Andrew Currin

Kazachstania bulderi is a non-conventional yeast species able to grow efficiently on glucose and δ-gluconolactone at low pH. These unique traits make K. bulderi an ideal candidate for use in sustainable biotechnology processes including low pH fermentations and the production of green chemicals including organic acids. To accelerate strain development with this species, detailed information of its genetics is needed. Here, by employing long read sequencing we report a high-quality phased genome assembly for three strains of K. bulderi species, including the type strain. The sequences were assembled into 12 chromosomes with a total length of 14 Mb, and the genome was fully annotated at structural and functional levels, including allelic and structural variants, ribosomal array and mating type locus. This high-quality reference genome provides a resource to advance our fundamental knowledge of biotechnologically relevant non-conventional yeasts and to support the development of genetic tools for manipulating such strains towards their use as production hosts in biotechnological processes.

The pathogenic fungus Aspergillus fumigatus is a major etiological agent of fungal invasive and chronic diseases affecting tens of millions of individuals worldwide. Draft genome sequences of two clinical isolates (Af293 and A1163) are commonly used as reference genomes for analyses of clinical and environmental strains. However, the reference sequences lack coverage of centromeres, an accurate sequence for ribosomal repeats, and a comprehensive annotation of chromosomal rearrangements such as translocations and inversions. Here, we used PacBio Single Molecule Real-Time (SMRT), Oxford Nanopore and Illumina HiSeq sequencing for de novo genome assembly and polishing of two laboratory reference strains of A. fumigatus, CEA10 (parental isolate of A1163) and its descendant A1160. We generated full length chromosome assemblies and a comprehensive telomere-to-telomere coverage for CEA10 and near complete assembly of A1160 including ribosomal repeats and the sequences of centromeres, which we discovered to be composed of long transposon elements. We envision these high-quality reference genomes will become fundamental resources to study A. fumigatus biology, pathogenicity and virulence, and to discover more effective treatments against diseases caused by this fungus.

Directed evolution is a powerful tool for the rapid improvement of a target protein toward a desired fitness criteria, such as activity, specificity, or stability. In order to achieve these desired improvements, it is often beneficial to subject the entirety of the protein to mutagenesis. However, the creation of such libraries by targeted methods (i.e. site-directed mutagenesis) can be a laborious and costly task. Here we outline the GeneORator method, which uses Boolean “OR” logic to introduce specific codon mutations at multiple loci in a single reaction, thereby greatly reducing the experimental workload. The method describes library synthesis using asymmetric PCR, in which mutagenic primers are designed to create OR-type mutations at multiple sites of variation in a two-step protocol. As an example, we show how this can be utilized for controlled and economical mutagenesis of every amino acid codon in a gene. Key words Directed evolution Synthetic biology DNA library assembly Variant libraries Asymmetric PCR

Synthetic biology holds great potential for the industrial production of many fine and speciality chemicals, including natural products. To achieve production, selected enzyme sequences are constructed into metabolic pathways such that the entire catalytic cascade can occur in vivo. Successful production with high yields is dependent on the selection of enzymes which exhibit the

Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets.

Background: Genetically encoded biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal. Results: Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions: This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its substrate specificity to generate a biosensor for aromatic aldehydes.

Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilise the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N (Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structure-containing regions (276 out of a total 495 amino acids) to screen for improvements in kcat. Secondly, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity towards novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 1010-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein’s sequence space. OR-type libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering and synthetic biology.

Synthetic biology utilises the Design-Build-Test-Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilise this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using PCR is introduced and sequencing data is analysed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 x 96 well plates) were processed in a single workflow in 72 hours (from E. coli colonies to analysed data). Given our procedure’s low hardware costs and highly multiplexed capability, this provides cost effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, SNP analysis and gene synthesis.