45 results found
Ledesma Amaro R, Selles Vidal L, Isalan M, et al., 2023, A primer to directed evolution: current methodologies and future directions, RSC Chemical Biology, Vol: 4, Pages: 271-291, ISSN: 2633-0679
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.
Little GT, Vidal LS, Steadman M, et al., 2022, Complete Genome Sequence of Paraclostridium bifermentans DSM 14991, MICROBIOLOGY RESOURCE ANNOUNCEMENTS, Vol: 11, ISSN: 2576-098X
Vidal LS, Murray JW, Heap JT, 2021, Versatile selective evolutionary pressure using synthetic defect in universal metabolism, Nature Communications, Vol: 12, Pages: 1-15, ISSN: 2041-1723
Versatile selective evolutionary pressure using synthetic defect in universal metabolism
Taylor GM, Hitchcock A, Heap JT, 2021, Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria, NUCLEIC ACIDS RESEARCH, Vol: 49, ISSN: 0305-1048
Thirumurthy MA, Hitchcock A, Cereda A, et al., 2020, Type IV Pili-Independent Photocurrent Production by the Cyanobacterium Synechocystis sp. PCC 6803, FRONTIERS IN MICROBIOLOGY, Vol: 11, ISSN: 1664-302X
Liu Z, Yoshihara A, Jenkinson SF, et al., 2020, Hanessian-Hullar reaction in the synthesis of highly substituted trans-3,4-dihydroxypyrrolidines: Rhamnulose iminosugar mimics inhibit alpha-glucosidase, TETRAHEDRON, Vol: 76, ISSN: 0040-4020
Taylor GM, Heap JT, 2020, Design and Implementation of Multi-protein Expression Constructs and Combinatorial Libraries using Start-Stop Assembly, DNA CLONING AND ASSEMBLY, Vol: 2205, Pages: 219-237, ISSN: 1064-3745
Kelly C, Taylor G, Satkute A, et al., 2019, Transcriptional terminators allow leak-free chromosomal integration of genetic constructs in cyanobacteria, Microorganisms, Vol: 7, Pages: 1-9, ISSN: 2076-2607
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.
Taylor G, Mordaka P, Heap J, 2019, Start-stop assembly: a functionally scarless DNA assembly system optimized for metabolic engineering, Nucleic Acids Research, Vol: 47, Pages: e17-e17, ISSN: 0305-1048
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 minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.
Little GT, Willson BJ, Heap JT, et al., 2018, The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools, BIOTECHNOLOGY JOURNAL, Vol: 13, ISSN: 1860-6768
Kelly CL, Harris AWK, Steel H, et al., 2018, Synthetic negative feedback circuits using engineered small RNAs, Nucleic Acids Research, Vol: 46, Pages: 9875-9889, ISSN: 0305-1048
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.
Kelly CL, Taylor GM, Hitchcock A, et al., 2018, A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria., ACS Synth Biol, Vol: 7, Pages: 1056-1066
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.
Taylor G, Mordaka P, Heap J, 2018, Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering.
Mordaka PM, Heap JT, 2018, Stringency of Synthetic Promoter Sequences in Clostridium Revealed and Circumvented by Tuning Promoter Library Mutation Rates, ACS Synthetic Biology, Vol: 7, Pages: 672-681
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.
Sellés Vidal L, Kelly CL, Mordaka PM, et al., 2018, Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application., Biochim Biophys Acta Proteins Proteom, Vol: 1866, Pages: 327-347, ISSN: 1570-9639
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.
Jain S, Smyth D, O'Hagan BMG, et al., 2017, Inactivation of the dnaK gene in Clostridium difficile 630 Δerm yields a temperature- sensitive phenotype and increases biofilm-forming ability, Scientific Reports, Vol: 7, ISSN: 2045-2322
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.
Miari VF, Solanki P, Hleba Y, et al., 2017, In vitro susceptibility to closthioamide among clinical and reference strains of Neisseria gonorrhoeae, Antimicrobial Agents and Chemotherapy, Vol: 10, ISSN: 0066-4804
Neisseria gonorrhoeae is one of the leading antimicrobial resistance threats worldwide. This study determined the minimum inhibitory concentrations of closthioamide to be 0.008-0.5 mg/L 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.
Kelly CL, Liu Z, Yoshihara A, et al., 2016, Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the rhaBAD Promoter., ACS Synth Biol, Vol: 5, Pages: 1136-1145
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.
