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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Microbiology".

Deadline for manuscript submissions: closed (25 February 2023) | Viewed by 29273

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Prof. Dr. Georg A. Sprenger

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Institute of Microbiology, University of Stuttgart, Stuttgart, Germany
Interests: carbon-carbon (C-C) bonding enzymes; amino acid biosynthesis; metabolic engineering

Special Issue Information

Dear Colleagues,

This issue of IJMS will be devoted to microbial enzymes and metabolites. Microorganisms host a plentitude of enzymes. Microbial enzymes play roles in specific biosynthetic and catabolic pathways. They are being used in vitro for diagnostic purposes, for the production of low-molecular-weight compounds (amino acids, peptides, organic acids, alcohols, antibiotics, sugars), in the production of biofuels a.o. Microbial enzymes have already become standard tools in bio-organic and pharmaceutical chemistry, as they enlarge the toolbox for organic chemists. The study of the structure–function relationships of these enzymes (enzymatic mechanisms, substrate scope and stereoselectivity, protein design) allows us to broaden their substrate ranges. New developments in enzyme design also deal with microbial enzymes to create reactions that are new to nature. Enzyme cascades involve several enzymes to achieve novel compounds. Finally, in metabolic engineering of microorganisms, novel metabolic pathways are opened up by coupling of microbial enzymes stemming from various origins.

Prof. Dr. Georg A. Sprenger
Guest Editor

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Keywords

Structure/function relationships
protein engineering/enzyme design
biocatalysis
metabolites

Published Papers (11 papers)

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Research

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18 pages, 3817 KiB

Open AccessArticle

Silencing of the Laccase (lacc2) Gene from Pleurotus ostreatus Causes Important Effects on the Formation of Toxocyst-like Structures and Fruiting Body

by Anahí Armas-Tizapantzi, José Luis Martínez y Pérez, Francisco José Fernández, Gerardo Mata, Laura V. Hernández-Cuevas, Elvia Ortiz Ortiz, Edelmira García Nieto, Araceli Tomasini, Edgar Sierra-Palacios, Jaime Marcial-Quino and Alba Mónica Montiel-González

Int. J. Mol. Sci. 2023, 24(9), 8143; https://doi.org/10.3390/ijms24098143 - 2 May 2023

Cited by 2 | Viewed by 1832

Abstract

A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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18 pages, 4340 KiB

Open AccessArticle

Skin Microbiome in Prurigo Nodularis

by Klaudia Tutka, Magdalena Żychowska, Anna Żaczek, Karolina Maternia-Dudzik, Jakub Pawełczyk, Dominik Strapagiel, Jakub Lach and Adam Reich

Int. J. Mol. Sci. 2023, 24(8), 7675; https://doi.org/10.3390/ijms24087675 - 21 Apr 2023

Cited by 3 | Viewed by 2945

Abstract

Prurigo nodularis (PN) is a chronic condition characterized by the presence of nodular lesions accompanied by intense pruritus. The disease has been linked to several infectious factors, but data on the direct presence of microorganisms in the lesions of PN are scarce. The aim of this study was to evaluate the diversity and composition of the bacterial microbiome in PN lesions by targeting the region V3-V4 of 16S rRNA. Skin swabs were obtained from active nodules in 24 patients with PN, inflammatory patches of 14 patients with atopic dermatitis (AD) and corresponding skin areas of 9 healthy volunteers (HV). After DNA extraction, the V3-V4 region of the bacterial 16S rRNA gene was amplified. Sequencing was performed using the Illumina platform on the MiSeq instrument. Operational taxonomic units (OTU) were identified. The identification of taxa was carried out using the Silva v.138 database. There was no statistically significant difference in the alpha-diversity (intra-sample diversity) between the PN, AD and HV groups. The beta-diversity (inter-sample diversity) showed statistically significant differences between the three groups on a global level and in paired analyses. Staphylococcus was significantly more abundant in samples from PN and AD patients than in controls. The difference was maintained across all taxonomic levels. The PN microbiome is highly similar to that of AD. It remains unclear whether the disturbed composition of the microbiome and the domination of Staphylococcus in PN lesions may be the trigger factor of pruritus and lead to the development of cutaneous changes or is a secondary phenomenon. Our preliminary results support the theory that the composition of the skin microbiome in PN is altered and justify further research on the role of the microbiome in this debilitating condition. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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13 pages, 2178 KiB

