Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina

https://doi.org/10.1016/j.fgb.2021.103572Get rights and content

Highlights

  • Expressing IPMDH increased total fatty acid by 20.2% compared with control.

  • IPMDH affected amino acid metabolism, TCA cycle and butanoate metabolism.

  • IPMDH provided α-ketoisocaproate and acetyl-CoA to influence the metabolism.

Abstract

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.

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)

  • X. Tang et al.

    Investigation of fatty acid accumulation in the engineered Saccharomyces cerevisiae under nitrogen limited culture condition

    Bioresour Technol

    (2014)
  • F. Wang et al.

    Metabolic engineering to enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty acids in Schizochytrium sp. S31

    Biotechnol Biofuels

    (2019)
  • S. Wang et al.

    Optimization of Agrobacterium tumefaciens-mediated transformation method of oleaginous filamentous fungus Mortierella alpina on co-cultivation materials choice

    J Microbiol Meth

    (2018)
  • A. Ando et al.

    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

    (2009)
  • L. Chang et al.

    Role of adenosine monophosphate deaminase during fatty acid accumulation in oleaginous fungus Mortierella alpina

    J Agric Food Chem

    (2019)
  • L. Chang et al.

    Improved lipogenesis in Mortierella alpina by abolishing the Snf4-mediated energy-saving mode under low glucose

    J Agric Food Chem

    (2020)
  • H. Chen et al.

    Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi

    Sci Rep

    (2015)
  • J. Chong et al.

    MetaboAnalystR: an R package for flexible and reproducible analysis of metabolomics data

    Bioinformatics

    (2018)
  • C. Ge et al.

    Application of a omega-3 Desaturase with an arachidonic acid preference to eicosapentaenoic acid production in Mortierella alpina

    Front Bioeng Biotechnol

    (2017)
  • Cited by (9)

    • Enhancing microbial lipids yield for biodiesel production by oleaginous yeast Lipomyces starkeyi fermentation: A review

      2022, Bioresource Technology
      Citation Excerpt :

      Nonetheless, the efficiency of adopting CRISPR/Cas9 for gene knockout remains unclear and requires further research. Frequently used metabolic engineering strategies include (1) overexpression of genes responsible for the key enzymes of lipid biosynthesis pathways (Jeffries et al., 2018); (2) expression of various exogenous genes encoding the related functional enzymes determining the speeds of biochemical reaction in the target strains (McNeil & Stuart, 2018b); (3) promoting objective metabolic flux through removing major competing metabolic pathways (Liu et al., 2019); and (4) improving lipid accumulation by regulating metabolic bypass and branched enzymes (Tang et al., 2021). Thus far, the positive correlation between the high number of copies of the genes encoding the subunits of fatty acid synthase and greater lipid content has been reported (Chen et al., 2020).

    View all citing articles on Scopus
    1

    Xin Tang and Lulu Chang contributed equally to this work.

    View full text