Results for “microbial manufacturing platform”

DOE OSTI2020

Towards renewable flavors, fragrances, and beyond

Esters demand a large space of unique molecules with broad range of applications as flavors, fragrances, pharmaceuticals, cosmetics, green solvents, and advanced biofuels. Global demand of natural esters in food, household cleaner, personal care, and perfume industries is increasing while the ester supply from natural sources has been limited. Development of novel microbial cell factories for ester production from renewable feedstocks can potentially provide an alternative and sustainable source of natural esters and hence help fulfill growing demand. In this work, we highlight recent advances in microbial production of esters and provide perspectives for improving its economic feasibility. As the field matures, microbial ester production platforms will enable renewable and sustainable production of flavors, fragrances, and open new market opportunities beyond what nature can offer.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2013

Cosmo Cassette: A Microfluidic Microgravity Microbial System For Synthetic Biology Unit Tests and Satellite Missions

Although methods in the design-build-test life cycle of the synthetic biology field have grown rapidly, the expansion has been non-uniform. The design and build stages in development have seen innovations in the form of biological CAD and more efficient means for building DNA, RNA, and other biological constructs. The testing phase of the cycle remains in need of innovation. Presented will be both a theoretical abstraction of biological measurement and a practical demonstration of a microfluidics-based platform for characterizing synthetic biological phenomena. Such a platform demonstrates a design of additive manufacturing (3D printing) for construction of a microbial fuel cell (MFC) to be used in experiments carried out in space. First, the biocompatibility of the polypropylene chassis will be demonstrated. The novel MFCs will be cheaper, and faster to make and iterate through designs. The novel design will contain a manifold switchingdistribution system and an integrated in-chip set of reagent reservoirs fabricated via 3D printing. The automated nature of the 3D printing yields itself to higher resolution switching valves and leads to smaller sized payloads, lower cost, reduced power and a standardized platform for synthetic biology unit tests on Earth and in space. It will be demonstrated that the application of unit testing in synthetic biology will lead to the automatic construction and validation of desired constructs. Unit testing methodologies offer benefits of preemptive problem identification, change of facility, simplicity of integration, ease of documentation, and separation of interface from implementation, and automated design.

Bioelectrochemical System↗

NASA NTRS2018

In-space Biomanufacturing Using CO2 with Methylobacterium Extorquens

Long duration missions to deep space will require new approaches for supplying astronauts. In-space microbial manufacturing could generate many important compounds (such as nutrients, pharmaceuticals and fuels) but there are significant barriers to deploying reliable bioproduction platforms to space. These include ensuring adequate production and proper purification of the desired product, especially in the unique radiation and microgravity environment. Here we are focused on developing methods and technologies to feed microbial factories using the resources available in space. CO2, found in abundance in spacecraft cabins and the Mars atmosphere, can be sequestered and converted into bioproducts. While autotrophic organisms can use CO2 directly, they are generally slow growing and have less-developed biotechnology toolkits. Therefore we are developing an alternative paradigm in which CO2 is first reduced to more energetic carbon compounds that can support more rapid growth of workhorse biotechnology platforms (E. coli, S. cerevisiae, P. pastoris).Various technologies exist or are being developed to convert CO2. For example, the Sabatier system currently installed on the ISS, reacts CO2 and H2 to generate CH4 and H2O. This methane could be consumed by engineered methanotrophic bacteria. Alternatively, electrochemical systems can convert CO2 into formate (CHO2) which could be consumed by formatotrophic bacteria. In either case, synthetic biology techniques allow these microbes to serve as reprogrammable biofactories capable of producing a vast number or products.

