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

Dong, Chang; Shi, Zhuwei; Huang, Lei; Zhao, Huimin; Xu, Zhinan; Lian, Jiazhang

doi:10.1002/bit.27896

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

Journal Article · Sat Jul 17 00:00:00 EDT 2021 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.27896· OSTI ID:1855988

Dong, Chang ^[1]; Shi, Zhuwei ^[1]; Huang, Lei ^[1]; Zhao, Huimin ^[2];

^[3];

^[3]

Zhejiang University, Hangzhou (China)
University of Illinois at Urbana-Champaign, IL (United States)
Zhejiang University, Hangzhou (China); University of Illinois at Urbana-Champaign, IL (United States)

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 alpha-santalene biosynthetic pathway consisting of 10 expression cassettes capable of converting acety coA to alpha-santalene was compartmentalized into the mitochondria, leading to a 3.7 fold improvement in the production of alpha-santalene. Furthermore, the newly characterized MTSs should contribute to the expanded metabolic engineering and synthetic biology toolbox for yeast mitochondrial compartmentalization engineering.

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Research Organization:: CABBI, Urbana, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018420

OSTI ID:: 1855988

Journal Information:: Biotechnology and Bioengineering, Vol. 118, Issue 11; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
Mitochondrial targeting sequences
Saccharomyces cerevisiae
Compartmentalization engineering
Metabolic engineering
α-Santalene

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

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