Elsevier

Journal of Biotechnology

Volume 307, 10 January 2020, Pages 148-163
Journal of Biotechnology

Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid

https://doi.org/10.1016/j.jbiotec.2019.10.022Get rights and content

Highlights

  • Compilation of detailed inventory of intrinsic pectinolytic enzymes of U. maydis.

  • Activation of intrinsic endo-polygalacturonase in the yeast form.

  • Complementation of the pectinolytic repertoire by bacterial and fungal enzymes.

  • Online evaluation method for enzymatic substrate degradation using respiratory data.

  • Co-fermentation of engineered strains for efficient growth on polygalacturonic acid.

Abstract

Microbial valorization of plant biomass is a key target in bioeconomy. A promising candidate for consolidated bioprocessing is the dimorphic fungus Ustilago maydis. It harbors hydrolytic enzymes to degrade biomass components and naturally produces valuable secondary metabolites like itaconic acid, malic acid or glycolipids. However, hydrolytic enzymes are mainly expressed in the hyphal form. This type of morphology should be prevented in industrial fermentation processes. Genetic activation of these enzymes can enable growth on cognate substrates also in the yeast form. Here, strains were engineered for growth on polygalacturonic acid as major component of pectin. Besides activation of intrinsic enzymes, supplementation with heterologous genes for potent enzymes was tested. The presence of an unconventional secretion pathway allowed exploiting fungal and bacterial enzymes. Growth of the engineered strains was evaluated by a recently developed method for online determination of residual substrates based on the respiration activity. This enabled the quantification of the overall consumed substrate as a key asset for the assessment of the enzyme degradation potential even on polymeric substrates. Co-fermentation of endo- and exo-polygalacturonase overexpression strains resulted in efficient growth on polygalacturonic acid. In the future, the approach will be extended to establish efficient degradation and valorization of pectin.

Introduction

One key step towards a sustainable bioeconomy is the transition away from fossil resources to the synthesis of chemicals or chemical building blocks from renewable bio-based substrates, waste materials, or low-value industrial by-products (Porro et al., 2014; Morais et al., 2015). Sugar beet pulp is an abundant but low-value by-product of the sugar industry which accumulates during sugar production in Europe and the United States. Worldwide about 8.6 million metric tons of dried fiber-rich sugar beet pulp are used as animal feed (web reference 1). However, the economic profit is limited due to the drying costs (Edwards and Doran-Peterson, 2012). Hence, it would be advantageous to use sugar beet pulp for production of high-value substances in a microbial consolidated bioprocess. Substrate costs of 123 US$ / t of sugar beet pulp dry matter (web reference 2) and a market price of 1500 US$ / t for itaconic acid (De Carvalho et al., 2018) as a potential product indicate the economic range for the microbial conversion of sugar beet pulp. The respective microorganisms must be able to enzymatically hydrolyze the (pretreated) pectin-rich pulp to release fermentable sugars and, at the same time, produce a valuable product.

Four structural classes of pectic polymers exist: homogalacturonan (HG), rhamnogalacturonan I (RGI), xylogalacturonan (XG), and rhamnogalacturonan II (RGII). HG and RGI are depicted in Fig. 1. HG is composed of partially esterified polygalacturonic acid, an α-1,4-linked galacturonic acid (galacturonic acid) polymer (Glass et al., 2013; Lampugnani et al., 2018). RGI, RGII and XG consist of a heteropolymer backbone and several different side chains including numerous different sugars (Glass et al., 2013). Due to its complex structure, a large set of carbohydrate-active enzymes (CAZymes) is needed for efficient pectin degradation (Jayani et al., 2005). HG represents the most abundant structural class with a share of about 60 % (w/w) (Caffall and Mohnen, 2009). Complete HG degradation requires the efficient interplay of exo- and endo-polygalacturonases that in concert hydrolyze the polygalacturonic acid backbone. Alternatively, pectate and pectin lyases can break the α-1-4 bonds by a trans-elimination mechanism yielding unsaturated (methyl)oligogalacturonates (Yadav et al., 2009). In addition, methyl-esterifications and O-acetylations of the polygalacturonic acid backbone result in the additional need for pectin methylesterases and pectin acetylesterases (Senechal et al., 2014). Hydrolysis of other structural classes containing, among others, arabinose, rhamnose, galactose, fucose and xylose moieties, requires diverse other hydrolytic enzymes (Glass et al., 2013).

