Elsevier

Food Microbiology

Volume 95, May 2021, 103689
Food Microbiology

Regulation of secondary metabolite biosynthesis in Monascus purpureus via cofactor metabolic engineering strategies

https://doi.org/10.1016/j.fm.2020.103689Get rights and content

Highlights

  • Addition of exogenous cofactor compounds enhanced Monascus pigment production.

  • Yellow pigment and free citrinin were produced during electrolytic fermentation.

  • Secondary metabolism was regulated by disturbing gene nuoⅠ.

  • A novel device was designed to adjust the color tone of the Monascus pigment.

Abstract

This study investigated the effects of cofactor metabolism on secondary metabolite production in M. purpureus through the application of different cofactor engineering strategies. Total pigment production dramatically increased by 39.08% and 40.89%, and yellow pigment production increased by 74.62% and 114.06% after the addition of 1.0 mg/L of the exogenous cofactor reagents methyl viologen and rotenone, respectively, in submerged batch-fermentation. The extracellular red pigment tone changed to yellow with the application of electrolytic stimulation at 800 mV/cm2, but almost no citrinin production was detected. In addition, the total pigment, yellow pigment and citrinin production increased by 35.46%, 54.89% and 6.27% after disruption of the nuoⅠ gene that encodes NADH-quinone oxidoreductase, respectively. Thus, cofactor metabolic engineering strategies could be extended to the industrial production of Monascus pigment or high yellow pigment with free citrinin production.

Introduction

Monascus spp., a genus of small filamentous saprophytic fungi, was nominated by van Tieghem in 1884. In Southeast Asian countries, Monascus spp. are best-known for being inoculated and cultivated in steamed polished rice to produce red mold rice (RMR) (Shao et al., 2014). RMR contains a variety of biologically active components (Patakova et al., 2017), such as Monascus pigments (MPs) (Feng et al., 2012), monacolin K (MK), and γ-aminobutyric acid (GABA) (Su et al., 2003). However, the presence of citrinin (CT), a mycotoxin produced by the Monascus spp. in RMR that is nephrotoxic to animals, limits its application (Jia et al., 2010). Because of their safety and practicability, MPs have been widely used in China as natural coloring agents for more than two thousand years and are also vital to the global food and drug industry (Chen et al., 2019). With the development of modern biotechnology, various processes can be performed during submerged batch-fermentation (SBF) to enhance the production of secondary metabolites by Monascus spp. (Liu et al., 2019a,b).

M. purpureus LQ-6, which does not produce MK, was subjected to whole-genome sequencing analysis in our lab, and it was concluded that the biosynthetic gene cluster encoding the MK is incomplete (Chai et al., 2020). The present study explored the regulation of the biosynthesis of MPs and CT production by M. purpureus LQ-6 in SBF. MPs are a mixture of azaphilones and are mainly composed of yellow MPs (Y-MPs, absorption at 330–450 nm), orange MPs (O-MPs, absorption at 460–480 nm), and red MPs (R-MPs, absorption at 490–530 nm) (Chen et al., 2017). Among them, the molecular structures of six types of classic hydrophobic pigments (Liu et al., 2019; Shi et al., 2015) and four kinds of classically red-toned hydrophilic MPs (Chen, 2015) are well known. Typically, a large amount of hydrophobic pigments, termed intracellular MPs (inMPs), and a small amount of extracellular hydrophilic MPs (exMPs) accumulate in the mycelium during SBF (Xiong et al., 2015b). It is well-known that MPs and CT share a biosynthetic pathway up to a certain branch point and are synthesized by acetyl-CoA and malonyl-CoA through the fatty acid synthesis (FAS) and polyketide synthesis (PKS) pathways. Acetyl-CoA and malonyl-CoA form β-ketoacid through the FAS pathway. Further, β-ketoacid combines with the polyketone chromophore catalyzed by the polyketide synthase to form an orange pigment (Hajjaj et al., 2000), which is then reduced to a yellow pigment through a hydrogenation reaction that depends upon the NADH/NADPH content. Meanwhile, the orange pigment and amino acid form a red pigment through an amination reaction (Shi et al., 2015), which is a facile non-enzymatic spontaneous reaction that occurs when pH > 6 (Li et al., 2019).

Cofactor balance plays a vital role in manipulating the metabolic flux during biosynthesis to favor the desired fermentation products or regulate their production (Tan et al., 2018). The biosynthesis of MPs, especially Y-MPs, occurs through the PKS and FAS pathways and requires several primary metabolites, such as acetyl-CoA, malonyl-CoA, and NADPH (Berríos-Rivera, 2002; Shao et al., 2014). Oxidation-reduction potential (ORP) has been identified as a parameter that can provide excellent control during the SBF process for the enhancement of the production of the desired product. It has been reported that the yields of spinosad and pseudoaglycone were enhanced 3.11-fold under oxidative conditions by adding 5 mM H2O2 (Zhang et al., 2014). Hu et al. (2020) utilized oxidative stressors by supplementing with 25 mM H2O2 to increase the production of the bioactive antioxidant secondary metabolite antrodin C in SBF by Antrodia cinnamomea (Hu et al., 2020). Huang et al. (Huang et al., 2017a,b) reported that the production of extracellular water-soluble Y-MPs increased significantly, and the maximum total MP (T-MPs) yield reached 209 AU when 10 mM H2O2 was added on the third day of fermentation. However, no related studies have directly utilized cofactor metabolic engineering or technologies to regulate the metabolism of MPs and CT in Monascus strains.

