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Article ; Online: Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform.

Arnolds, Kathleen L / Higgins, Riley C / Crandall, Jennifer / Li, Gabriella / Linger, Jeffrey G / Guarnieri, Michael T

2023 Volume 87, Issue 1, Page(s) 12

Abstract: Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related ...

Abstract	Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae. We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.
MeSH term(s)	Ecosystem ; Saccharomyces cerevisiae/genetics ; Soil Microbiology ; Microbiota ; Soil ; Risk Assessment
Chemical Substances	Soil
Language	English
Publishing date	2023-12-11
Publishing country	United States
Document type	Journal Article
ZDB-ID	1462065-0
ISSN	1432-184X ; 0095-3628
ISSN (online)	1432-184X
ISSN	0095-3628
DOI	10.1007/s00248-023-02321-8
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Article ; Online: Improved Combinatorial Assembly and Barcode Sequencing for Gene-Sized DNA Constructs.

Hernandez Hernandez, Diana / Ding, Lin / Murao, Ayako / Dahlin, Lukas R / Li, Gabriella / Arnolds, Kathleen L / Amezola, Melissa / Klein, Amit / Mitra, Aishwarya / Mecacci, Sonia / Linger, Jeffrey G / Guarnieri, Michael T / Suzuki, Yo

ACS synthetic biology

2023 Volume 12, Issue 9, Page(s) 2778–2782

Abstract: Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The ... ...

Abstract	Synergistic and supportive interactions among genes can be incorporated in engineering biology to enhance and stabilize the performance of biological systems, but combinatorial numerical explosion challenges the analysis of multigene interactions. The incorporation of DNA barcodes to mark genes coupled with next-generation sequencing offers a solution to this challenge. We describe improvements for a key method in this space, CombiGEM, to broaden its application to assembling typical gene-sized DNA fragments and to reduce the cost of sequencing for prevalent small-scale projects. The expanded reach of the method beyond currently targeted small RNA genes promotes the discovery and incorporation of gene synergy in natural and engineered processes such as biocontainment, the production of desired compounds, and previously uncharacterized fundamental biological mechanisms.
MeSH term(s)	High-Throughput Nucleotide Sequencing ; DNA/genetics
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2023-08-15
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, Non-U.S. Gov't
ISSN	2161-5063
ISSN (online)	2161-5063
DOI	10.1021/acssynbio.3c00183
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Article ; Online: Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Brunecky, Roman / Knott, Brandon C / Subramanian, Venkataramanan / Linger, Jeffrey G / Beckham, Gregg T / Amore, Antonella / Taylor, Larry E / Vander Wall, Todd A / Lunin, Vladimir V / Zheng, Fei / Garrido, Mercedes / Schuster, Logan / Fulk, Emily M / Farmer, Samuel / Himmel, Michael E / Decker, Stephen R

The Journal of biological chemistry

2024 Volume 300, Issue 3, Page(s) 105749

Abstract: Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically ... ...

Abstract	Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.
MeSH term(s)	Aspergillus oryzae/enzymology ; Aspergillus oryzae/genetics ; Cellulose 1,4-beta-Cellobiosidase/chemistry ; Cellulose 1,4-beta-Cellobiosidase/classification ; Cellulose 1,4-beta-Cellobiosidase/genetics ; Cellulose 1,4-beta-Cellobiosidase/metabolism ; Enzyme Assays ; Genome, Fungal/genetics ; Mutation ; Protein Engineering/methods ; Substrate Specificity ; Talaromyces/enzymology ; Talaromyces/genetics ; Trichoderma/enzymology ; Trichoderma/genetics ; Trichoderma/metabolism ; Biocatalysis
Chemical Substances	Cellulose 1,4-beta-Cellobiosidase (EC 3.2.1.91) ; lignocellulose (11132-73-3)
Language	English
Publishing date	2024-02-13
Publishing country	United States
Document type	Comparative Study ; Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2024.105749
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Article ; Online: Biotechnology for secure biocontainment designs in an emerging bioeconomy.

Arnolds, Kathleen L / Dahlin, Lukas R / Ding, Lin / Wu, Chao / Yu, Jianping / Xiong, Wei / Zuniga, Cristal / Suzuki, Yo / Zengler, Karsten / Linger, Jeffrey G / Guarnieri, Michael T

Current opinion in biotechnology

2021 Volume 71, Page(s) 25–31

Abstract: Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology ... ...

