Synergistic Rewiring of Carbon Metabolism and Redox
Metabolism in Cytoplasm and Mitochondria of <i>Aspergillus oryzae</i> for Increased l‑Malate Production
Jingjing Liu
Jianghua Li
Yanfeng Liu
Hyun-dong Shin
Rodrigo Ledesma-Amaro
Guocheng Du
Jian Chen
Long Liu
10.1021/acssynbio.8b00130.s001
https://acs.figshare.com/articles/journal_contribution/Synergistic_Rewiring_of_Carbon_Metabolism_and_Redox_Metabolism_in_Cytoplasm_and_Mitochondria_of_i_Aspergillus_oryzae_i_for_Increased_l_Malate_Production/6990947
l-Malate is an important platform chemical that has extensive
applications in the food, feed, and wine industries. Here, we synergistically
engineered the carbon metabolism and redox metabolism in the cytosol
and mitochondria of a previously engineered <i>Aspergillus oryzae</i> to further improve the l-malate titer and decrease the
byproduct succinate concentration. First, the accumulation of the
intermediate pyruvate was eliminated by overexpressing a pyruvate
carboxylase from <i>Rhizopus oryzae</i> in the cytosol and
mitochondria of <i>A. oryzae</i>, and consequently,
the l-malate titer increased 7.5%. Then, malate synthesis <i>via</i> glyoxylate bypass in the mitochondria was enhanced,
and citrate synthase in the oxidative TCA cycle was downregulated
by RNAi, enhancing the l-malate titer by 10.7%. Next, the
exchange of byproducts (succinate and fumarate) between the cytosol
and mitochondria was regulated by the expression of a dicarboxylate
carrier Sfc1p from <i>Saccharomyces cerevisiae</i> in the
mitochondria, which increased l-malate titer 3.5% and decreased
succinate concentration 36.8%. Finally, an NADH oxidase from <i>Lactococcus lactis</i> was overexpressed to decrease the NADH/NAD<sup>+</sup> ratio, and the engineered <i>A. oryzae</i> strain produced 117.2 g/L l-malate and 3.8 g/L succinate,
with an l-malate yield of 0.9 g/g corn starch and a productivity
of 1.17 g/L/h. Our results showed that synergistic engineering of
the carbon and redox metabolisms in the cytosol and mitochondria of <i>A. oryzae</i> effectively increased the l-malate
titer, while simultaneously decreasing the concentration of the byproduct
succinate. The strategies used in our work may be useful for the metabolic
engineering of fungi to produce other industrially important chemicals.
2018-08-09 00:00:00
cytosol
byproduct succinate concentration
. oryzae
dicarboxylate carrier Sfc 1p
NADH
oxidative TCA cycle
. oryzae strain
metabolism
Aspergillus oryzae
mitochondria
l-malate titer