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Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, and parity of deamination for four different MutaT7GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology, with its ability to target a specific DNA locus using guide RNAs (gRNAs), is particularly suited for targeted mutagenesis. The targeted diversification of nucleotides in Saccharomyces cerevisiae using a CRISPR-guided error-prone DNA polymerase─called yEvolvR─was recently reported. Here, we investigate the effect of multiplexed expression of gRNAs flanking a short stretch of DNA on reversion and mutation frequencies using yEvolvR. Phenotypic assays demonstrate that higher reversion frequencies are observed when expressing multiple gRNAs simultaneously. Next generation sequencing reveals a synergistic effect of multiple gRNAs on mutation frequencies, which is more pronounced in a mutant with a partially defective DNA mismatch repair system. Additionally, we characterize a galactose-inducible yEvolvR, which enables temporal control of mutagenesis. This study demonstrates that multiplex expression of gRNAs and induction of mutagenesis greatly improves the capabilities of yEvolvR for generation of genetic libraries in vivo.

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The eukaryotic yeast Saccharomyces cerevisiae is a model host utilized for whole cell biocatalytic conversions, protein evolution, and scientific inquiries into the pathogenesis of human disease. Over the past decade, the scale and pace of such studies has drastically increased alongside the advent of novel tools for both genome-wide studies and targeted genetic mutagenesis. In this review, we will detail past and present (e.g., CRISPR/Cas) genome-scale screening platforms, typically employed in the context of growth-based selections for improved whole cell phenotype or for mechanistic interrogations. We will further highlight recent advances that enable the rapid and often continuous evolution of biomolecules with improved function. Additionally, we will detail the corresponding advances in high throughput selection and screening strategies that are essential for assessing or isolating cellular and protein improvements. Finally, we will describe how future developments can continue to advance yeast high throughput engineering.

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Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with in vivo continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. IMPORTANCE Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid in vivo mutagenesis. The combination of growth-based screening with in vivo mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis.

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Adaptive laboratory evolution (ALE) has served as a historic microbial engineering method that mimics natural selection to obtain desired microbes. The past decade has witnessed improvements in all aspects of ALE workflow, in terms of growth coupling, genotypic diversification, phenotypic selection, and genotype-phenotype mapping. The developing growth-coupling strategies facilitate ALE to a wider range of appealing traits. In vivo mutagenesis methods and multiplexed automated culture platforms open new gates to streamline its execution. Meanwhile, the application of multi-omics analyses and multiplexed genetic engineering promote efficient knowledge mining. This article provides a comprehensive and updated review of these advances, highlights newest significant applications, and discusses future improvements, aiming to provide a practical guide for implementation of novel, effective, and efficient ALE experiments.

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Kirkland et al. [Mutation Research/Genetic Toxicology and Environmental Mutagenesis 847 (2019) 403035, https://doi.org/10.1016/j.mrgentox.2019.03.008; Mutation Research/Genetic Toxicology and Environmental Mutagenesis 839 (2019): 21-35, https://doi.org/10.1016/j.mrgentox.2019.01.007] made recommendations on the use of the in vivo comet and transgenic rodent (TGR) gene mutation assays to screen for in vivo mutagenicity. Although it is not directly stated in either of these publications, we are concerned that the reports could potentially be used to support assertions that it is equally acceptable to follow up a positive bacterial reverse mutation (Ames) finding for an investigational drug with either the in vivo TGR mutation assay or an in vivo comet assay. For regulatory genotoxicity assessment, the in vivo follow-up for an in vitro bacterial mutation-positive drug, drug-related metabolite, or impurity should be based upon evaluating a similar endpoint (i.e., mutagenicity) as the intent is to determine if the findings of in vitro gene mutation correlate with findings of in vivo gene mutation (i.e., biologically relevant to the in vitro results). Thus, the most scientifically appropriate in vivo assays would be the TGR mutation assay or, in some circumstances, the in vivo Pig-a assay. An in vivo rodent comet assay or combination of the in vivo micronucleus and in vivo rodent comet assays would generally not be an appropriate follow-up test.

