Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina
Graphical abstract
Introduction
Mortierella alpina is an oleaginous filamentous fungus with considerable oil-producing capacity (Sakuradani & Shimizu, 2009). Under the appropriate fermentation conditions, the total lipid content of this strain can reach 50% of its dry cell weight, which is rich in long-chain polyunsaturated fatty acids (LC-PUFAs) such as dihomo-gamma-linolenic acid (DGLA), alpha linolenic acid (ALA), arachidonic acid (ARA) and eicosapentaenoic acid (EPA) (Kikukawa et al., 2018). The lipid fraction produced by M. alpina is commonly used as dietary supplement because of its high ARA content (>50% of total fatty acid content). This has in turn promoted research interest in the lipid metabolism of M. alpina to enable the exploitation of this species for the production of specific fatty acids (Ge et al., 2017, Hao et al., 2015).
Since the establishment of efficient genetic toolbox, biotechnology-enabled genetic modification of M. alpina has further enhanced its lipid accumulation capacity and PUFAs production (Ando et al., 2009, Hao et al., 2014). Multi-omics studies have provided a broader and clearer view of microbial lipid metabolism, indicating that lipogenesis is regulated by global physiological and biochemical pathways (Chen et al., 2015, Lu et al., 2020). To improve the total lipid content in oleaginous microorganisms, including that of M. alpina, genetic modification is often carried out to enhance the supply of fatty acid precursors, such as acetyl-CoA, and the generation of reducing power via nicotinamide adenine dinucleotide phosphate (NADPH) (Hao et al., 2016, Liu et al., 2019, Wang et al., 2019, Yao et al., 2017). Generally, genes related to acetyl-CoA generation and distribution, de novo fatty acid synthesis and PUFA biosynthesis (i.e. those coding for the key enzymes fatty acid desaturase and elongase) have attracted researchers’ attention to construct useful genetically engineered strains (Ando et al., 2009, Ge et al., 2017, Hao et al., 2014). In recent years, upstream regulatory factors, such as some signaling molecule and protein kinase, have also attracted research interests for global regulation of lipid metabolism in M. alpina (Chang et al., 2019, Chang et al., 2020, Lu et al., 2020).
In the microbial biosynthesis of natural products, acyl-coenzymes A (such as acetyl-CoA, propionyl-CoA, and malonyl-CoA) are important precursors for several types of desired products, such as polyketides, alkaloids, biofuels and LC-PUFAs (Krivoruchko et al., 2015). Therefore, enhancing the supply and production of acyl-CoA is a commonly used strategy in metabolic engineering to increase the yield of the desired products (Xu et al., 2018). In fatty acid biosynthesis, acetyl-CoA and malonyl-CoA are used as direct precursors, which function as the start unit and the extender unit for the biosynthesis, respectively. Acetyl-CoA is mainly produced during carbohydrate catabolism, tricarboxylic acid cycle, and branched-chain amino acids (BCAAs) degradation (Krivoruchko et al., 2015, Xu et al., 2018). Among these pathways, BCAAs catabolism plays a vital role in acetyl-CoA supply (Han et al., 2012, Jiao et al., 2016, Kerkhoven et al., 2016). A recent study reported that BCAAs, including leucine, isoleucine and valine, are involved in lipid biosynthesis in triacylglycerol-rich Dunaliella tertiolecta, wherein they function in refilling the acetyl-CoA pool (Tan et al., 2016). More importantly, the contribution of BCCAs to lipid biosynthesis is not limited to their role as alternative source of acetyl-CoA; BCCAs can also affect lipid metabolism by acting as signaling molecules by stimulating the intracellular energy sensor mammal target protein of the rapamycin complex 1 (mTORC1) pathway, which may therefore affect lipogenesis and autophagy-based lipidolysis (Chantranupong et al., 2016, Han et al., 2012, Jiao et al., 2016, Regnacq et al., 2016).
BCAAs transport and metabolism are governed by branched-chain aminotransferases (BCATs), including the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) and glutamate dehydrogenase, and the redox state of the cell is coupled with phosphorylation/dephosphorylation of BCKDC (He et al., 2011, He et al., 2009, Slocombe et al., 2008). Among the BCAAs, leucine has been reported to participate in lipid metabolism in many organisms. LeuB-encoded β-isopropylmalate dehydrogenase (IPMDH, E.C 1.1.1.85) is an important enzyme in leucine metabolism, which is involved in the conversion from α-ketoisovalerate to α-ketoisocaproate in the leucine anabolic pathway (He et al., 2009, Li et al., 2018). Our previous study showed the IPMDH protein level was higher in a high lipid-producing strain of the oleaginous fungus Mucor circinelloides than that in the low lipid-producing strain, also we overexpressed the IPMDH gene from high lipid-producing strain in low lipid-producing strain and found its fatty acids increased by approximately 70%, indicating that IMPDH play a positive role in lipid accumulation of M. circinelloides (Tang et al., 2017, Tang et al., 2020).
