Heterologous Expression of Two Malate Transporters From an Oleaginous Fungus Mucor circinelloides Improved the Lipid Accumulation in Mucor lusitanicus.

Xiuwen Wang,Junhuan Yang,Hassan Mohamed,Chen Wu,Yonghong Bao,Wenyue Shi,Yuanda Song

doi:10.3389/fmicb.2021.774825

Xiuwen Wang, Junhuan Yang + Show 5 more

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https://doi.org/10.3389/fmicb.2021.774825

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Abstract

The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

Highlights

Oleaginous microorganisms, generally producing cellular lipids up to 20–80% of cell dry weight (CDW), can accumulate lipids when grown in the media with high C/N ratio, since the excess carbon sources can be converted into fatty acids, and stored in the form of triacylglycerols (TAGs) in the cells (Ratledge and Cohen, 2008)
The cytosolic malate can be converted to pyruvate by malic enzyme and produce NADPH, the essential supply of reducing power for fatty acid biosynthesis (Vongsangnak et al, 2012; Rodriguez-Frometa et al, 2013), which was once considered as a rate-limiting step in fatty acid synthesis of oleaginous microorganisms (Wynn et al, 1999; Song et al, 2001; Zhang et al, 2007)
These results indicated that the intracellular glycolysis, lipid biosynthesis and citrate metabolism had been affected by the heterologous expression of mt and sodit genes and led to the increased accumulation of lipid in M. lusitanicus

Summary

Introduction

Oleaginous microorganisms, generally producing cellular lipids up to 20–80% of cell dry weight (CDW), can accumulate lipids when grown in the media with high C/N ratio, since the excess carbon sources can be converted into fatty acids, and stored in the form of triacylglycerols (TAGs) in the cells (Ratledge and Cohen, 2008). Lusitanicus (Wagner et al, 2020), is a model organism to study the mechanism of lipid accumulation (Ratledge and Wynn, 2002), gene silence (Nicolas et al, 2007), and light response (Silva et al, 2006). It is high in γ-linolenic acid (GLA, C18:3) production, which has many beneficial effects for human (Ratledge, 2013). Some studies have proposed that malate is the key metabolite in a wide range of central metabolic processes, such as the maintenance of cellular osmolarity, pH, and stomatal regulation (Emmerlich et al, 2003; Fernie and Martinoia, 2009)

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