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At least 109 records · Page 6

Engineered plants for the production of the antioxidants arbutin and gallate

The shikimate pathway is a crucial metabolic route for the biosynthesis of numerous valuable chemicals. In this study, we engineered the shikimate pathway in plants via expression of microbial enzymes to produce the two important antioxidants gallate and arbutin. The engineered pathways utilize the aromatics protocatechuate and 4-hydroxybenzoate as metabolic intermediates. Through transient expression in Nicotiana benthamiana leaves, we first identified biosynthetic routes for the production of gallate from either chorismate or 3-dehydroshikimate. Gallate production was then achieved in Arabidopsis using a genetic background that overproduces protocatechuate and via expression of a mutated version of the 4-hydroxybenzoate hydroxylase PobA from Pseudomonas sp. Arbutin production was obtained in Arabidopsis using a genetic background that overproduces 4-hydroxybenzoate and via expression of the monooxygenase MNX1 from Candida parapsilosis. The best Arabidopsis transgenic lines accumulated gallate and arbutin in the range of 0.25 and 0.93 dry weight % (dwt%), respectively. Using sorghum for large-scale in planta production, the titers of gallate and arbutin produced from the intermediate 4-hydroxybenzoate reached 0.58 dwt% and 0.50 dwt%, respectively, in mature transgenic plants, surpassing levels typically observed in plants that naturally produce these compounds. Gallate and arbutin were readily extracted from plant tissues using methanol solvent. Analysis of extractive-free biomass showed only trace amounts of gallate and its precursors 4-hydroxybenzoate and protocatechuate crosslinked to cell walls, suggesting that they mainly occur as soluble conjugated forms stored in the vacuole. This study presents alternative synthesis routes using plant hosts for the eco-friendly production of gallate and arbutin.

Antioxidant

Engineering Escherichia coli for Urease-Driven Synthesis of Metal Oxide Nanomaterials

The development of functional nanomaterials with controlled morphologies is essential for advancements in medicine, electronics and computing, energy, catalysis, and environmental applications. However, conventional synthesis methods often demand high energy input and pose significant environmental challenges. Urease-based biomineralization presents an efficient, eco-friendly alternative for nanomaterial production under mild conditions. In this study, we engineered Escherichia coli ( E. coli ) to express a urease gene cluster from Sporosarcina pasteurii using CRAGE-Duet technology. The engineered strain successfully synthesized calcium carbonate and calcium phosphate crystals. Expanding the approach, we synthesized metal oxide nanoparticles, including hematite (Fe 2 O 3 ), and nanocrystalline anatase titanium dioxide (TiO 2 ). These nanomaterials were characterized by electron microscopy, demonstrating the potential of E. coli as a sustainable and versatile platform for green nanomaterial synthesis.

bacteria

Engineering an Extremely Hybrid PKS for Adipic Acid Production

Polyketide synthases (PKSs) are modular enzymes with exceptional potential as biocatalysts for producing non-native compounds. Here, we report the first PKS-based pathway for adipic acid (AA), an industrial monomer for nylon production, by engineering one of the most extensively hybridized PKS systems to date. Using a retrobiosynthetic approach, we identified EtnB, a succinyl-CoA-loading module that uniquely retains the terminal carboxyl group, enabling access to dicarboxylic polyketide products, rarely produced by canonical PKSs. EtnB was coupled to a fully reducing extension module through an engineered communication linker, which improved ACP–KS interactions, enhanced titers, and demonstrated selective succinyl-CoA loading in vivo . This construct integrates genes from five organisms─with domains from seven PKS modules joined across six non-natural junctions─and functions in both Escherichia coli and Pseudomonas putida . Additional engineering that included AT domain exchanges, optimization of extender unit supply, and host strain metabolic rewiring further increased AA production. Together, this work demonstrates that highly chimeric PKSs can be rendered functional through rational design, expands the PKS toolkit with a carboxyl-retaining loading module, and establishes a versatile platform for engineering diacids and other noncanonical products through PKS pathways.

