Biology subjects

Strucko, T.

Publications and source records attributed to Strucko, T..

3 recordsLinked to original sources

bioRxiv2023

Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii

Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bio-production of valuable compounds is not yet fully exploited. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of non-conventional species including K. phaffii. Yet, improvements in gene-editing methods are desirable to further accelerate engineering of industrially and scientifically relevant K. phaffii strains. In this study, we have developed a versatile one vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences. Importantly, we show that by using our setup it is possible to catalyze single-stranded oligonucleotide-mediated mutagenesis and marker-free gene integrations. Notably, we performed site-specific point mutations and full gene deletions using single stranded 90-mers at very high efficiencies. Lastly, we present a strategy for transient inactivation of non-homologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). The latter system enables precise CRISPR-Cas9 based editing (including multiplexing) and accelerates selection of the mutants that have simultaneously undergone a desired genetic modification(s) and restored NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and could potentially be coupled with high-throughput automated workflows.

synthetic biology↗

bioRxiv2023

Microbial cell factory optimisation using genome-wide host-pathway interaction screens

The ubiquity of genetic interactions in living cells challenges the concept of parts orthogonality, which is a cornerstone of synthetic biology. Parts, such as heterologously expressed genes, draw from shared pools of limited cellular resources and interactions between parts themselves and their host are inevitable. Instead of trying to eliminate or disregard these interactions, we propose to leverage them to promote desirable phenotypes. We recently described CRI-SPA, a method for high-throughput genome-wide gene delivery and screening of host:pathway interactions in Saccharomyces cerevisiae. In this study, we combine this method with biosensor-based high-throughput screening and high-density colony image analysis to identify lead engineering targets for optimising cis-cis-muconic acid (CCM) production in yeast cell factories. Using the biosensor screen, we phenotype >9,700 genotypes for their interaction with the heterologously expressed CCM biosynthesis pathway, including both gene knock-out and overexpression, and identify novel metabolic targets belonging to sulphur assimilation and methionine synthesis, as well as cellular redox homeostasis, positively impacting CCM biosynthesis by up to 280%. Our genome-wide exploration of host pathway interaction opens novel strategies for the metabolic engineering of yeast cell factories.

synthetic biology↗

bioRxiv2022

CRI-SPA, a mating based CRISPR-Cas9 assisted method for high-throughput genetic modification of yeast strain libraries

AbstractBiological functions are orchestrated by intricate networks of interacting genetic elements. Predicting the interaction landscape remains a challenge for systems biology and the identification of phenotypic maximas would be of great benefit to synthetic biology. Thus, new research tools allowing simple and rapid mapping of sequence to function are required to forward these research fields. Here, we describe CRI-SPA, a method allowing the transfer of a chromosomal genetic feature from a donor strain to arrayed strains in large libraries of Saccharomyces cerevisiae. CRI-SPA is based on mating, CRISPR-Cas9-induced gene conversion and Selective Ploidy Ablation and is executed within a week. We demonstrate the power of CRI-SPA by transferring four genes responsible for the production of betaxanthin, a yellow biosensor for the morphine precursor L-DOPA, into each strain of the yeast knock-out collection ({approx}4800 strains), providing a genome-wide overview of the genetic requirements for betaxanthin production. CRI-SPA is fast, highly reproducible, can be massively parallelized with automation and does not require selection for the transferred genetic feature.

systems biology↗