Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae.
ACS Synth Biol
; 9(3): 546-555, 2020 03 20.
Article
em En
| MEDLINE
| ID: mdl-32049515
ABSTRACT
Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 ± 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Pentanóis
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Engenharia Metabólica
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Mitocôndrias
Idioma:
En
Revista:
ACS Synth Biol
Ano de publicação:
2020
Tipo de documento:
Article
País de afiliação:
Estados Unidos