Enhanced single-base mutation diversity by the combination of cytidine deaminase with DNA-repairing enzymes in yeast.

The occurrence of random mutations can increase the diversity of the genome and promote the evolutionary process of organisms. High efficiency mutagenesis techniques significantly accelerate the evolutionary process. In this work, we describe a targeted mutagenesis system named MutaT7trans to significantly increase mutation rate and generate mutations across all four nucleotides in yeast. We constructed different DNA-repairing enzyme-PmCDA1-T7 RNA polymerase (T7 RNAP) fusion proteins, achieved targeted mutagenesis by flanking the target gene with T7 promoters, and tuned the mutation spectra by introducing different DNA-repairing enzymes. With this mutagenesis tool, the proportion of non-C â†’ T mutations was 10-11-fold higher than the cytidine deaminase-based evolutionary tools, and the transversion mutation frequency was also elevated. The mutation rate of the target gene was significantly increased to 5.25 × 10-3 substitutions per base (s. p. b.). We also demonstrated that MutaT7trans could be used to evolve the CrtE, CrtI, and CrtYB gene in the ß-carotene biosynthesis process and generate different types of mutations.

Genome engineering on size reduction and complexity simplification: A review.

BACKGROUND: Genome simplification is an important topic in the field of life sciences that has attracted attention from its conception to the present day. It can help uncover the essential components of the genome and, in turn, shed light on the underlying operating principles of complex biological systems. This has made it a central focus of both basic and applied research in the life sciences. With the recent advancements in related technologies and our increasing knowledge of the genome, now is an opportune time to delve into this topic. AIM OF REVIEW: Our review investigates the progress of genome simplification from two perspectives: genome size reduction and complexity simplification. In addition, we provide insights into the future development trends of genome simplification. KEY SCIENTIFIC CONCEPTS OF REVIEW: Reducing genome size requires eliminating non-essential elements as much as possible. This process has been facilitated by advances in genome manipulation and synthesis techniques. However, we still need a better and clearer understanding of living systems to reduce genome complexity. As there is a lack of quantitative and clearly defined standards for this task, we have opted to approach the topic from various perspectives and present our findings accordingly.

Cross-species microbial genome transfer: a Review.

Synthetic biology combines the disciplines of biology, chemistry, information science, and engineering, and has multiple applications in biomedicine, bioenergy, environmental studies, and other fields. Synthetic genomics is an important area of synthetic biology, and mainly includes genome design, synthesis, assembly, and transfer. Genome transfer technology has played an enormous role in the development of synthetic genomics, allowing the transfer of natural or synthetic genomes into cellular environments where the genome can be easily modified. A more comprehensive understanding of genome transfer technology can help to extend its applications to other microorganisms. Here, we summarize the three host platforms for microbial genome transfer, review the recent advances that have been made in genome transfer technology, and discuss the obstacles and prospects for the development of genome transfer.

The TelN/tos-assisted precise targeting of chromosome segments (TAPE).

INTRODUCTION: Performing genomic large segmentation experiments will promote the annotation of complex genomic functions and contribute to the synthesis of designed genomes. It is challenging to obtain and manipulate large or complex DNA sequences with high efficiency. OBJECTIVES: This study aims to develop an effective method for direct cloning of target genome sequences from different species. METHODS: The TelN/tos system and a linear plasmid vector were first used to directly clone the large genomic segments in E. coli. For the in vitro cloning reaction, two telomeric sites were developed using TelN protelomerase at the end of the linear plasmid vector. The target DNA sequence can be easily hooked with the homology arms and maintained as a linear artificial chromosome with arbitrary restriction sites in a specific E. coli strain. RESULTS: Using the linear cloning strategy, we successfully cloned the bacterial DNA fragment of 156 kb, a yeast genomic fragment of 124 kb and mammalian mitochondrial fragment of 16 kb. The results showed a considerable improvement in cloning efficiency and demonstrated the important role of vector ratio in the cloning process. CONCLUSION: Due to the high efficiency and stability, TAPE is an effective technique for DNA cloning and fundamental molecular biotechnology method in synthetic biology.