Improvement of isobutanol production in Saccharomyces cerevisiae by increasing mitochondrial import of pyruvate through mitochondrial pyruvate carrier

Abstract

Subcellular compartmentalization of the biosynthetic enzymes is one of the limiting factors for isobutanol production in Saccharomyces cerevisiae. Previously, it has been shown that mitochondrial compartmentalization of the biosynthetic pathway through re-locating cytosolic Ehrlich pathway enzymes into the mitochondria can increase isobutanol production. In this study, we improved mitochondrial isobutanol production by increasing mitochondrial pool of pyruvate, a key substrate for isobutanol production. Mitochondrial isobutanol biosynthetic pathway was introduced into bat1Δald6Δlpd1Δ strain, where genes involved in competing pathways were deleted, and MPC1, MPC2, and MPC3 genes encoding the subunits of mitochondrial pyruvate carrier (MPC) hetero-oligomeric complex were overexpressed with different combinations. Overexpression of Mpc1 and Mpc3 forming high-affinity MPCOX was more effective in improving isobutanol production than overexpression of Mpc1 and Mpc2 forming low-affinity MPCFERM. The final engineered strain overexpressing MPCOX produced 330.9 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared to wild type.

Development of an efficient cytosolic isobutanol production pathway in Saccharomyces cerevisiae by optimizing copy numbers and expression of the pathway genes based on the toxic effect of α-acetolactate

“…To overcome this compartmentalization problem, we relocated llv2, Ilv5, and Ilv3 to the cytosol by deleting the N-terminal mitochondrial targeting sequence as reported previously 8 . ILV2ΔN54 , ILV5ΔN48 , and ILV3ΔN19 were expressed under the control of a strong constitutive promoter, P TDH3 , on CEN-based plasmids, in bat1Δald6Δ strain (JHY43), where the competing pathway genes, BAT1 and ALD6 involved in valine biosynthesis from 2-KIV and oxidation of isobutyraldehyde, respectively, were deleted 17 . JHY43 overexpressing ILV2ΔN54 , ILV5ΔN48 , and ILV3ΔN19 (JHY4301) produced 71.8 mg/L isobutanol (Fig.…”

“…Cell growth was determined by the measurement of an optical density at 600 (OD 600 ) with spectrophotometer (Varian Cary® 50 UV-Vis). To determine isobutanol, glucose, and ethanol concentrations, 500 μL of culture supernatants filtered through a 0.22 μm syringe filter were analyzed by High performance liquid chromatography (HPLC) following a previously described procedure 17 .…”

“…To overcome this limitation, the whole isobutanol biosynthetic pathway has been relocated either to the cytosol by expressing the mitochondrial enzymes (Ilv2, Ilv3, and Ilv5) in the cytosol 8,13 or to the mitochondria by expressing the cytosolic enzymes (KDCs and ADHs) in the mitochondria 16 . Previously, we tried to improve mitochondrial pathway by increasing mitochondrial pyruvate pool via overexpression of the MPC complex 17 . However, the majority of pyruvate still existed in the cytoplasm and converted to ethanol.…”

Optogenetic regulation of engineered cellular metabolism for microbial chemical production

“…4d, e ). To further enhance isobutanol production, we deleted the mitochondrial branched chain amino acid aminotransferase, BAT1 , which competes for the α-ketoisovalerate precursor 26 , 27 , resulting in strain YEZ167-4 ( Supplementary Table 2 ). YEZ167-4 contains six copies of P C120 - PDC1 ( Extended Data Fig.…”

Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production

“…Furthermore, a pathway for isobutanol production was also improved by localizing biosynthetic enzymes to the mitochondria of S. cerevisiae . Park et al ( 2016 ) increased the pool of mitochondrial pyruvate by overexpressing the subunits of the hetero-oligomeric mitochondrial pyruvate carrier. Higher titers of isobutanol production were evident in this case when compared with the wild type and to previous reports.…”