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  • 1
    Online Resource
    Online Resource
    Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation
    Proceedings of the National Academy of Sciences ; 2023
    In:  Proceedings of the National Academy of Sciences Vol. 120, No. 4 ( 2023-01-24)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 120, No. 4 ( 2023-01-24)
    Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans . The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2212246120
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2023
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  • 2
    Online Resource
    Online Resource
    Rapid, Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans
    American Society for Microbiology ; 2018
    In:  Applied and Environmental Microbiology Vol. 84, No. 22 ( 2018-11-15)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 84, No. 22 ( 2018-11-15)
    Abstract: Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here, we show that when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously hypothetical enzymes in an unbiased genome-scale search. We used this technique to identify two genes, desC and desD , which are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the nonmodel bacterium Novosphingobium aromaticivorans . We show that DesC is a methyl esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. This approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering. IMPORTANCE The identification of the genes involved in specific biochemical transformations is a key step in predicting microbial function from nucleic acid sequences and in engineering microbes to endow them with new functions. We have shown that new techniques for transposon mutagenesis can dramatically simplify this process and enable the rapid identification of genes in uncharacterized pathways. These techniques provide the necessary scale to fully elucidate complex biological networks such as those used to degrade mixtures of lignin-derived aromatic compounds.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01185-18
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
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  • 3
    Online Resource
    Online Resource
    Protoplast fusion in Bacillus species produces frequent, unbiased, genome-wide homologous recombination
    Oxford University Press (OUP) ; 2022
    In:  Nucleic Acids Research Vol. 50, No. 11 ( 2022-06-24), p. 6211-6223
    Details
    In: Nucleic Acids Research, Oxford University Press (OUP), Vol. 50, No. 11 ( 2022-06-24), p. 6211-6223
    Abstract: In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.
    Type of Medium: Online Resource
    ISSN: 0305-1048 , 1362-4962
    URL: Article
    DOI: 10.1093/nar/gkac025
    RVK:
    WA 15000
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2022
    detail.hit.zdb_id: 1472175-2
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  • 4
    Online Resource
    Online Resource
    Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases
    Proceedings of the National Academy of Sciences ; 2012
    In:  Proceedings of the National Academy of Sciences Vol. 109, No. 47 ( 2012-11-20), p. 19504-19509
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 47 ( 2012-11-20), p. 19504-19509
    Abstract: Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae . Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1212287109
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2012
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    SSG: 11
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  • 5
    Online Resource
    Online Resource
    Construction and Optimization of a Heterologous Pathway for Protocatechuate Catabolism in Escherichia coli Enables Bioconversion of Model Aromatic Compounds
    American Society for Microbiology ; 2017
    In:  Applied and Environmental Microbiology Vol. 83, No. 18 ( 2017-09-15)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 83, No. 18 ( 2017-09-15)
    Abstract: The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01313-17
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2017
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    detail.hit.zdb_id: 1478346-0
    SSG: 12
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  • 6
    Online Resource
    Online Resource
    CropPol : A dynamic, open and global database on crop pollination
    Wiley ; 2022
    In:  Ecology Vol. 103, No. 3 ( 2022-03)
    Details
    In: Ecology, Wiley, Vol. 103, No. 3 ( 2022-03)
    Abstract: Seventy five percent of the world's food crops benefit from insect pollination. Hence, there has been increased interest in how global change drivers impact this critical ecosystem service. Because standardized data on crop pollination are rarely available, we are limited in our capacity to understand the variation in pollination benefits to crop yield, as well as to anticipate changes in this service, develop predictions, and inform management actions. Here, we present CropPol, a dynamic, open, and global database on crop pollination. It contains measurements recorded from 202 crop studies, covering 3,394 field observations, 2,552 yield measurements (i.e., berry mass, number of fruits, and fruit density [kg/ha], among others), and 47,752 insect records from 48 commercial crops distributed around the globe. CropPol comprises 32 of the 87 leading global crops and commodities that are pollinator dependent. Malus domestica is the most represented crop (32 studies), followed by Brassica napus (22 studies), Vaccinium corymbosum (13 studies), and Citrullus lanatus (12 studies). The most abundant pollinator guilds recorded are honey bees (34.22% counts), bumblebees (19.19%), flies other than Syrphidae and Bombyliidae (13.18%), other wild bees (13.13%), beetles (10.97%), Syrphidae (4.87%), and Bombyliidae (0.05%). Locations comprise 34 countries distributed among Europe (76 studies), North America (60), Latin America and the Caribbean (29), Asia (20), Oceania (10), and Africa (7). Sampling spans three decades and is concentrated on 2001–2005 (21 studies), 2006–2010 (40), 2011–2015 (88), and 2016–2020 (50). This is the most comprehensive open global data set on measurements of crop flower visitors, crop pollinators and pollination to date, and we encourage researchers to add more datasets to this database in the future. This data set is released for non‐commercial use only. Credits should be given to this paper (i.e., proper citation), and the products generated with this database should be shared under the same license terms (CC BY‐NC‐SA).
    Type of Medium: Online Resource
    ISSN: 0012-9658 , 1939-9170
    URL: Issue
    URL: Article
    DOI: 10.1002/ecy.v103.3
    DOI: 10.1002/ecy.3614
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2022
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    SSG: 12
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