In this research, we explored the role of IPMDH derived from the oleaginous fungus M. alpina. By means of homologous overexpression, combined with cell growth, lipid accumulation and intracellular metabolite analysis to explore in depth the function of IPMDH and explain how it mediated lipid biosynthesis in oleaginous fungus M. alpina.
Section snippets
Strains and plasmids
M. alpina CCFM 501 (ura5−) was used as the transformation recipient. M. alpina CCFM 505 (ura5+) was used as the control. Agrobacterium tumefaciens CCFM 834 was used as the transfer DNA (T-DNA) donor for A. tumefaciens-mediated transformation (ATMT). The binary expression vector pBIG2-ura5s-ITs was used as the gene expression vector. The binary expression vector pBIG2-ura5s-MaLeuB was constructed in this study and used for MaLeuB overexpression. All strains and vectors were constructed as
Screening and identification of the MaLeuB recombinant strain
The coding sequence of MaLeuB was obtained from the basic local alignment search tool (BLAST) using the amino acid sequence of McIPMDH from M. circinelloides as a template (Tang et al., 2020). According to the genome information of M. alpina ATCC 32222, only one sequence showed considerable homology with McIPMDH with 65% identity, whereas the other four sequences showed < 25% identity (Wang et al., 2011). The candidate protein contained 1146 base pairs and corresponded to 381 amino acids and
Discussion
Improving the supply of precursors of a targeted product is a commonly used method to increase the yield of acyl-CoA based natural production in metabolic engineering. In addition to providing exogenous substrates that can be assimilated and converted to the precursors, researchers often enhance the generation and conversion rate of the precursors through genetic modification of the endogenous feeder pathway (Xu et al., 2018). Generally, a targeted gene will be selected because of its
CRediT authorship contribution statement
Xin Tang: Conceptualization, Formal analysis, Writing - review & editing, Funding acquisition. Lulu Chang: Investigation, Data curation, Writing - original draft. Shujie Gu: Investigation, Data curation. Hao Zhang: Supervision. Yong Q. Chen: Methodology. Haiqin Chen: Conceptualization, Funding acquisition. Jianxin Zhao: Conceptualization. Wei Chen: Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (38)
- et al.
The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway
Cell
(2016) - et al.
Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway
Cell
(2012) - et al.
Decentralised manufacturing of cell and gene therapy products: Learning from other healthcare sectors
Biotechnol Adv
(2018) - et al.
Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana
J Biol Chem
(2011) - et al.
Arachidonic acid production by the oleaginous fungus Mortierella alpina 1S–4: A review
J Adv Res
(2018) - et al.
Microbial acetyl-CoA metabolism and metabolic engineering
Metab Eng
(2015) - et al.
Engineering acetyl-CoA metabolic shortcut for eco-friendly production of polyketides triacetic acid lactone in Yarrowia lipolytica
Metab Eng
(2019) - et al.
The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms
Advances Appl Microbiol
(2002) - et al.
Increased fatty acid synthesis inhibits nitrogen starvation-induced autophagy in lipid droplet-deficient yeast
Biochem Biophys Res Commun
(2016) - et al.
Single cell oil production by Mortierella alpina
J Biotechnol
(2009)
Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition
Bioresour Technol
Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31
Biotechnol Biofuels
Optimization of Agrobacterium tumefaciens-mediated transformation method of oleaginous filamentous fungus Mortierella alpina on co-cultivation materials choice
J Microbiol Meth
Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S–4, and its application for eicosapentaenoic acid producer breeding
Appl Environ Microbiol
Role of adenosine monophosphate deaminase during fatty acid accumulation in oleaginous fungus Mortierella alpina
J Agric Food Chem
Improved lipogenesis in Mortierella alpina by abolishing the Snf4-mediated energy-saving mode under low glucose
J Agric Food Chem
Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi
Sci Rep
MetaboAnalystR: an R package for flexible and reproducible analysis of metabolomics data
Bioinformatics
Application of a omega-3 Desaturase with an arachidonic acid preference to eicosapentaenoic acid production in Mortierella alpina
Front Bioeng Biotechnol
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Xin Tang and Lulu Chang contributed equally to this work.