyanobacterial inducible promoter systems, such as a non-toxic, photostable, non-metabolizable inducer, a linear response to inducer concentration and crucially no basal transcription in the absence of inducer.

Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates.

Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (Pthl) except for the consensus -35 and -10 elements. In each synthetic promoter, the chance of each degenerate position matching Pthl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching Pthl was 79% or 58%, instead of 25% as before. Synthetic promoters from these "stringent" libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected "stringency" of promoter sequences in Clostridium, not reported previously.

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application.

NAD(P)H-dependent oxidoreductases catalyze the reduction or oxidation of a substrate coupled to the oxidation or reduction, respectively, of a nicotinamide adenine dinucleotide cofactor NAD(P)H or NAD(P)+. NAD(P)H-dependent oxidoreductases catalyze a large variety of reactions and play a pivotal role in many central metabolic pathways. Due to the high activity, regiospecificity and stereospecificity with which they catalyze redox reactions, they have been used as key components in a wide range of applications, including substrate utilization, the synthesis of chemicals, biodegradation and detoxification. There is great interest in tailoring NAD(P)H-dependent oxidoreductases to make them more suitable for particular applications. Here, we review the main properties and classes of NAD(P)H-dependent oxidoreductases, the types of reactions they catalyze, some of the main protein engineering techniques used to modify their properties and some interesting examples of their modification and application.

Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature-sensitive phenotype and increases biofilm-forming ability.

Clostridium difficile infection is a growing problem in healthcare settings worldwide and results in a considerable socioeconomic impact. New hypervirulent strains and acquisition of antibiotic resistance exacerbates pathogenesis; however, the survival strategy of C. difficile in the challenging gut environment still remains incompletely understood. We previously reported that clinically relevant heat-stress (37-41 °C) resulted in a classical heat-stress response with up-regulation of cellular chaperones. We used ClosTron to construct an insertional mutation in the dnaK gene of C. difficile 630 Δerm. The dnaK mutant exhibited temperature sensitivity, grew more slowly than C. difficile 630 Δerm and was less thermotolerant. Furthermore, the mutant was non-motile, had 4-fold lower expression of the fliC gene and lacked flagella on the cell surface. Mutant cells were some 50% longer than parental strain cells, and at optimal growth temperatures, they exhibited a 4-fold increase in the expression of class I chaperone genes including GroEL and GroES. Increased chaperone expression, in addition to the non-flagellated phenotype of the mutant, may account for the increased biofilm formation observed. Overall, the phenotype resulting from dnaK disruption is more akin to that observed in Escherichia coli dnaK mutants, rather than those in the Gram-positive model organism Bacillus subtilis.

In Vitro Susceptibility to Closthioamide among Clinical and Reference Strains of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is one of the leading antimicrobial resistance threats worldwide. This study determined the MICs of closthioamide to be 0.008 to 0.5 mg/liter for clinical N. gonorrhoeae strains and related species. Cross-resistance with existing antimicrobial resistance was not detected, indicating that closthioamide could be used to treat drug-resistant N. gonorrhoeae.

A genetic assay for gene essentiality in Clostridium.

Essential genes of pathogens are potential therapeutic targets, but are difficult to verify. Here, gene essentiality was determined by targeted knockout following engineered gene duplication. Null mutants of candidate essential genes of Clostridium difficile were viable only in the presence of a stable second copy of the gene.

6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology.

In the search for alternative non-metabolizable inducers in the l-rhamnose promoter system, the synthesis of fifteen 6-deoxyhexoses from l-rhamnose demonstrates the value of synergy between biotechnology and chemistry. The readily available 2,3-acetonide of rhamnonolactone allows inversion of configuration at C4 and/or C5 of rhamnose to give 6-deoxy-d-allose, 6-deoxy-d-gulose and 6-deoxy-l-talose. Highly crystalline 3,5-benzylidene rhamnonolactone gives easy access to l-quinovose (6-deoxy-l-glucose), l-olivose and rhamnose analogue with C2 azido, amino and acetamido substituents. Electrophilic fluorination of rhamnal gives a mixture of 2-deoxy-2-fluoro-l-rhamnose and 2-deoxy-2-fluoro-l-quinovose. Biotechnology provides access to 6-deoxy-l-altrose and 1-deoxy-l-fructose.

Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter.

External control of gene expression is crucial in synthetic biology and biotechnology research and applications, and is commonly achieved using inducible promoter systems. The E. coli rhamnose-inducible rhaBAD promoter has properties superior to more commonly used inducible expression systems, but is marred by transient expression caused by degradation of the native inducer, l-rhamnose. To address this problem, 35 analogues of l-rhamnose were screened for induction of the rhaBAD promoter, but no strong inducers were identified. In the native configuration, an inducer must bind and activate two transcriptional activators, RhaR and RhaS. Therefore, the expression system was reconfigured to decouple the rhaBAD promoter from the native rhaSR regulatory cascade so that candidate inducers need only activate the terminal transcription factor RhaS. Rescreening the 35 compounds using the modified rhaBAD expression system revealed several promising inducers. These were characterized further to determine the strength, kinetics, and concentration-dependence of induction; whether the inducer was used as a carbon source by E. coli; and the modality (distribution) of induction among populations of cells. l-Mannose was found to be the most useful orthogonal inducer, providing an even greater range of induction than the native inducer l-rhamnose, and crucially, allowing sustained induction instead of transient induction. These findings address the key limitation of the rhaBAD expression system and suggest it may now be the most suitable system for many applications.