A multitude of pectinolytic enzymes is produced by ascomycete filamentous fungi like Aspergillus niger (de Vries, 2003), basidiomycete mushrooms like Schizophyllum commune (Ohm et al., 2010) and various bacteria (Jayani et al., 2005). Potent enzymes can be heterologously produced in Saccharomyces cerevisiae and are currently used in different industrial applications, with fruit juice extraction and clarification as the most important process (Blanco et al., 1999).

With respect to consolidated bioprocessing, recent research has focused on converting the established fungal model S. cerevisiae into a host for bioethanol production from pectin. S. cerevisiae does not produce intrinsic CAZymes for biomass degradation and does not naturally grow on many of the pectin components like galacturonic acid. Therefore, genetic engineering for substrate hydrolysis and uptake was conducted (Benz et al., 2014; Biz et al., 2016). Another study describes the expression of an endo-polygalacturonase in S. cerevisiae, but the aim was not on substrate consumption (Glauche et al., 2017). To our knowledge, no efficient process using S. cerevisiae simultaneously as CAZyme production host and for galacturonic acid metabolization has been published yet. As mentioned above, some filamentous fungi are naturally equipped for pectin degradation and hence constitute promising alternatives to the yeast system (Lara-Marquez et al., 2011). However, suitable high-value products are lacking and efficient culturing in bioreactors remains a challenge (Klement et al., 2012; Papagianni et al., 1998).

In the past years, the corn smut fungus Ustilago maydis has emerged as an attractive candidate for consolidated bioprocessing (Geiser et al., 2016a; Feldbrügge et al., 2013). The basidiomycete is mostly known for its ability to cause corn smut disease in its host maize and has been studied for decades. By now, it has developed into a fungal model that is prominent for research on host-pathogen interaction, cell and RNA biology as well as homologous recombination (Vollmeister et al., 2012; Djamei and Kahmann, 2012). Handling of U. maydis in the laboratory is very well established and includes a versatile toolset for efficient genetic manipulation yielding genetically stable strains (Brachmann et al., 2004; Terfrüchte et al., 2014; Schuster et al., 2016; Khrunyk et al., 2010; Stock et al., 2012). The duplication time is less than two hours in the presence of glucose as sole carbon source (Feldbrügge et al., 2013; Terfrüchte et al., 2018). The dimorphic fungus can be grown in the laboratory in a haploid yeast form that duplicates by budding. These haploid cells are very robust and, in contrast to filamentous fungi, can easily be cultivated in submerged culture, including large-scale cultivation in bioreactors (Klement et al., 2012; Carstensen et al., 2013). In nature, after mating of compatible yeast cells, infectious hyphae are formed that penetrate the plant surface and cause infection (Brefort et al., 2009). However, pure axenic cultures of haploid cells without a suitable mating partner are considered harmless, and since the crucial genetic factors for infection like the mating genes are known, the fungus can easily be transformed into a non-infectious, safe-to-use form (Feldbrügge et al., 2013; Kahmann et al., 1995). In submerged culture of haploid cells, a morphological switch to hyphal growth can also occur under the influence of stresses such as low pH or nutrient limitations (Klose et al., 2004), but this switch can be avoided through disruption of the mitogen-activated protein kinase (MAPK) signal transduction pathway that regulates the sexual cycle (Kahmann and Kämper, 2004). Such a modification does not affect the fitness of the cell under biotechnologically relevant conditions (Hosseinpour Tehrani et al., 2019). Of note, U. maydis has a very narrow host range infecting only Zea mays and its ancestor Teosinte. It is innocuous to humans and infected plant parts are even relished as a delicacy called Huitlacoche in Central America (Feldbrügge et al., 2013; Valverde et al., 1995).

