TY - JOUR
T1 - Cloning and characterization of a panel of mitochondrial targeting sequences for compartmentalization engineering in Saccharomyces cerevisiae
AU - Dong, Chang
AU - Shi, Zhuwei
AU - Huang, Lei
AU - Zhao, Huimin
AU - Xu, Zhinan
AU - Lian, Jiazhang
N1 - This study was supported by the National Key Research and Development Program of China (2018YFA0901800 to JL), the Natural Science Foundation of Zhejiang Province (LR20B060003 to JL), the Natural Science Foundation of China (21808199 to JL), and the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (DE‐SC0018420 to HZ).
PY - 2021/11
Y1 - 2021/11
N2 - 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.
AB - 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.
KW - Saccharomyces cerevisiae
KW - compartmentalization engineering
KW - metabolic engineering
KW - mitochondrial targeting sequences
KW - α-santalene
UR - http://www.scopus.com/inward/record.url?scp=85111092098&partnerID=8YFLogxK
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U2 - 10.1002/bit.27896
DO - 10.1002/bit.27896
M3 - Article
C2 - 34273106
AN - SCOPUS:85111092098
SN - 0006-3592
VL - 118
SP - 4269
EP - 4277
JO - Biotechnology and bioengineering
JF - Biotechnology and bioengineering
IS - 11
ER -