In our previous study, we explored the effects of MPs and CT biosynthesis on the development and metabolism of M. purpureus LQ-6 via genetic engineering technology. We found that the intracellular levels of cofactors NADH and NADPH were altered to varying degrees as the SBF progressed (Chai et al., 2020). The anabolism of NADH and NADPH involves many NADPH-dependent redox enzymes in the FAS and PKS pathways. We speculated that cofactor metabolic engineering strategies, such as supplementing exogenous factors, providing electrolytic stimulation during the SBF, or inactivating the NADH-quinone oxidoreductase in the electron transport chain complex I, would regulate the biosynthesis of MP and CT.

Section snippets

Microorganisms and culture conditions

Monascus purpureus LQ-6 (CCTCC M 2018600, China Central for Type Culture Collection (CCTCC), Wuhan, China) obtained from RMR was used as the parent strain. M. purpureus Δ4971 was generated from this parent strain by disrupting the gene monascus_4971. Strains were cultured at 30 °C for seven days in potato dextrose broth (PDB, potato (200 g) and dextrose (20 g) in 1000 mL pure water) or on potato dextrose agar (PDA, PDB with 20 g/L agar) medium supplemented, as needed, with 50 μg/mL of geneticin

Biochemical engineering strategy

Biochemical engineering technology involves the addition of exogenous compounds to alter cell metabolism and facilitate the regulation of the biosynthesis of target products during SBF. The biosynthesis of Monascus secondary metabolites is strongly dependent upon intracellular cofactors and energy metabolism; therefore, disrupting the original balance of the cofactors such as NADPH and NADH by supplementing exogenous cofactor compounds would modify the MP and CT production. In this study, MV,

Conclusion

This study implemented cofactor engineering technologies in an SBF system to regulate the secondary metabolism of Monascus, resulting in increased production of MPs, especially Y-MPs. Among them, disruption of the ETC in aerobic metabolism provided the most dramatic up-regulation of MP and CT biosynthesis. We are the first to report that ES can promote the biosynthesis of Y-MPs but almost completely inhibit R-MPs and CT production in SBF systems, which is advantageous for the high-yield,

Declaration of competing interest

The authors declared that they have no conflicts of interest to this work.

Acknowledgements

The authors would like to thank Prof. Tiangang Liu from Wuhan University, China, for kindly providing the pXS deletion vector;and thank ph. D Zuotao Tan from Nanjing Tech University for kindly providing the synthetic cofactor compound;We would like to thank Editage (www.editage.cn) for English language editing.

This work was supported by National Natural Science Foundation of China (Grant No. 31571874); Research and Development Plan in Key Areas of Hunan Province (No. 2019NK2111).

References (45)

  • S. Roy et al.

    Electro-stimulated microbial factory for value added product synthesis

    Bioresour. Technol.

    (2016)
  • A. Schievano et al.

    Electro-fermentation – merging electrochemistry with fermentation in industrial applications

    Trends Biotechnol.

    (2016)
  • P. She et al.

    Electrolytic stimulation of bacteria Enterobacter dissolvens by a direct current

    Biochem. Eng. J.

    (2006)
  • K. Shi et al.

    Controlling composition and color characteristics of Monascus pigments by pH and nitrogen sources in submerged fermentation

    J. Biosci. Bioeng.

    (2015)
  • R.N.F. Thorneley

    A convenient electrochemical preparation of reduced methyl viologen and a kinetic study of the reaction with oxygen using an anaerobic stopped-flow apparatus

    Biochim. Biophys. Acta Bioenerg.

    (1974)
  • X. Xiong et al.

    Coupled aminophilic reaction and directed metabolic channeling to red Monascus pigments by extractive fermentation in nonionic surfactant micelle aqueous solution

    Process Biochem.

    (2015)
  • Z. Bai et al.

    Physiological responses of chemostat cultures of Aspergillus Niger (B1-D) to simulated and actual oxidative stress

    Biotechnol. Bioeng.

    (2003)
  • S. Berríos-Rivera

    The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures

    Metab. Eng.

    (2002)
  • Z. Bo et al.

    Optimization of culture medium for yellow pigments production withMonascus ankamutant using response surface methodology

    Eur. Food Res. Technol.

    (2009)
  • X. Chai et al.

    Effects of pigment and citrinin biosynthesis on the metabolism and morphology of Monascus purpureus in submerged fermentation

    Food Sci Biotechnol

    (2020)
  • W. Chen et al.

    edible filamentous fungi from the species monascus: early traditional fermentations, modern molecular biology, and future genomics

    Comprehensive reviews in food science & food safety

    (2015)
  • W. Chen et al.

    Biosynthesis of azaphilone pigments in Monascus fungi

    Chem. Sci.

    (2017)
  • Cited by (24)

    • Electrolytic stimulation in aid of poly(β-L-malic acid) production by Aureobasidium melanogenum ipe-1

      2022, International Journal of Biological Macromolecules
      Citation Excerpt :

      Similar to the production of polyhydroxyalkanoate (PHB) (i.e., a structural analog of PMLA) [2], the PMLA production is also strongly affected by the reducing power of the electron acceptor [14]. Moreover, the redox mediators, such as anthraquinone-2-sulfonate [15], methyl viologen and rotenone [16], and neutral red [17,18], can mediate microbial metabolism resulting in a high production. To our knowledge, few studies concerned the effect of the redox mediator on the production of PMLA.

    View all citing articles on Scopus
    1

    Jun Liu and Jingyan Wu contributed equally to this work.

    View full text