From a biotechnological perspective, U. maydis is very interesting because it naturally produces valuable secondary metabolites, including organic acids like itaconic and malic acid as well as the glycolipids ustilagic acid and mannosylerythrytol lipids (Bölker et al., 2008; Geiser et al., 2014; Khachatryan et al., 2015; Regestein et al., 2018; Geiser et al., 2016b). As a plant pathogen, U. maydis also contains a limited but potent set of conventionally secreted hydrolytic enzymes including several CAZymes such as multiple xylanases, endoglucanases, β-glucosidases and oxidoreductases (Geiser et al., 2016a; Doehlemann et al., 2008; Mueller et al., 2008; Couturier et al., 2012; Cano-Canchola et al., 2000; Geiser et al., 2013). Hence, it would be perfectly suited for biomass valorization. Unfortunately, the CAZymes are mainly produced during the infections stage in the plant and not during the biotechnologically relevant yeast phase (Doehlemann et al., 2008). We recently addressed this problem and activated several CAZymes in the yeast phase by exchanging the native promoters of the respective genes by a strong artificial promoter (Hartmann et al., 1999). This enabled the secretion of active enzymes during yeast-like growth and resulted in degradation of novel simple biomass-related substrates like cellobiose (Geiser et al., 2016a). Additionally, as a proof of principle, itaconic acid was produced using cellobiose as sole carbon source (Geiser et al., 2016a).

U. maydis not only harbors the conventional secretion pathway for protein export via the endomembrane system but also an unconventional secretion route (Dimou and Nickel, 2018; Reindl et al., 2019). Chitinase Cts1 has been identified as a target protein that is exported via the fragmentation zone of dividing cells in a novel lock-type mechanism (Reindl et al., 2019; Koepke et al., 2011; Aschenbroich et al., 2018). Export of heterologous proteins via unconventional secretion using Cts1 as a carrier has been established a few years ago (Stock et al., 2012; Sarkari et al., 2014; Terfrüchte et al., 2017). Importantly, unconventional secretion circumvents the endomembrane system and consequently the cognate post-translational modifications. This can be essential for secretion of bacterial proteins coincidentally containing detrimental eukaryotic N-glycosylation sites (Stock et al., 2012; Koepke et al., 2011).

U. maydis is able to grow on monomeric galacturonic acid, the most abundant sugar in pectin, as we have shown recently in a parallel methodological study (Müller et al., 2018). This ability has been proven by application of the respiration activity monitoring system (RAMOS), enabling the development of a methodology for the online quantification of consumed galacturonic acid. It is based on the stoichiometric linkage between galacturonic acid consumption, oxygen consumption and carbon dioxide release. Extension of this methodology towards polymeric substrates even enabled the determination of enzymatic activity in the culture supernatant (Müller et al., 2018). Thus, the RAMOS technology provides a powerful tool for the characterization of CAZyme producing strains based on their metabolic activity on complex substrates.

In this work we extended our previous approach of activating intrinsic CAZymes for the degradation of plant biomass components (Geiser et al., 2016a) to perform the first important step towards valorization of pectin-rich biomass. We focused on the major pectin component, polygalacturonic acid, and used a combination of genetic methods and bioprocess engineering to establish its decomposition and consumption. By application of online monitoring tools, we compared the hydrolytic activity of different intrinsic and heterologous polygalacturonases on polygalacturonic acid. To assess additive effects, we co-cultivated U. maydis strains expressing endo- and exo-polygalacturonase and identified a suitable combination for complete substrate conversion.

Section snippets

Plasmids, strains and media

All plasmid vectors were generated using standard molecular cloning methods including Golden Gate cloning (Terfrüchte et al., 2014; Green and Sambrook, 2012). Plasmids were propagated in Escherichia coli Top10 cells. The vector for intrinsic gene activation with the strong Poma promoter was assembled by Golden Gate cloning (Geiser et al., 2016a; Terfrüchte et al., 2014) using approximately 1 kb flanking regions in the 5´ region of the intrinsic open reading frame thereby deleting the putative

Inventory of intrinsic enzymes for pectin degradation and metabolization

Earlier studies suggested that some pectinolytic enzymes are present in U. maydis (Doehlemann et al., 2008; Mueller et al., 2008; Cano-Canchola et al., 2000; Kämper et al., 2006). To inspect its natural abilities to degrade pectin in more detail, a list of potentially relevant enzymes was collected and carefully re-evaluated bioinformatically. Therefore, the proteins were inspected for the presence of enzymatic domains, especially of glycoside hydrolase (GH) domains and conserved domains of

Conclusions

In the present study, bioinformatic analyses, sophisticated strain generation and online monitoring techniques were combined to establish and follow polygalacturonic acid degradation and subsequent metabolization of its monomer galacturonic acid by U. maydis growing in a yeast-like morphology. This is a first and essential step towards pectin valorization. Intriguingly, bacterial enzymes were shown to be exported in an active state by unconventional secretion. However, yields are yet limiting.