Open AccessArticle

Characterization of a Novel Myrosinase with High Activity from Marine Bacterium Shewanella baltica Myr-37

by Qinwen Ye, Yaowei Fang, Mengjiao Li, Haoyu Mi, Shu Liu, Guang Yang, Jing Lu, Yaling Zhao, Qitong Liu, Wei Zhang and Xiaoyue Hou

Int. J. Mol. Sci. 2022, 23(19), 11258; https://doi.org/10.3390/ijms231911258 - 24 Sep 2022

Cited by 4 | Viewed by 1832

Abstract

Myrosinase can hydrolyze glucosinolates to generate isothiocyanates, which have cancer prevention and anti-cancer properties. The main sources of myrosinase are cruciferous plants. To further improve the efficiency of isothiocyanates preparation, it is necessary to explore novel sources of myrosinases. In this study, we described a bacterium, Shewanella baltica Myr-37, isolated from marine mud, capable of producing a novel myrosinase (Smyr37) with a molecular weight of 100 kDa. The crude enzyme of Smyr37 showed the highest activity at 50 °C and pH 8.0. The sinigrin- and glucoraphanin-hydrolyzing activities of Smyr37 were 6.95 and 5.87 U/mg, respectively. Moreover, when the reaction temperature was 40 °C and pH was 7.0, the crude enzyme of Smyr37 could efficiently degrade glucoraphanin into sulforaphane within 25 min with a yield of 0.57 mg/mL. The corresponding conversion efficiency of sulforaphane from glucoraphanin was 89%. In summary, S. baltica Myr-37 myrosinase Smyr37, a novel myrosinase, can be used in the preparation of isothiocyanates. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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15 pages, 1551 KiB

Open AccessArticle

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems

by Tatiana Karaseva, Dmitry Fedorov, Sophia Baklagina, Olga Ponamoreva, Sergey Alferov, Galina Ekimova, Azat Abdullatypov, Liubov Trubitsina and Ildar Mustakhimov

Int. J. Mol. Sci. 2022, 23(18), 10337; https://doi.org/10.3390/ijms231810337 - 7 Sep 2022

Viewed by 2132

Abstract

(Ca²⁺)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6β6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9–10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (K_M = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002–0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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18 pages, 7071 KiB

Open AccessArticle

Optimization of Signal Peptide via Site-Directed Mutagenesis for Enhanced Secretion of Heterologous Proteins in Lactococcus lactis

by Nur Aqlili Riana Alias, Adelene Ai-Lian Song, Noorjahan Banu Alitheen, Raha Abdul Rahim, Siti Sarah Othman and Lionel Lian Aun In

Int. J. Mol. Sci. 2022, 23(17), 10044; https://doi.org/10.3390/ijms231710044 - 2 Sep 2022

Cited by 4 | Viewed by 2183

Abstract

Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) Lactococcus lactis is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of Staphylococcus aureus. A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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16 pages, 3792 KiB

Open AccessArticle

A Novel Pathway of Chlorimuron-Ethyl Biodegradation by Chenggangzhangella methanolivorans Strain CHL1 and Its Molecular Mechanisms

by Zhixiong Yu, Yumeng Dai, Tingting Li, Wu Gu, Yi Yang, Xiang Li, Pai Peng, Lijie Yang, Xinyu Li, Jian Wang, Zhencheng Su, Xu Li, Mingkai Xu and Huiwen Zhang

Int. J. Mol. Sci. 2022, 23(17), 9890; https://doi.org/10.3390/ijms23179890 - 31 Aug 2022

Cited by 3 | Viewed by 1537

Abstract

Chlorimuron-ethyl is a widely used herbicide in agriculture. However, uncontrolled chlorimuron-ethyl application causes serious environmental problems. Chlorimuron-ethyl can be effectively degraded by microbes, but the underlying molecular mechanisms are not fully understood. In this study, we identified the possible pathways and key genes involved in chlorimuron-ethyl degradation by the Chenggangzhangella methanolivorans strain CHL1, a Methylocystaceae strain with the ability to degrade sulfonylurea herbicides. Using a metabolomics method, eight intermediate degradation products were identified, and three pathways, including a novel pyrimidine-ring-opening pathway, were found to be involved in chlorimuron-ethyl degradation by strain CHL1. Transcriptome sequencing indicated that three genes (atzF, atzD, and cysJ) are involved in chlorimuron-ethyl degradation by strain CHL1. The gene knock-out and complementation techniques allowed for the functions of the three genes to be identified, and the enzymes involved in the different steps of chlorimuron-ethyl degradation pathways were preliminary predicted. The results reveal a previously unreported pathway and the key genes of chlorimuron-ethyl degradation by strain CHL1, which have implications for attempts to enrich the biodegradation mechanism of sulfonylurea herbicides and to construct engineered bacteria in order to remove sulfonylurea herbicide residues from environmental media. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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28 pages, 34319 KiB

