The influence of oxygen limitation for the production of succinic acid with recombinant strains of Yarrowia lipolytica

Abstract

The yeast Yarrowia lipolytica is able to produce high amounts of several organic acids such as pyruvic, citric, isocitric, alpha-ketoglutaric, and succinic acid. Here we report on the influence of the reduced activity of succinate dehydrogenase in Y. lipolytica on its ability to produce succinate. The recombinant strains Y. lipolytica H222-AZ1 and H222-AZ2 were created by exchange of the native promoter of the succinate dehydrogenase subunit 2 encoding gene by inducible promoters. During the cultivation of the strain Y. lipolytica H222-AZ1 in shaking flask experiments, it was found that the promoter exchange resulted in an increase in succinic acid (SA) production. Moreover, it was found that the production of SA depends on an additional limitation of oxygen. Fed-batch cultivations in 1-l bioreactors confirmed this fundamental finding. Y. lipolytica H222-AZ1 produced 2 g l(-1) of SA with oxygen supply and 9.2 g l(-1) under the limitation of oxygen after 165 h. By using a less active promoter in Y. lipolytica H222-AZ2, the production of SA was increased to 25 g l(-1) with a productivity of 0.152 g (l*h)(-1) and a selectivity of 67 % after 165 h. Yields of 2.39 g SA per gram biomass and 0.26 g SA per gram glycerol were found.

Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica

“…The fifth subunit SDH5, a highly conserved mitochondrial protein in SDH complex, is required for SDH activity and stability [18]. Recently, reduction or loss of SDH enzyme activity in Y. lipolytica was explored through impairing sdh1 / sdh2 gene or exchanging native promoter of sdh2 gene with a weak promoter [5, 20]. These recombinant strains can accumulate about 4–5 g L −1 SA using glycerol as substrate in unbaffled flasks.…”

“…Apart from SA, the PGC01003 strain also accumulated 5.7 g L −1 acetic acid, which was not found in the control strain. Jost et al [5] also reported the sdh2 deleted strain secreted acetic acid. This acetic acid overflow was expected due to the metabolic flux between glycolysis and TCA cycle became imbalance.…”

“…Although SA production by engineered Y. lipolytica was reported before, no more than 50 g L −1 initial glycerol concentration was used [5, 20]. The initial concentration of crude glycerol was optimized from 75 to 200 g L −1 in batch fermentations to improve SA fermentation performance.…”

“…Moreover, crude glycerol concentration was controlled under 5 g L −1 by continuous cultivation approach when using Basfia succiniciproducens DD1 [26]. Jost et al [5] pointed out that glycerol concentration should not exceed 40 g L −1 for an engineered Y. lipolytica in SA fermentation. The experiment indicated that Y. lipolytica PGC01003 is able to grow well under high glycerol concentration, which would increase the SA productivity and facilitate the production process.…”

Physiologo-biochemical characteristics of citrate-producing yeast Yarrowia lipolytica grown on glycerol-containing waste of biodiesel industry

“…Glycerol-based microbiological processes have been developed which aimed at production of 1,3-propanediol (Papanikolaou et al 2008), poly-3-hydroxybutyrate (Mothes et al 2007;Ibrahim and Steinbüchel 2010), lipids (Papanikolaou and Aggelis 2010;Papanikolaou and Aggelis 2002;Dedyukhina et al 2012), polyols (Rywińska et al 2013;Mirończuk et al 2014), arachidonic acid (Dedyukhina et al 2014), pyruvic acid (Morgunov et al 2004a), succinic acid (Yuzbashev et al 2010;Jost et al 2014), α-ketoglutaric acid Yin et al 2012;Otto et al 2012), and citric acid (Rymowicz et al 2006;Förster et al 2007a;Levinson et al 2007;Rymowicz et al 2006Rymowicz et al , 2010Holz et al 2009;Kamzolova et al 2011a;Rywińska et al 2009Rywińska et al , 2013Morgunov et al 2013). Citric acid (CA) is considered to be the most economically feasible product with the annual world production of 1.4 million ton (Anastassiadis et al 2008).…”