Funding information

The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Culture and Science within the framework of the NRW Strategieprojekt BioSC (No. 313/323‐400‐002 13). The work was funded in part by grants from the Deutsche Forschungsgemeinschaft to MF, CEPLAS EXC1028.

Author contributions

P.S., M.M., S.SC. and S.ST. designed and performed the experiments. K.S., M.T., N.I., N.W., J.B., and M.F. directed the study. K.S. wrote the manuscript with input of all co-authors.

Declaration of Competing Interest

The authors have no competing interests to declare.

Acknowledgements

We thank B. Axler for excellent technical support and E. Geiser for initial assessments of growth on galacturonate. F. Finkernagel, M. Vraneš and J. Kämper provided a bioinformatics tool for automated generation of U. maydis dicodon-optimized sequences. We thank T. Häßler and A. Schonhoff for their support in the economic evaluation of future industrial processes.

References (84)

  • R.S. Jayani et al.

    Microbial pectinolytic enzymes: a review

    Process Biochem.

    (2005)
  • R. Kahmann et al.

    Control of mating and development in Ustilago maydis

    Curr. Opin. Genet. Dev.

    (1995)
  • E.S. Martens-Uzunova et al.

    An evolutionary conserved d-galacturonic acid metabolic pathway operates across filamentous fungi capable of pectin degradation

    Fungal Genet. Biol.

    (2008)
  • A.R.C. Morais et al.

    Chemical and biological-based isoprene production: green metrics

    Catal. Today

    (2015)
  • M. Papagianni et al.

    Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

    Biochem. Eng. J.

    (1998)
  • D. Porro et al.

    Old obstacles and new horizons for microbial chemical production

    Curr. Curr. Opin. Biotechnol.

    (2014)
  • P. Sarkari et al.

    Improved expression of single-chain antibodies in Ustilago maydis

    J. Biotechnol.

    (2014)
  • M. Schuster et al.

    Genome editing in Ustilago maydis using the CRISPR-Cas system

    Fungal Genet. Biol.

    (2016)
  • J. Stock et al.

    Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis

    J. Biotechnol.

    (2012)
  • M. Terfrüchte et al.

    Establishing a versatile Golden Gate cloning system for genetic engineering in fungi

    Fungal Genet. Biol.

    (2014)
  • M. Terfrüchte et al.

    Tackling destructive proteolysis of unconventionally secreted heterologous proteins in Ustilago maydis

    J. Biotechnol.

    (2018)
  • S. Yadav et al.

    Pectin lyase: a review

    Process Biochem.

    (2009)
  • P. Yuan et al.

    A protease-resistant exo-polygalacturonase from Klebsiella sp. Y1 with good activity and stability over a wide pH range in the digestive tract

    Bioresour. Technol.

    (2012)
  • D. Abdulrachman et al.

    Heterologous expression of Aspergillus aculeatus endo-polygalacturonase in Pichia pastoris by high cell density fermentation and its application in textile scouring

    BMC Biotechnol.

    (2017)
  • E. Antonov et al.

    Efficient evaluation of cellulose digestibility by Trichoderma reesei Rut-C30 cultures in online monitored shake flasks

    Microb. Cell Fact.

    (2016)
  • E. Antonov et al.

    Process relevant screening of cellulolytic organisms for consolidated bioprocessing

    Biotechnol. Biofuels

    (2017)
  • J. Aschenbroich et al.

    The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis

    Biochim. Biophys. Acta Proteins Proteom.

    (2018)
  • J.P. Benz et al.

    Identification and characterization of a galacturonic acid transporter from Neurospora crassa and its application for Saccharomyces cerevisiae fermentation processes

    Biotechnol. Biofuels

    (2014)
  • A. Biz et al.

    The introduction of the fungal D-galacturonate pathway enables the consumption of D-galacturonic acid by Saccharomyces cerevisiae

    Microb. Cell Fact.

    (2016)
  • K. Bosch et al.

    Genetic manipulation of the plant pathogen Ustilago maydis to study fungal biology and plant microbe interactions

    J. Vis. Exp.

    (2016)
  • A. Brachmann et al.

    A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

    Mol. Genet. Genomics

    (2004)
  • A. Brachmann et al.

