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2. Complete integration of carbene-transfer chemistry into biosynthesis

Author

Huang, Jing, Quest, Andrew, Cruz-Morales, Pablo, Deng, Kai, Pereira, Jose Henrique, Van Cura, Devon, Kakumanu, Ramu, Baidoo, Edward E. K., Dan, Qingyun, Chen, Yan, Petzold, Christopher J., Northen, Trent R., Adams, Paul D., Clark, Douglas S., Balskus, Emily P., Hartwig, John F., Mukhopadhyay, Aindrila, and Keasling, Jay D.

Published
2023
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13. Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries.

Author

Shrestha, Shilva, Goswami, Shubhasish, Banerjee, Deepanwita, Garcia, Valentina, Zhou, Elizabeth, Olmsted, Charles N., Majumder, Erica L.‐W., Kumar, Deepak, Awasthi, Deepika, Mukhopadhyay, Aindrila, Singer, Steven W., Gladden, John M., Simmons, Blake A., and Choudhary, Hemant

Abstract

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino‐refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin‐utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino‐chemical industry. [ABSTRACT FROM AUTHOR]

Published
2024
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14. Integration of Genome-Scale Metabolic Model with Biorefinery Process Model Reveals Market-Competitive Carbon-Negative Sustainable Aviation Fuel Utilizing Microbial Cell Mass Lipids and Biogenic CO2.

Author

Baral, Nawa Raj, Banerjee, Deepanwita, Mukhopadhyay, Aindrila, Simmons, Blake A., Singer, Steven W., and Scown, Corinne D.

Subjects
MICROBIAL lipids, FOSSIL fuels, CARBON sequestration, MICROBIAL fuel cells, AIRCRAFT fuels, BIOCONVERSION
Abstract

Producing scalable, economically viable, low-carbon biofuels or biochemicals hinges on more efficient bioconversion processes. While microbial conversion can offer robust solutions, the native microbial growth process often redirects a large fraction of carbon to CO2 and cell mass. By integrating genome-scale metabolic models with techno-economic and life cycle assessment models, this study analyzes the effects of converting cell mass lipids to hydrocarbon fuels, and CO2 to methanol on the facility's costs and life-cycle carbon footprint. Results show that upgrading microbial lipids or both microbial lipids and CO2 using renewable hydrogen produces carbon-negative bisabolene. Additionally, on-site electrolytic hydrogen production offers a supply of pure oxygen to use in place of air for bioconversion and fuel combustion in the boiler. To reach cost parity with conventional jet fuel, renewable hydrogen needs to be produced at less than $2.2 to $3.1/kg, with a bisabolene yield of 80% of the theoretical yield, along with cell mass and CO2 yields of 22 wt% and 54 wt%, respectively. The economic combination of cell mass, CO2, and bisabolene yields demonstrated in this study provides practical insights for prioritizing research, selecting suitable hosts, and determining necessary engineered production levels. [ABSTRACT FROM AUTHOR]

Published
2024
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20. Machine learning for metabolic engineering: A review

Author

Lawson, Christopher E., Martí, Jose Manuel, Radivojevic, Tijana, Jonnalagadda, Sai Vamshi R., Gentz, Reinhard, Hillson, Nathan J., Peisert, Sean, Kim, Joonhoon, Simmons, Blake A., Petzold, Christopher J., Singer, Steven W., Mukhopadhyay, Aindrila, Tanjore, Deepti, Dunn, Joshua G., and Garcia Martin, Hector

Published
2021
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24. IMG/PR: a database of plasmids from genomes and metagenomes with rich annotations and metadata.

Author

Camargo, Antonio Pedro, Call, Lee, Roux, Simon, Nayfach, Stephen, Huntemann, Marcel, Palaniappan, Krishnaveni, Ratner, Anna, Chu, Ken, Mukherjeep, Supratim, Reddy, T B K, Chen, I-Min A, Ivanova, Natalia N, Eloe-Fadrosh, Emiley A, Woyke, Tanja, Baltrus, David A, Castañeda-Barba, Salvador, de la Cruz, Fernando, Funnell, Barbara E, Hall, James P J, and Mukhopadhyay, Aindrila

Published
2024
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27. A model for improving microbial biofuel production using a synthetic feedback loop

Author

Dunlop, Mary J., Keasling, Jay D., and Mukhopadhyay, Aindrila

Subjects
Biomedicine, Biomedicine general, Feedback control, Biofuels, Biological control systems, Transcriptional regulation, Efflux pump, Feed forward loop
Abstract

Cells use feedback to implement a diverse range of regulatory functions. Building synthetic feedback control systems may yield insight into the roles that feedback can play in regulation since it can be introduced independently of native regulation, and alternative control architectures can be compared. We propose a model for microbial biofuel production where a synthetic control system is used to increase cell viability and biofuel yields. Although microbes can be engineered to produce biofuels, the fuels are often toxic to cell growth, creating a negative feedback loop that limits biofuel production. These toxic effects may be mitigated by expressing efflux pumps that export biofuel from the cell. We developed a model for cell growth and biofuel production and used it to compare several genetic control strategies for their ability to improve biofuel yields. We show that controlling efflux pump expression directly with a biofuel-responsive promoter is a straightforward way of improving biofuel production. In addition, a feed forward loop controller is shown to be versatile at dealing with uncertainty in biofuel production rates.