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We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

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L-Tryptophan (Trp) is a high-value aromatic amino acid with diverse applications in food and pharmaceutical industries. Although production of Trp by engineered Escherichia coli has been extensively studied, the need of multiple precursors for its synthesis and the complex regulations of the biosynthetic pathways make the achievement of a high product yield still very challenging. Metabolic flux analysis suggests that the use of a phosphoenolpyruvate:sugar phosphotransferase system (PTS) independent glucose uptake system, i.e. the galactose permease/glucokinase (GalP/Glk) system, can theoretically double the Trp yield from glucose. To explore this possibility, a PTS- and GalP/Glk-dependent E. coli strain was constructed from a previously rationally developed Trp producer strain S028. However, the growth rate of the S028 mutant was severely impaired. To overcome this problem, promoter screening for modulated gene expression of GalP/Glk was carried out, following by a batch mode of adaptive laboratory evolution (ALE) which resulted in a strain K3 with a similar Trp yield and concentration as S028. In order to obtain a more efficient Trp producer, a novel continuous ALE system was developed by combining CRISPR/Cas9-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of Trp-mediated fluorescence intensity. With the aid of this automatic system (auto-CGSS), a promising strain T5 was obtained and fed-batch fermentations showed an increase of Trp yield by 19.71% with this strain compared with that obtained by the strain K3 (0.164 vs. 0.137 â€‹g/g). At the same time, the specific production rate was increased by 52.93% (25.28 vs. 16.53 â€‹mg/g DCW/h). Two previously engineered enzyme variants AroGD6G-D7A and AnTrpCR378F were integrated into the strain T5, resulting in a highly productive strain T5AA with a Trp yield of 0.195 â€‹g/g and a specific production rate of 28.83 â€‹mg/g DCW/h.

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The in vivo mutagenicity of hexavalent chromium in the small intestine, the target organ of tumorgenicity, was examined by means of a transgenic mouse gene mutation assay. Sodium dichromate dihydrate was administered orally in drinking water to male gpt delta mice at a dose of 85.7 or 257.4 mg/L for 28 days or at a dose of 8.6, 28.6 or 85.7 mg/L for 90 days. No significant increase in gpt mutant frequency relative to that in control mice was observed in the small intestine in either the 28- or 90-day study, whereas 28-day oral administration of potassium bromate, a positive control substance, increased mutant frequency.

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The yeast cytoplasmically localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically increased without influencing genomic replication, thereby supporting in vivo continuous evolution. Here, we report that the pGKL2/TP-DNAP2 plasmid/DNA polymerase pair forms a second orthogonal replication system. We show that custom genes can be encoded and expressed from pGKL2, that error-prone TP-DNAP2s can be engineered, and that pGKL2 replication by TP-DNAP2 is both orthogonal to genomic replication in Saccharomyces cerevisiae and mutually orthogonal with pGKL1 replication by TP-DNAP1. This demonstration of two mutually orthogonal DNA replication systems with tunable error rates and properties should enable new applications in cell-based continuous evolution, genetic recording, and synthetic biology at large.

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This chapter presents the three-dimensional (3D) model of the Sigma1 receptor protein as obtained from homology modeling techniques. We show the applicability of this structure to docking-based virtual screening and discuss combined in silico/in vitro mutagenesis studies performed to validate the structural features of the Sigma1 receptor model and to qualify/quantify the prominent role of specific amino acid residues in ligand binding. The validation of the virtual 3D Sigma1 receptor model and its reliable applicability to docking-based virtual screening is of significance for rational ligand design, even in light of the recently reported crystal structure for the Sigma1 receptor.

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Furan is an abundant food and environmental contaminant that is a potent liver carcinogen in rodent models. To determine if furan is genotoxic in vivo, female B6C3F1 Big Blue transgenic mice were treated with 15 mg/kg bw furan by gavage 5 days a week for 6 weeks, or once weekly for 3 weeks. Liver cII transgene mutation-frequency and mutation spectra were determined. Furan did not increase the mutation frequency under either treatment condition. In the 6-week treatment regimen, there was a change in the cII transgene mutation-spectrum, with the fraction of GC to AT transitions significantly reduced. The only other significant change was an increase in GC to CG transversions; these represented a minor contribution to the overall mutation spectrum. A much larger furan-dependent shift was observed in the 3-week study. There was a significant increase in transversion mutations, predominantly GC to TA transversions as well as smaller non-significant changes in GC to CG and AT to TA transversions. To determine if these mutations were caused by cis-2-butene-1,4-dial (BDA), a reactive metabolite of furan, the mutagenic activity and the mutation spectrum of BDA was determined in vitro, in Big Blue mouse embryonic fibroblasts. This compound did not increase the cII gene mutation-frequency but caused a substantial increase in AT to CG transversions. This increase, however, lost statistical significance when adjusted for multiple comparisons. Together, these findings suggest that BDA may not be directly responsible for the in-vivo effects of furan on mutational spectra. Histopathological analysis of livers from furan-treated mice revealed that furan induced multifocal, hepatocellular necrosis admixed with reactive leukocytes and pigment-laden Kupffer cells, enhanced oval-cell hyperplasia, and increased hepatocyte mitoses, some of which were atypical. An indirect mechanism of genotoxicity is proposed in which chronic toxicity followed by inflammation and secondary cell proliferation triggers cancer development in furan-exposed rodents.

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