Open AccessArticle

Enzymatic Pretreatment with Laccases from Lentinus sajor-caju Induces Structural Modification in Lignin and Enhances the Digestibility of Tropical Forage Grass (Panicum maximum) Grown under Future Climate Conditions

by Emanuelle Neiverth de Freitas, Robson Carlos Alnoch, Alex Graça Contato, Karoline Maria V. Nogueira, Eduardo José Crevelin, Luiz Alberto Beraldo de Moraes, Roberto Nascimento Silva, Carlos Alberto Martínez and Maria de Lourdes T. M. Polizeli

Int. J. Mol. Sci. 2021, 22(17), 9445; https://doi.org/10.3390/ijms22179445 - 31 Aug 2021

Cited by 10 | Viewed by 2710

Abstract

Since laccase acts specifically in lignin, the major contributor to biomass recalcitrance, this biocatalyst represents an important alternative to the pretreatment of lignocellulosic biomass. Therefore, this study investigates the laccase pretreatment and climate change effects on the hydrolytic performance of Panicum maximum. Through a Trop-T-FACE system, P. maximum grew under current (Control (C)) and future climate conditions: elevated temperature (2 °C more than the ambient canopy temperature) combined with elevated atmospheric CO₂ concentration(600 μmol mol⁻¹), name as eT+eC. Pretreatment using a laccase-rich crude extract from Lentinus sajor caju was optimized through statistical strategies, resulting in an increase in the sugar yield of P. maximum biomass (up to 57%) comparing to non-treated biomass and enabling hydrolysis at higher solid loading, achieving up to 26 g L⁻¹. These increments are related to lignin removal (up to 46%) and lignin hydrophilization catalyzed by laccase. Results from SEM, CLSM, FTIR, and GC-MS supported the laccase-catalyzed lignin removal. Moreover, laccase mitigates climate effects, and no significant differences in hydrolytic potential were found between C and eT+eC groups. This study shows that crude laccase pretreatment is a potential and sustainable method for biorefinery solutions and helped establish P. maximum as a promising energy crop. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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22 pages, 2968 KiB

Open AccessArticle

Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Escherichia coli Mutants through Expression of a Gene Variant (fsaA^A129S) for Fructose 6-Phosphate Aldolase

by Emma Guitart Font and Georg A. Sprenger

Int. J. Mol. Sci. 2020, 21(24), 9625; https://doi.org/10.3390/ijms21249625 - 17 Dec 2020

Cited by 5 | Viewed by 4780

Abstract

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes pfkA, pfkB (encoding the two PFK isoenzymes), and zwf (glucose 6-phosphate dehydrogenase) in Escherichia coli K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the fsaA wild type gene (encoding E. coli fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P). Although FSAA enzyme activity was detected, growth on glucose was not reestablished. A mutant allele encoding for FSAA with an amino acid exchange (Ala129Ser) which showed increased catalytic efficiency for F6P, allowed growth on glucose with a µ of about 0.12 h⁻¹. A GL3 derivative with a chromosomally integrated copy of fsaA^A129S (GL4) grew with 0.05 h⁻¹ on glucose. A mutant strain from GL4 where dhaKLM genes were deleted (GL5) excreted DHA. By deletion of the gene glpK (glycerol kinase) and overexpression of gldA (of glycerol dehydrogenase), a strain (GL7) was created which showed glycerol formation (21.8 mM; yield approximately 70% of the theoretically maximal value) as main end product when grown on glucose. A new-to-nature pathway from glucose to glycerol was created. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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15 pages, 2988 KiB

Open AccessArticle

Assessing the Thiamine Diphosphate Dependent Pyruvate Dehydrogenase E1 Subunit for Carboligation Reactions with Aliphatic Ketoacids

by Stefan R. Marsden, Duncan G. G. McMillan and Ulf Hanefeld

Int. J. Mol. Sci. 2020, 21(22), 8641; https://doi.org/10.3390/ijms21228641 - 16 Nov 2020