Walker DJ, Heap JT, Winzer K, et al., 2016, A genetic assay for gene essentiality in Clostridium, Anaerobe, Vol: 42, Pages: 40-43, ISSN: 1095-8274
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.
Liu Z, Yoshihara A, Kelly C, et al., 2016, 6-Deoxyhexoses from l-Rhamnose in the Search for Inducers of the Rhamnose Operon: Synergy of Chemistry and Biotechnology, Chemistry, Vol: 22, Pages: 12557-12565, ISSN: 1521-3765
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.
Ehsaan M, Kuit W, Zhang Y, et al., 2016, Mutant generation by allelic exchange and genome resequencing of the biobutanol organism Clostridium acetobutylicum ATCC 824, Biotechnology for Biofuels, Vol: 9, Pages: 4-4, ISSN: 1754-6834
BACKGROUND: Clostridium acetobutylicum represents a paradigm chassis for the industrial production of the biofuel biobutanol and a focus for metabolic engineering. We have previously developed procedures for the creation of in-frame, marker-less deletion mutants in the pathogen Clostridium difficile based on the use of pyrE and codA genes as counter selection markers. In the current study we sought to test their suitability for use in C. acetobutylicum. RESULTS: Both systems readily allowed the isolation of in-frame deletions of the C. acetobutylicum ATCC 824 spo0A and the cac824I genes, leading to a sporulation minus phenotype and improved transformation, respectively. The pyrE-based system was additionally used to inactivate a putative glycogen synthase (CA_C2239, glgA) and the pSOL1 amylase gene (CA_P0168, amyP), leading to lack of production of granulose and amylase, respectively. Their isolation provided the opportunity to make use of one of the key pyrE system advantages, the ability to rapidly complement mutations at appropriate gene dosages in the genome. In both cases, their phenotypes were restored in terms of production of granulose (glgA) and amylase (amyP). Genome re-sequencing of the ATCC 824 COSMIC consortium laboratory strain used revealed the presence of 177 SNVs and 49 Indels, including a 4916-bp deletion in the pSOL1 megaplasmid. A total of 175 SNVs and 48 Indels were subsequently shown to be present in an 824 strain re-acquired (Nov 2011) from the ATCC and are, therefore, most likely errors in the published genome sequence, NC_003030 (chromosome) and NC_001988 (pSOL1). CONCLUSIONS: The codA or pyrE counter selection markers appear equally effective in isolating deletion mutants, but there is considerable merit in using a pyrE mutant as the host as, through the use of ACE (Allele-Coupled Exchange) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic eff
Heap JT, Theys J, Ehsaan M, et al., 2014, Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo, ONCOTARGET, Vol: 5, Pages: 1761-1769
Kovacs K, Willson BJ, Schwarz K, et al., 2013, Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824, BIOTECHNOLOGY FOR BIOFUELS, Vol: 6, ISSN: 1754-6834
Jabbari S, Steiner E, Heap JT, et al., 2013, The putative influence of the agr operon upon survival mechanisms used by Clostridium acetobutylicum, MATHEMATICAL BIOSCIENCES, Vol: 243, Pages: 223-239, ISSN: 0025-5564
Zhang Z, Korkeala H, Dahlsten E, et al., 2013, Two-Component Signal Transduction System CBO0787/CBO0786 Represses Transcription from Botulinum Neurotoxin Promoters in Clostridium botulinum ATCC 3502, PLOS PATHOGENS, Vol: 9, ISSN: 1553-7366
Lindstrom M, Dahlsten E, Soderholm H, et al., 2012, Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 78, Pages: 5466-5470, ISSN: 0099-2240
Cartman ST, Kelly ML, Heeg D, et al., 2012, Precise Manipulation of the Clostridium difficile Chromosome Reveals a Lack of Association between the tcdC Genotype and Toxin Production, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol: 78, Pages: 4683-4690, ISSN: 0099-2240
Saad NY, Schiel B, Braye M, et al., 2012, Riboswitch (T-box)-mediated Control of tRNA-dependent Amidation in Clostridium acetobutylicum Rationalizes Gene and Pathway Redundancy for Asparagine and Asparaginyl-tRNA(Asn) Synthesis, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 287, Pages: 20382-20394
Heap JT, Ehsaan M, Cooksley CM, et al., 2012, Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker, NUCLEIC ACIDS RESEARCH, Vol: 40, ISSN: 0305-1048
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