Overexpression of Citrate Synthase Increases Isocitric Acid Biosynthesis in the Yeast Yarrowia lipolytica

Piotr Hapeta,Magdalena Rakicka-Pustułka,Małgorzata Robak,Zbigniew Lazar,Piotr Juszczyk,Waldemar Rymowicz

doi:10.3390/su12187364

Piotr Hapeta, Magdalena Rakicka-Pustułka + Show 4 more

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https://doi.org/10.3390/su12187364

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Abstract

Yarrowia lipolytica is a non-conventional yeast producing valuable compounds, such as citric acids, from renewable raw materials. This study investigated the impact of citrate synthase overexpression on the biosynthesis of citric and isocitric acid in Y. lipolytica. Two transformants of Y. lipolytica A101.1.31 strain (efficient citric acid producer), overexpressing CIT1 or CIT2 gene (encoding proteins with citrate synthase activity), were constructed. The results revealed that overexpression of either of these genes enhances citrate synthase activity. Additionally, the cit1 knockout strain was unable to use propionate as the sole carbon source, which proves that CIT1 gene encodes a dual activity protein–citrate and 2-methylcitrate synthase. In the overexpressing mutants, a significant increase in isocitric acid biosynthesis was observed. Both CIT1 and CIT2 overexpressing strains produced citric and isocitric acid from vegetable oil in a ratio close to 1 (CA/ICA ratio for wild-type strain was 4.12).

Highlights

Bulk chemicals such as organic acids can be readily produced with the use of microorganisms
The yeast Y. lipolytica is a highly appreciated industrial microorganism due to its wide range of applications in biotechnology. This is the first study on the identification of genes encoding the citrate synthase in the yeast Y. lipolytica
The activity assays showed that both genes encode proteins with citrate synthase activities; YlCit2 proved to be the main citrate synthase, whereas the protein encoded by the CIT1 gene possessed dual citrate and 2-methylcitrate synthase activity

Summary

Introduction

Bulk chemicals such as organic acids can be readily produced with the use of microorganisms. Due to its natural origin, low toxicity and chemical properties, CA has found many applications in food and pharmaceutical industries as an acidifying and flavoring agent, antioxidant, preservative and stabilizer This compound is used as a cleaning agent in household chemicals as a replacement for environmentally hazardous, phosphate-containing cleaners. The demand for CA is constantly growing, especially for the food and beverages industry (57.9% of the CA market in 2016), where a CA growth rate of 5% from 2017 to 2025 is expected. This high demand can be met through its production in microbial-based processes. The well-established technology harnessing the fungus Aspergillus niger to produce CA from glucose or molasses provides high product yields [2]; it causes some environmental issues [3,4]

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