PCR & Go: A Pre-installed Expression Chassis for Facile Integration of Multi-Gene Biosynthetic Pathways

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

Debugging: putting the synthetic yeast chromosome to work

This review summarizes strategies used to map and repair various bugs in synthetic genomic sequences and provides guidance for the construction of synthetic yeast chromosomes that are capable of maintaining cell fitness.

Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

A multi-scale model of the yeast chromosome-segregation system

ABSTRACTIn dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome-segregation machinery at molecular resolution in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer-kinetochore assemblies.Dam1C/DASH only contacts the flat surface of the microtubule and does so with its flexible “bridges”. In metaphase, 40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study supports a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.