Cited by 6 | Viewed by 2803

Abstract

The synthetic properties of the Thiamine diphosphate (ThDP)-dependent pyruvate dehydrogenase E1 subunit from Escherichia coli (EcPDH E1) was assessed for carboligation reactions with aliphatic ketoacids. Due to its role in metabolism, EcPDH E1 was previously characterised with respect to its biochemical properties, but it was never applied for synthetic purposes. Here, we show that EcPDH E1 is a promising biocatalyst for the production of chiral α-hydroxyketones. WT EcPDH E1 shows a 180–250-fold higher catalytic efficiency towards 2-oxobutyrate or pyruvate, respectively, in comparison to engineered transketolase variants from Geobacillus stearothermophilus (TK_GST). Its broad active site cleft allows for the efficient conversion of both (R)- and (S)-configured α-hydroxyaldehydes, next to linear and branched aliphatic aldehydes as acceptor substrates under kinetically controlled conditions. The alternate, thermodynamically controlled self-reaction of aliphatic aldehydes was shown to be limited to low levels of conversion, which we propose to be due to their large hydration constants. Additionally, the thermodynamically controlled approach was demonstrated to suffer from a loss of stereoselectivity, which makes it unfeasible for aliphatic substrates. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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Review

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23 pages, 2299 KiB

Open AccessReview

New Insights into the Modification of the Non-Core Metabolic Pathway of Steroids in Mycolicibacterium and the Application of Fermentation Biotechnology in C-19 Steroid Production

by Yang Zhang, Peiyao Xiao, Delong Pan and Xiuling Zhou

Int. J. Mol. Sci. 2023, 24(6), 5236; https://doi.org/10.3390/ijms24065236 - 9 Mar 2023

Cited by 3 | Viewed by 1919

Abstract

Androsta-4-ene-3,17-dione (AD), androsta-1,4-diene-3,17-dione (ADD), and 9α-hydroxy-4-androstene-3,17-dione (9-OHAD), which belong to C-19 steroids, are critical steroid-based drug intermediates. The biotransformation of phytosterols into C-19 steroids by Mycolicibacterium cell factories is the core step in the synthesis of steroid-based drugs. The production performance of engineered mycolicibacterial strains has been effectively enhanced by sterol core metabolic modification. In recent years, research on the non-core metabolic pathway of steroids (NCMS) in mycolicibacterial strains has made significant progress. This review discusses the molecular mechanisms and metabolic modifications of NCMS for accelerating sterol uptake, regulating coenzyme I balance, promoting propionyl-CoA metabolism, reducing reactive oxygen species, and regulating energy metabolism. In addition, the recent applications of biotechnology in steroid intermediate production are summarized and compared, and the future development trend of NCMS research is discussed. This review provides powerful theoretical support for metabolic regulation in the biotransformation of phytosterols. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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31 pages, 4514 KiB

Open AccessReview

Fungal Secondary Metabolites as Inhibitors of the Ubiquitin–Proteasome System

by Magdalena Staszczak

Int. J. Mol. Sci. 2021, 22(24), 13309; https://doi.org/10.3390/ijms222413309 - 10 Dec 2021

Cited by 10 | Viewed by 3156

Abstract

The ubiquitin–proteasome system (UPS) is the major non-lysosomal pathway responsible for regulated degradation of intracellular proteins in eukaryotes. As the principal proteolytic pathway in the cytosol and the nucleus, the UPS serves two main functions: the quality control function (i.e., removal of damaged, misfolded, and functionally incompetent proteins) and a major regulatory function (i.e., targeted degradation of a variety of short-lived regulatory proteins involved in cell cycle control, signal transduction cascades, and regulation of gene expression and metabolic pathways). Aberrations in the UPS are implicated in numerous human pathologies such as cancer, neurodegenerative disorders, autoimmunity, inflammation, or infectious diseases. Therefore, the UPS has become an attractive target for drug discovery and development. For the past two decades, much research has been focused on identifying and developing compounds that target specific components of the UPS. Considerable effort has been devoted to the development of both second-generation proteasome inhibitors and inhibitors of ubiquitinating/deubiquitinating enzymes. With the feature of unique structure and bioactivity, secondary metabolites (natural products) serve as the lead compounds in the development of new therapeutic drugs. This review, for the first time, summarizes fungal secondary metabolites found to act as inhibitors of the UPS components. Full article

(This article belongs to the Special Issue Microbial Enzymes and Metabolites)

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