Biology subjects

Spinck, M.

Publications and source records attributed to Spinck, M..

4 recordsLinked to original sources

bioRxiv2026

Continuous target-specific mutagenesis and rapid gene evolution by diversity-generating retroelements in Escherichia coli

Sequence-programmable directed evolution systems have great potential to accelerate bioengineering. Diversity-generating retroelements (DGRs) are natural hypermutation systems widely distributed in prokaryotes and bacteriophages with the capacity to introduce diverse mutations at template-specified sites of target genes. Here, we show that DGRs can be installed in E. coli and reprogrammed for the continuous, iterative mutagenesis of user-defined target genes. We show that the DGR template RNA can be reprogrammed for gene- and residue- specific mutagenesis, leaving untargeted, adjacent residues unchanged. Furthermore, we establish continuous DGR-enabled mutagenesis with F-plasmid-mediated horizontal gene transfer of target genes (HGT-DGR) into a new host for the progressive accumulation of target- specific mutations. Iterative HGT-DGR mutagenesis over seven cycles yielded an average mutation load of approximately 6% across adenine positions in the target segment, generating a diverse library of variants comprising 40% mutant sequences, with a median pairwise Hamming distance of 4 among mutant variants. HGT-DGR enables iterative diversification of either the same or different user-specified segments of the target gene, as demonstrated with the directed evolution of the M. mazei pyrrolysyl-tRNA synthetase for non-canonical amino acid incorporation. HGT-DGR provides a simple, low-cost, sequence-programmable system that enables iterative, position-specific and tunable in vivo mutagenesis of any target sequence for applications in biotechnology and medicine.

synthetic biology↗

bioRxiv2026

Continuous site-directed mutagenesis and selection in Escherichia coli

The programmed diversification of genes and other encoded genetic elements, through site directed- and site saturation-mutagenesis, underpins approaches to learning the relationship between DNA sequence and function, and forms a foundation for creating new function through directed evolution. However, current approaches for generating large genetic libraries commonly generate diversity in vitro and then transform the resulting library into cells; this multistep process is inefficient and this paradigm places limits on the scale of diversity that can be achieved and the size of diversified genetic elements that can be introduced into cells. Here we demonstrate a distinct paradigm for library generation through: 1) efficient transfer of genetic elements, as cargos in F plasmids, to recipient cells bearing libraries of retron editors, 2) efficient editing of cargo genes in recipient cells, and 3) continuous iteration of the conjugation-editing cycles with selection for recipients in sequential cycles using three selection markers in series. In this paradigm, the library diversity emerges multiplicatively through the iteration of conjugation-editing cycles. Using this paradigm, we generated substantial libraries that enabled the selection of new phenotypes, with library members containing up to six distinct edits and edits arising from several conjugation-editing cycles.

synthetic biology↗

bioRxiv2025

Escherichia coli with a 57-codon genetic code

The near-universal genetic code of living organisms uses 64 codons to encode the 20 canonical amino acids in protein synthesis. Here we design and generate a variant of Escherichia coli with a 4 Mb synthetic genome in which we replace every known occurrence of six sense codons and a stop codon with synonymous codons. We thereby recode 105 codons to create an organism with a 57-codon genetic code; this organism - which we name Syn57 - uses 55 codons to encode the 20 canonical amino acids.

synthetic biology↗

bioRxiv2019

Evolved, Selective Erasers of Distinct Lysine Acylations

Lysine acetylation, including related lysine modifications such as butyrylation and crotonylation, is a widespread post-translational modification with important roles in many important physiological processes. However, uncovering the regulatory mechanisms that govern the reverse process, deacylation, has been challenging to address, in great part because the small set of lysine deacetylases (KDACs) that remove the modifications are promiscuous in their substrate and acylation-type preference. This lack of selectivity hinders a broader understanding of how deacylation is regulated at the cellular level and how it is correlated with lysine deacylation-related diseases. To facilitate the dissection of KDACs with respect to substrate specificity and modification type, it would be beneficial to re-engineer KDACs to be selective towards a given substrate and/or modification. To dissect the differential contributions of various acylations to cell physiology, we developed a novel directed evolution approach to create selective KDAC variants that are up to 400-fold selective towards butyryl- over crotonyl-lysine substrates. Structural analyses of this non-promiscuous KDAC revealed unprecedented insights regarding the conformational changes mediating the gain in specificity. As a second case study to illustrate the power of this approach, we re-engineer the human SirT1 to increase its selectivity towards acetylated versus crotonylated substrates. These new enzymes, as well as the generic approach that we report here, will greatly facilitate the dissection of the differential roles of lysine acylation in cell physiology.\n\nSignificance StatementAcetylation of lysine residues features numerous roles in diverse physiological processes and correlates with the manifestation of metabolic diseases, cancer and ageing. The already huge diversity of the acetylome is multiplied by variations in the types of acylation. This complexity is in stark contrast to the small set of lysine deacetylases (KDACs) present in human cells, anticipating a pronounced substrate promiscuity.\n\nWe device a strategy to tackle this disarray by creating KDAC variants with increased selectivity towards particular types of lysine acylations using a novel selection system. The variants facilitate the dissection of the differential contributions of particular acylations to gene expression, development and disease. Our structural analyses shed light on the mechanism of substrate discrimination by Sirtuin-type KDACs.

biochemistry↗