    Identification of genes in the bW/bE regulatory cascade in Ustilago maydis

    Mol. Microbiol.

    (2001)
  • T. Brefort et al.

    Ustilago maydis as a pathogen

    Annu. Rev. Phytopathol.

    (2009)
  • R. Brunecky et al.

    High activity CAZyme cassette for improving biomass degradation in thermophiles

    Biotechnol. Biofuels

    (2018)
  • R.C. Carere et al.

    Third generation biofuels via direct cellulose fermentation

    Int. J. Mol. Sci.

    (2008)
  • CAZypedia Consortium

    Ten years of CAZypedia: a living encyclopedia of carbohydrate-active enzymes

    Glycobiology

    (2018)
  • M. Couturier et al.

    Post-genomic analyses of fungal lignocellulosic biomass degradation reveal the unexpected potential of the plant pathogen Ustilago maydis

    BMC Genom.

    (2012)
  • J. De Carvalho et al.

    Biobased itaconic acid market and research trends - is it really a promising chemical?

    Chim Oggi – Chem. Today

    (2018)
  • R.P. de Vries

    Regulation of Aspergillus genes encoding plant cell wall polysaccharide-degrading enzymes; relevance for industrial production

    Appl. Microbiol. Biotechnol.

    (2003)
  • E. Dimou et al.

    Unconventional mechanisms of eukaryotic protein secretion

    Curr. Biol.

    (2018)
  • A. Djamei et al.

    Ustilago maydis: dissecting the molecular interface between pathogen and plant

    PLoS Pathog.

    (2012)
  • M. Edwards et al.

    Pectin-rich biomass as feedstock for fuel ethanol production

    Appl. Microbiol. Biotechnol.

    (2012)
  • Cited by (17)

    • Renewable carbon sources to biochemicals and -fuels: contributions of the smut fungi Ustilaginaceae

      2023, Current Opinion in Biotechnology
      Citation Excerpt :

      Another approach dealt with the overall goal of pectin valorization. In this regard, a crucial step was achieved by realizing polygalacturonic acid degradation and metabolizing the generated monomer galacturonic acid by U. maydis [80]. From crude glycerol, U. maydis can efficiently produce various products, for example, 32 g L−1 glycolipids from 50 g L−1 substrate [78].

    • Production of an endo-polygalacturonase from Fusarium proliferatum isolated from agro-industrial waste

      2021, Biocatalysis and Agricultural Biotechnology
      Citation Excerpt :

      The RG-I and RG-II regions in the hairy regions consist of mono-sugars, such as d-xylose, d-glucose, l-rhamnose, l-arabinose or d-galactose (Cao et al., 2020). Due to the complexity and heterogeneity of pectin, the complete degradation of its structure depends on the combined action of several carbohydrate-active enzymes (CAZymes); these pectinases are classified as esterases, lyases and hydrolases (Stoffels et al., 2020; Xu et al., 2020). Among these pectinolytic enzymes, polygalacturonases (PGAses) stand out due to their ability of depolymerization, as this performs the hydrolysis and division of the glycosidic chain (Satapathy et al., 2020).

    • Reviewing the recent advances in application of pectin for technical and health promotion purposes: From laboratory to market

      2021, Carbohydrate Polymers
      Citation Excerpt :

      There are four structures in pectin based on the units involved in the molecule. They include homo-galacturonan (approximately 60 % w/w of pectin by α-1 4 linking of galacturonic acid), and hetero structures of rhamno-galacturonan I (RGI) (galacturonic acid and rhamnose in the backbone attached by other sugars in branches), xylo-galacturonan (XG) (galacturonic acid in the backbone attached by xylose in branches), and rhamno-galacturonan II (RGII) (galacturonic acid in the backbone attached by rhamnose and other sugars in branches) (Stoffels et al., 2020; Tan, Chen, Liu, Yang, & Li, 2018). The acidic backbone esterifies to different levels and the degree of esterification (DE%) will be reduced after extraction resulting in higher charge density of the biopolymer under treatments (Munarin et al., 2012).

    • Fungal bioprocessing of lignocellulosic materials for biorefinery

      2021, Recent Advancement in Microbial Biotechnology: Agricultural and Industrial Approach
    View all citing articles on Scopus
    1

    The authors contributed equally.

    View full text