Abstract	Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.
MeSH term(s)	Agriculture ; Biotechnology ; Ecosystem ; Genome ; Synthetic Biology
Language	English
Publishing date	2021-06-03
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2021.05.004
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Article: Biotechnology for secure biocontainment designs in an emerging bioeconomy

Arnolds, Kathleen L / Dahlin, Lukas R / Ding, Lin / Wu, Chao / Yu, Jianping / Xiong, Wei / Zuniga, Cristal / Suzuki, Yo / Zengler, Karsten / Linger, Jeffrey G / Guarnieri, Michael T

Current opinion in biotechnology. 2021 Oct., v. 71

2021

Abstract	Genetically modified organisms (GMOs) have emerged as an integral component of a sustainable bioeconomy, with an array of applications in agriculture, bioenergy, and biomedicine. However, the rapid development of GMOs and associated synthetic biology approaches raises a number of biosecurity concerns related to environmental escape of GMOs, detection thereof, and impact upon native ecosystems. A myriad of genetic safeguards have been deployed in diverse microbial hosts, ranging from classical auxotrophies to global genome recoding. However, to realize the full potential of microbes as biocatalytic platforms in the bioeconomy, a deeper understanding of the fundamental principles governing microbial responsiveness to biocontainment constraints, and interactivity of GMOs with the environment, is required. Herein, we review recent analytical biotechnological advances and strategies to assess biocontainment and microbial bioproductivity, as well as opportunities for predictive systems biodesigns towards securing a viable bioeconomy.
Keywords	biocontainment ; bioeconomics ; bioenergy ; biosecurity ; biotechnology ; genome ; medicine ; synthetic biology
Language	English
Dates of publication	2021-10
Size	p. 25-31.
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2021.05.004
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: COVID-19 infection and transmission includes complex sequence diversity.

Chan, Ernest R / Jones, Lucas D / Linger, Marlin / Kovach, Jeffrey D / Torres-Teran, Maria M / Wertz, Audric / Donskey, Curtis J / Zimmerman, Peter A

PLoS genetics

2022 Volume 18, Issue 9, Page(s) e1010200

Abstract: ... identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed ...

Abstract	SARS-CoV-2 whole genome sequencing has played an important role in documenting the emergence of polymorphisms in the viral genome and its continuing evolution during the COVID-19 pandemic. Here we present data from over 360 patients to characterize the complex sequence diversity of individual infections identified during multiple variant surges (e.g., Alpha and Delta). Across our survey, we observed significantly increasing SARS-CoV-2 sequence diversity during the pandemic and frequent occurrence of multiple biallelic sequence polymorphisms in all infections. This sequence polymorphism shows that SARS-CoV-2 infections are heterogeneous mixtures. Convention for reporting microbial pathogens guides investigators to report a majority consensus sequence. In our study, we found that this approach would under-report sequence variation in all samples tested. As we find that this sequence heterogeneity is efficiently transmitted from donors to recipients, our findings illustrate that infection complexity must be monitored and reported more completely to understand SARS-CoV-2 infection and transmission dynamics. Many of the nucleotide changes that would not be reported in a majority consensus sequence have now been observed as lineage defining SNPs in Omicron BA.1 and/or BA.2 variants. This suggests that minority alleles in earlier SARS-CoV-2 infections may play an important role in the continuing evolution of new variants of concern.
MeSH term(s)	COVID-19/genetics ; Genome, Viral/genetics ; Humans ; Pandemics ; SARS-CoV-2/genetics
Language	English
Publishing date	2022-09-08
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2186725-2
ISSN	1553-7404 ; 1553-7390
ISSN (online)	1553-7404
ISSN	1553-7390
DOI	10.1371/journal.pgen.1010200
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Article ; Online: Conversion of levoglucosan and cellobiosan by

Linger, Jeffrey G / Hobdey, Sarah E / Franden, Mary Ann / Fulk, Emily M / Beckham, Gregg T

Metabolic engineering communications

2016 Volume 3, Page(s) 24–29

Abstract: Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of ...

