Effects of pyruvate dehydrogenase subunits overexpression on the α-ketoglutarate production in Yarrowia lipolytica WSH-Z06

Abstract

Yarrowia lipolytica WSH-Z06 harbours a promising capability to oversynthesize α-ketoglutarate (α-KG). Its wide utilization is hampered by the formation of high concentrations of pyruvate. In this study, a metabolic strategy for the overexpression of the α and β subunits of pyruvate dehydrogenase E1, E2 and E3 components was designed to reduce the accumulation of pyruvate. Elevated expression level of α subunit of E1 component improved the α-KG production and reduced the pyruvate accumulation. Due to a reduction in the acetyl-CoA supply, neither the growth of cells nor the synthesis of α-KG was restrained by the overexpression of β subunit of E1, E2 and E3 components. Furthermore, via the overexpression of these thiamine pyrophosphate (TPP)-binding subunits, the dependency of pyruvate dehydrogenase on thiamine was diminished in strains T1 and T2, in which α and β subunits of E1 component were separately overexpressed. In these two recombinant strains, the accumulation of pyruvate was insensitive to variations in exogenous thiamine. The results suggest that α-KG production can be enhanced by altering the dependence on TPP of pyruvate dehydrogenase and that the competition for the cofactor can be switched to ketoglutarate dehydrogenase via separate overexpression of the TPP-binding subunits of pyruvate dehydrogenase. The results presented here provided new clue to improve α-KG production.

Enhanced pyruvate production in Candida glabrata by carrier engineering

“…Reference and recombinant strain cells in pyruvate fermentation medium were collected at the beginning of the stationary phase by centrifugation and immediately frozen in liquid nitrogen. Total RNA was extracted using a previously described protocol (Guo, Madzak, Du, Zhou, & Chen, ). Gene expression profiles were measured by RT‐qPCR on a LightCycler 480 II RT‐PCR instrument (Roche Applied Science, Mannheim, Germany) with PrimeScript RT Reagent and the SYBR Premix Ex Taq ™ Kit (TaKaRa, Dalian, China); all primers are shown in Table S4.…”

“…Reference and recombinant strain cells in pyruvate fermentation medium were collected at the beginning of the stationary phase by centrifugation and immediately frozen in liquid nitrogen. Total RNA was extracted using a previously described protocol (Guo, Madzak, Du, Zhou, & Chen, 2014 (Herzig et al, 2012). Previous reports showed that the expression levels of MPC1 or MPC2 were different in human cells with different functions, such as mice liver, kidneys, lung, brain, and hearts (Li, Li, et al, 2016b).…”

Rational modular design of metabolic network for efficient production of plant polyphenol pinosylvin

“…The specificity of primers were verified by running the resulting amplifying DNA products in agarose gels. RT-PCR assays were conducted by SYBR green PCR Master Mix (Takara, Dalian, China) on a LightCycler 480 II thermal cycler system (Roche, Mannheim, Germany) 34 . Five dilutions of the obtaining cDNAs were used to verify the efficiencies of RT-PCR and a negative control were conducting without cDNA added to confirm purity of the sample.…”

De novo Biosynthesis of Odd-Chain Fatty Acids in Yarrowia lipolytica Enabled by Modular Pathway Engineering

“…A few studies have examined the functional expression of the PDH complex in Y. lipolytica. One study attempted to overexpress the direct pathway from pyruvate to acetyl-CoA in a coordinated manner (Markham et al, 2018), and another study showed that the individual overexpression of PDA1 (shared subunits with α-ketoglutarate dehydrogenase) improved α-ketoglutarate production (Guo et al, 2014).…”