Aconitase overexpression changes the product ratio of citric acid production by Yarrowia lipolytica

Abstract

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric acid (CA) and isocitric acid (ICA) under an excess of carbon source and several conditions of growth limitation. Depending on the carbon source used, Y. lipolytica strains produce a mixture of CA and ICA in a characteristic ratio. To examine whether this CA/ICA product ratio can be influenced by gene-dose-dependent overexpression of aconitase (ACO)-encoding gene ACO1, a recombinant Y. lipolytica strain was constructed containing multiple copies of ACO1. The high-level expression of ACO in the ACO1 multicopy integrative transformant resulted in a shift of the CA/ICA product pattern into the direction of ICA. On sunflower oil, a striking increase of the ICA proportion from 35-49% to 66-71% was observed compared to wild-type strains without influencing the total amount of acids (CA and ICA) produced. On glycerol, glucose or sucrose, the ICA proportion increased only moderately from 10-12% to 13-17%. This moderate shift into the direction of ICA was also observed in an icl1-defective strain.

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

“…Cell-free supernatants were prepared as described in Holz et al (2009), using 0.1 M TrisHCl, pH 7.4 and 5 mM MgCl 2 as the cell disruption buffer. Protein content was determined by the method of Mauersberger et al (Bradford 1976), using bovine serum albumin as standard.…”

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).…”

Production of citric and isocitric acid by Yarrowia lipolytica strains grown on different carbon sources

“…Considering the rising market of biotechnological processes for the production of various chemicals, the increasing interest in microbially produced organic acids as a key group among the building-block chemicals is not surprising [1]. With a global annual production exceeding 1.7 million tons, citric acid (CA) ranks first among the organic acid synthesized by microorganisms [2] and its production volume is second only to industrial ethanol as a fermentation product [3].…”

“…Because of the above-mentioned ecological problems of Aspergillus process, it is of interest to develop alternative processes using yeast as organism-producing agent [8]. A bioprocess for citric acid production using Yarrowia lipolytica would have several additional advantages compared to the Aspergillus process, including a larger substrate variety (using raw materials such as hydrocarbons, carbohydrates, plant oils and glycerol), higher maximal product formation rate, higher substrate concentrations and yield, a lower sensitivity to low dissolved oxygen concentrations and heavy metals, simple process control, and waste and sewage minimization [1,[7][8][9][10][11][12]. A disadvantage of using Y. lipolytica wild-type strains for the commercial production of citric acid is the simultaneous secretion of isocitric acid, which has an inferior buffer capacity and chelating ability compared to citric acid.…”

“…Monopotassium salt of threo-D-(S)-(+)-isocitric acid is used in several biochemical experiments (measurement of aconitate hydratase, NAD-isocitrate dehydrogenase, NADP-isocitrate dehydrogenase, isocitrate lyase) [5]. Additionally, there is obviously a growing interest in ICA as a useful pharmaceutical additive, food and beverage additive, in cosmetics and detergents, as well as a new chiral building block for chemical syntheses [1].…”