Abstract	Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe
Language	English
Publishing date	2016-02-02
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2821894-2
ISSN	2214-0301 ; 2214-0301
ISSN (online)	2214-0301
ISSN	2214-0301
DOI	10.1016/j.meteno.2016.01.005
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Article: Eliminating a global regulator of carbon catabolite repression enhances the conversion of aromatic lignin monomers to muconate in

Johnson, Christopher W / Abraham, Paul E / Linger, Jeffrey G / Khanna, Payal / Hettich, Robert L / Beckham, Gregg T

Metabolic engineering communications

2017 Volume 5, Page(s) 19–25

Abstract: Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural ... ...

Abstract	Carbon catabolite repression refers to the preference of microbes to metabolize certain growth substrates over others in response to a variety of regulatory mechanisms. Such preferences are important for the fitness of organisms in their natural environments, but may hinder their performance as domesticated microbial cell factories. In a
Language	English
Publishing date	2017-05-31
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	2821894-2
ISSN	2214-0301
ISSN	2214-0301
DOI	10.1016/j.meteno.2017.05.002
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Article ; Online: Development of a high-productivity, halophilic, thermotolerant microalga

Dahlin, Lukas R / Gerritsen, Alida T / Henard, Calvin A / Van Wychen, Stefanie / Linger, Jeffrey G / Kunde, Yuliya / Hovde, Blake T / Starkenburg, Shawn R / Posewitz, Matthew C / Guarnieri, Michael T

Communications biology

2019 Volume 2, Page(s) 388

Abstract: Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga, ...

Abstract	Microalgae are promising biocatalysts for applications in sustainable fuel, food, and chemical production. Here, we describe culture collection screening, down-selection, and development of a high-productivity, halophilic, thermotolerant microalga,
MeSH term(s)	Biocatalysis ; Biomass ; Biotechnology ; Chlorophyta/genetics ; Chlorophyta/growth & development ; Chlorophyta/metabolism ; Gene Expression Profiling ; Genetic Engineering ; Genome, Chloroplast ; Genome, Microbial ; Industrial Microbiology/methods ; Microalgae/genetics ; Microalgae/growth & development ; Microalgae/metabolism ; Phototrophic Processes ; Salt Tolerance/genetics ; Thermotolerance/genetics
Language	English
Publishing date	2019-10-23
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-019-0620-2
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Article: Chromatin disassembly and reassembly during DNA repair.

Linger, Jeffrey G / Tyler, Jessica K

Mutation research

2007 Volume 618, Issue 1-2, Page(s) 52–64

Abstract: Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for ... ...

Abstract	Current research is demonstrating that the packaging of the eukaryotic genome together with histone proteins into chromatin is playing a fundamental role in DNA repair and the maintenance of genomic integrity. As is well established to be the case for transcription, the chromatin structure dynamically changes during DNA repair. Recent studies indicate that the complete removal of histones from DNA and their subsequent reassembly onto DNA accompanies DNA repair. This review will present evidence indicating that chromatin disassembly and reassembly occur during DNA repair and that these are critical processes for cell survival after DNA repair. Concomitantly, candidate proteins utilized for these processes will be highlighted.
MeSH term(s)	Adenosine Triphosphate/chemistry ; Animals ; Chromatin/genetics ; Chromatin/metabolism ; Chromatin/physiology ; Chromatin Assembly and Disassembly ; DNA/chemistry ; DNA Repair ; Histones/chemistry ; Humans ; Models, Biological ; Models, Genetic ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Chromatin ; Histones ; Adenosine Triphosphate (8L70Q75FXE) ; DNA (9007-49-2)
Language	English
Publishing date	2007-05-01
Publishing country	Netherlands
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	206607-5
ISSN	1873-135X ; 0027-5107 ; 1383-5718 ; 0165-1110 ; 0165-1161 ; 0165-7992 ; 0921-8777 ; 0165-1218 ; 1383-5726 ; 0167-8817 ; 0921-8734 ; 1383-5742
ISSN (online)	1873-135X
ISSN	0027-5107 ; 1383-5718 ; 0165-1110 ; 0165-1161 ; 0165-7992 ; 0921-8777 ; 0165-1218 ; 1383-5726 ; 0167-8817 ; 0921-8734 ; 1383-5742
DOI	10.1016/j.mrfmmm.2006.05.039
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