Engineering adaptive alleles for Escherichia coli growth on sucrose using the EasyGuide CRISPR system

Author(s)

Barreto, Joneclei Alves ; Lacorte e Silva, Matheus Victor Maso ; Marin, Danieli Canaver ; Brienzo, Michel ; Jacobus, Ana Paula ; Contiero, Jonas ; Gross, Jeferson

• Document type: Journal article
• Source: Journal of Biotechnology; v. 403, p. 14-pg., 2025-04-24.

Abstract

Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits E. coli's natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40-60mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in E. coli DH5 alpha, most of which corresponded to alleles mapped in E. coli DH5 alpha and E2348/69 ALE populations selected for sucrose propagation. For DH5 alpha ALE, sucrose consumption was supported by the cscBKA operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the pcnB deletion, conferred a 30-35 % fitness gain, as demonstrated by CRISPR-engineered strains. A similar to 5 % advantage was also associated with a similar to 40.4 kb deletion involving fli operons for flagella assembly. In E2348/69 ALE, inactivation of the hfl system suggested selection pressures for maintaining lambda-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using costeffective carbon sources. (AU)

• FAPESP's process: 17/50249-6 - A novel strategy for anaerobic production of polyhydroxyalkanoates (PHA)
• Grantee: Jonas Contiero
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 17/24453-5 - Collaborative network: modern genetic approaches to bust yeast tolerance to inhibitor-rich lignocellulosic hydrolysates
• Grantee: Ana Paula Jacobus
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Young Investigators Grants
• FAPESP's process: 17/22401-8 - Fruit-refinery: obtention processes, characterization methods and generation of products derived from residues of the fruitculture
• Grantee: Nelson Ramos Stradiotto
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 20/02246-0 - Evolutionary engineering of Escherichia coli for anaerobic production of polyhydroxyalkanoates from sugarcane-derived sucrose
• Grantee: Joneclei Alves Barreto
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 23/04162-7 - EasyGuide expanded: CRISPR applications in Saccharomyces cerevisiae supported by in vivo assembly of gRNAs and oligo pool libraries
• Grantee: Jeferson Gross
• Support Opportunities: Regular Research Grants