Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae

• Published in: Biotechnology and bioengineering 2021-11, Vol.118 (11), p.4269-4277
• Main Authors: Dong, Chang, Shi, Zhuwei, Huang, Lei, Zhao, Huimin, Xu, Zhinan, Lian, Jiazhang
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; Cassettes; Cloning; Cloning, Molecular; Compartmentalization engineering; Complementation; Confocal microscopy; E coli; Engineering; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescence; Metabolic Engineering; Metabolic Networks and Pathways; Metabolic pathways; Metabolism; Mitochondria; Mitochondria - genetics; Mitochondria - metabolism; Mitochondrial targeting sequences; Proteins; Proteomes; Pyruvic acid; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Yeast; Yeasts; α-Santalene
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27896

Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. Much progress has been made in reconstituting whole metabolic pathways in the mitochondria of yeast to harness the precursor pools (i.e., pyruvate and acetyl‐CoA), bypass competing pathways, and minimize transportation limitations. However, only a few mitochondrial targeting sequences (MTSs) have been characterized (i.e., MTS of COX4), limiting the application of compartmentalization engineering for multigene biosynthetic pathways in the mitochondria of yeast. In the present study, based on the mitochondrial proteome, a total of 20 MTSs were cloned and the efficiency of these MTSs in targeting heterologous proteins, including the Escherichia coli FabI and enhanced green fluorescence protein (EGFP) into the mitochondria was evaluated by growth complementation and confocal microscopy. After systematic characterization, six of the well‐performed MTSs were chosen for the colocalization of complete biosynthetic pathways into the mitochondria. As proof of concept, the full α‐santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acetyl‐coA to α‐santalene was compartmentalized into the mitochondria, leading to a 3.7‐fold improvement in the production of α‐santalene. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering. Mitochondrion is generally considered as the most promising subcellular organelle for compartmentalization engineering. The authors systematically characterized a panel of mitochondrial targeting sequences (MTSs), many of which demonstrated good mitochondrial targeting performance. The newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.