biomanufacturing

Retrobiosynthesis of unnatural lactams via reprogrammed polyketide synthase

Engineered polyketide synthases (PKSs) have great potential as biocatalysts. These unnatural enzymes are capable of synthesizing molecules that are either not amenable to biosynthesis or are extremely challenging to access chemically. PKSs can thus be a powerful platform to expand the chemical landscape beyond the limits of conventional metabolic engineering. Here we employ a retrobiosynthesis approach to design and construct PKSs to produce δ-valerolactam (VL) and three enantiopure α-substituted VL analogues that have no known biosynthetic route. We introduce the engineered PKSs and pathways for various malonyl-CoA derivatives into Pseudomonas putida and use proteomics, metabolomics and culture condition optimization to improve the production of our target compounds. These α-substituted VLs are polymerized into polyamides (nylon-5) or converted into their N-acryloyl derivatives. RAFT polymerization produces bio-derived polymers with potential biomedical applications. Overall, this interdisciplinary effort highlights the versatility and effectiveness of a PKS-based retrobiosynthesis approach in exploring and developing innovative biomaterials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH

Using the optimal combined index weight ratio to improve the probability of anomaly detection in big area additive manufacturing

Big Area Additive Manufacturing (BAAM) of composites requires significant time, energy, and material, so it is critical to reduce production inefficiencies to make functional parts without multiple iterations. Statistical process control coupled with Principal Component Analysis (PCA) is a powerful technique that provides a quick, computationally inexpensive, and intuitive way for operators to detect defects that form in a manufacturing process without massive datasets. Recently, a combined index that is a weighted sum of the Hotelling's T 2 and squared residual error statistics has been proposed that can be monitored in one chart, improving interpretation accuracy and simplicity. However, the literature does not offer a formal method to optimise the weights. Here, we introduce two new approaches to the traditional weight selection approach using simulated and BAAM image data. Approach 1 uses a theoretically motivated optimum inspired by probabilistic principal component analysis. Approach 2 systematically varies the ratio of the weights to find the optimum. We show that approach 1 delivers optimal anomaly detection performance in select cases while approach 2 fares better in practice. Surprisingly, we also show that choosing a more complex PCA model has a minimal negative impact on anomaly detection performance compared to a more simplistic model.

3-dimensional printing

Biosensors for the detection of chorismate and cis,cis -muconic acid in Corynebacterium glutamicum

Abstract Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate–pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. One-Sentence Summary High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and β-ketoadipate pathways.

59 BASIC BIOLOGICAL SCIENCES

MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

59 BASIC BIOLOGICAL SCIENCES

Exabiome: Advancing Microbial Science through Exascale Computing

The Exabiome project seeks to improve the understanding of microbiomes through the development of methods for accelerating metagenomic science using exascale computing. This article gives an overview of scientific impact of the three components of the project: metagenome assembly, protein family detection, and comparative analysis of metagenomes. Exabiome developed MetaHipMer, the only metagenome assembler capable of scaling to full exascale systems. MetaHipMer has enabled ground-breaking assemblies on the Frontier supercomputer, with many scientific benefits, such as the discovery of rare species and viral genomes. To investigate protein families, Exabiome developed two exascale tools, PASTIS and HipMCL. Together, these can utilize exascale resources to understand the functional diversity of billions of dark matter proteins and novel protein families. For comparative analysis, Exabiome developed kmerprof, a tool that can be used to compare huge metagenomes for many different scientific purposes, for example, grouping human microbiomes according to body location.

59 BASIC BIOLOGICAL SCIENCES

Synthetic Biology to Support Human Exploration of Deep Space

The International Space Station (ISS) has enabled a continuous human presence in space since November 2000. The ISS is in low Earth orbit, facilitating regular resupply missions to deliver air, water, food, spare parts, and science experiments. NASA’s Moon to Mars campaign seeks to return humans to the Moon and prepare for crewed missions to Mars. Increased distance from Earth poses logistical challenges to provide all resources needed by humans for deep space missions. NASA Ames Research Center is conducting a series of synthetic biology projects to test the use of microbes for on-demand biosynthesis of human micronutrients. The BioNutrients spaceflight experiments test an implementation concept to produce fermented food products in which microbe growth enhances micronutrient content. On-demand production of carotenoids was engineered into two yeast (Saccharomyces cerevisiae) strains that have been tested in nearly 5 years of storage. One of the BioNutrients strains will be incorporated into the Lunar Explorer Instrument for Space Biology Applications (LEIA) project. LEIA is developing an instrument suite to be delivered to the lunar south pole region by the Commercial Lunar Payload Services (CLPS) program. The LEIA instrument suite will be used to measure multiple yeast strains for growth, metabolic activity, and synthetic biology-enabled production of carotenoids, while taking real time measurements of biologically relevant radiation exposure on the lunar surface. LEIA data will be used to assess the impact of lunar surface radiation and reduced gravity on the production of engineered traits.

Synthetic Biology