Published
2010

35. Enhanced microbial production of protocatechuate from engineered sorghum using an integrated feedstock-to-product conversion technology.

Author

Garcia, Valentina E., Pidatala, Venkataramana, Barcelos, Carolina A., Liu, Dupeng, Otoupal, Peter, Wendt, Oliver, Choudhary, Hemant, Sun, Ning, Eudes, Aymerick, Sundstrom, Eric R., Scheller, Henrik V., Putnam, Daniel H., Mukhopadhyay, Aindrila, Gladden, John M., Simmons, Blake A., and Rodriguez, Alberto

Subjects
SORGHUM, CARBON nanofibers, SUSTAINABLE development, GENETIC engineering, BIOLOGICAL products, BIOMASS, FEEDSTOCK
Abstract

Building a stronger bioeconomy requires production capabilities that are largely generated through microbial genetic engineering. Plant feedstocks can additionally be genetically engineered to generate desirable feedstock traits and provide precursors for direct microbial conversion into desired products. The oleaginous yeast Rhodosporidium toruloides is a promising organism for this type of conversion as it can grow on a wide range of deconstructed biomass and consume a variety of carbon sources. Here, we leveraged R. toruloides native p-coumaric acid consumption pathway to accumulate protocatechuate (PCA) from 4-hydroxybenzoate (4HBA) released from a sorghum feedstock line genetically engineered to overproduce 4HBA. We did so by generating and evaluating an R. toruloides strain that accumulates PCA, RSΔ12623. We then show that at two scales a cholinium lysinate pretreatment with enzymatic saccharification successfully extracts 95% of the 4HBA from the engineered sorghum biomass while producing deconstructed lignin that can be more efficiently depolymerized in a subsequent thermochemical reaction. We also demonstrate that strain RSΔ12623 can convert more than 95% of 4HBA to PCA while consuming >95% of the glucose and >80% of the xylose present in sorghum hydrolysates. Finally, to evaluate the scalability of such fermentations, we conducted the conversion of 4HBA to PCA in a 2 L bioreactor under controlled conditions. This work demonstrates the potential of purposefully producing aromatic precursors in planta that can be liberated during biomass deconstruction for direct microbial conversion to desirable bioproducts. [ABSTRACT FROM AUTHOR]

Published
2023
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42. Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida.

Author

Gauttam, Rahul, Eng, Thomas, Zhao, Zhiying, ul ain Rana, Qurrat, Simmons, Blake A., Yoshikuni, Yasuo, Mukhopadhyay, Aindrila, and Singer, Steven W.

Subjects
PSEUDOMONAS putida, BIOMASS conversion, PROTEIN expression, GENOME editing, GENE expression, BINDING sites
Abstract

Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts. [ABSTRACT FROM AUTHOR]

Published
2023
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43. Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

Author

Park, Mee‐Rye, Gauttam, Rahul, Fong, Bonnie, Chen, Yan, Lim, Hyun Gyu, Feist, Adam M., Mukhopadhyay, Aindrila, Petzold, Christopher J., Simmons, Blake A., and Singer, Steven W.

Subjects
PSEUDOMONAS putida, PLANT biomass, BIOMASS conversion, ARABINOSE, METABOLISM, HEMICELLULOSE
Abstract

The Pseudomonas putida group in the Gammaproteobacteria has been intensively studied for bioremediation and plant growth promotion. Members of this group have recently emerged as promising hosts to convert intermediates derived from plant biomass to biofuels and biochemicals. However, most strains of P. putida cannot metabolize pentose sugars derived from hemicellulose. Here, we describe three isolates that provide a broader view of the pentose sugar catabolism in the P. putida group. One of these isolates clusters with the well‐characterized P. alloputida KT2440 (Strain BP6); the second isolate clustered with plant growth‐promoting strain P. putida W619 (Strain M2), while the third isolate represents a new species in the group (Strain BP8). Each of these isolates possessed homologous genes for oxidative xylose catabolism (xylDXA) and a potential xylonate transporter. Strain M2 grew on arabinose and had genes for oxidative arabinose catabolism (araDXA). A CRISPR interference (CRISPRi) system was developed for strain M2 and identified conditionally essential genes for xylose growth. A glucose dehydrogenase was found to be responsible for initial oxidation of xylose and arabinose in strain M2. These isolates have illuminated inherent diversity in pentose catabolism in the P. putida group and may provide alternative hosts for biomass conversion. [ABSTRACT FROM AUTHOR]

Published
2023
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