# Alien Chromosome Serves as a Novel Platform for Multiple Gene Expression in Kluyveromyces marxianus

## TL;DR

Scientists used a yeast species to express multiple genes by inserting a foreign chromosome, enabling stable and efficient gene expression for biotechnology applications.

## Contribution

The study introduces an alien chromosome as a stable platform for multi-gene expression in Kluyveromyces marxianus.

## Key findings

- Replacing genes on the alien chromosome with heterologous genes led to active expression and enhanced enzyme production.
- Multiple gene insertions at specific loci resulted in higher expression levels compared to single insertions.
- The alien chromosome platform supports stable propagation without selective pressure.

## Abstract

Kluyveromyces marxianus is an emerging yeast cell host for diverse products, but multiple-gene expression in K. marxianus faces challenges due to limited current knowledge of cis-regulatory elements and insertion loci. Our previous study transferred an alien Saccharomyces cerevisiae chromosome I (R1) into K. marxianus, resulting in the creation of the monochromosomal hybrid yeast KS-R1. All R1 genes were actively transcribed, providing a series of loci with varying transcriptional activities. Here, we explore the use of R1 as a novel platform for stable, multi-gene integration and expression. By deleting three essential K. marxianus genes while complementing their functions with orthologs on R1, we achieved stable propagation of R1 in the absence of selective pressure. We characterized several loci on R1 that exhibit stable transcriptional activities under various conditions. GFP inserted in place of genes at six such loci demonstrated varying expression levels. Strains with GFP at two loci exhibited significantly higher expression than those with GFP at a single locus. Furthermore, we replaced five R1 genes with disulfide bond formation genes from Pichia pastoris at distinct loci, resulting in the active expression of all five genes and significantly enhanced production of heterologous glucoamylases BadGLA and TeGlaA. Our findings demonstrate that alien chromosomes offer a stable and versatile platform for the coordinated expression of multiple heterologous genes, serving as valuable tools for metabolic engineering and synthetic biology.