Synthetic biology; Formate metabolism↗

NASA NTRSDate not supplied

Biomanufacturing in Space: BioNutrients and CO2-Based Manufacturing

Biomanufacturing can provide on-demand production of mission-critical compounds and materials to support long-duration space exploration while circumventing the challenges of transporting materials from Earth. Synthetic Biology Project is developing two biomanufacturing capabilities: BioNutrients and CO2-Based Manufacturing. BioNutrients is an ongoing mission aboard the International Space Station focused on the production of perishable nutrients in an on demand for direct for consumption. The first flight experiment of this project targeted the production of carotenoids: β-carotene and zeaxanthin, in recombinant yeast strains. Since then, the project has expanded to encompass the production of the fermented consumables like yogurt and kefir for use as a nutrient delivery mechanism. The CO2-Based Manufacturing system aims to use in situ resources to allow for biomanufacturing with minimal re-supply required. The manufacturing platform is combined with an electrochemical CO2 conversion system which can produce simple carbon substrates to support microbial based biomanufacturing. A comprehensive ground-based platform for recombinant protein purification is in development with the goal of producing a thermostable carbonic anhydrase enzyme from E. coli utilizing CO2-derived acetate. Our group hopes to propel advancements in space biomanufacturing for long duration space flight by harnessing the tools of synthetic biology.

Matthew Brian Paddock↗

NASA NTRSDate not supplied

Harnessing Synthetic Biology for Biomanufacturing in Space: from Fermented Foods to Industrial Enzymes

Biomanufacturing can provide on-demand production of mission-critical compounds and materials to support long-duration space exploration while circumventing the challenges of transporting materials from Earth. Synthetic Biology Project is developing two biomanufacturing capabilities: BioNutrients and CO2-Based Manufacturing. BioNutrients is an ongoing mission aboard the International Space Station focused on the production of perishable nutrients in an on demand for direct for consumption. The first flight experiment of this project targeted the production of carotenoids: β-carotene and zeaxanthin, in recombinant yeast strains. Since then, the project has expanded to encompass the production of the fermented consumables like yogurt and kefir for use as a nutrient delivery mechanism. The CO2-Based Manufacturing system aims to use in situ resources to allow for biomanufacturing with minimal re-supply required. The manufacturing platform is combined with an electrochemical CO2 conversion system which can produce simple carbon substrates to support microbial based biomanufacturing. A comprehensive ground-based platform for recombinant protein purification is in development with the goal of producing a thermostable carbonic anhydrase enzyme from E. coli utilizing CO2-derived acetate. Our group hopes to propel advancements in space biomanufacturing for long duration space flight by harnessing the tools of synthetic biology.

synthetic biology↗

DOE OSTI2020

A yeast platform for high-level synthesis of tetrahydroisoquinoline alkaloids

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate ( S )-reticuline to 4.6 g L –1 , a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Development of spirulina for the manufacture and oral delivery of protein therapeutics

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in Escherichia coli

Microbial bioproduction using one-carbon (C1) feedstocks has the potential to decarbonize the manufacturing of materials, fuels, and chemicals. Formate is a promising C1 feedstock, and the realization of industrial, formatotrophic platform organisms is a key goal for C1-based bioproduction. So far, a major limitation for synthetic formatotrophy has been slow energy supply due to slow formate dehydrogenase activity. Here, we implement a fast, metal-dependent formate dehydrogenase complex in a synthetic formatotrophic Escherichia coli utilizing the reductive glycine pathway. After a short-term evolution, we demonstrate formatotrophic growth of E. coli with a doubling time of less than 4.5 h, comparable to the fastest natural formatotrophs. To further explore the potential of a formate-based bioeconomy, this strain is engineered to produce mevalonate, as well as the terpenoid and aviation fuel precursor isoprenol, using formate we generate directly from the electrochemical reduction of CO2. This work demonstrates an improvement in bioproduct titer from formate, achieving the production of 3.8 g/L of mevalonate. Additionally, the abundant and recalcitrant polymer lignin is chemically decomposed into a formate-rich mixture of small organic acids and subsequently bioconverted into mevalonate. Overall, the described fast-growing, formatotrophic bioproduction strain demonstrates that a sustainable formate bioeconomy is within reach.

Cowan, Aidan E↗

DOE OSTI2026

Data for High Yield Production of 3-Hydroxypropionic Acid Using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Bioproducts↗

DOE OSTI2023

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported, and the highest titer produced from lignocellulosic hydrolysate to the best of our knowledge. This work demonstrates microbial production of 3HP in R. toruloides from lignocellulosic hydrolysate at high titers, and it represents a significant step toward enabling industrial production of 3HP in the future.

3-Hydroxypropionic acid↗

DOE OSTI2024

Developing multi-gene CRISPRa/i programs to accelerate DBTL cycles in ABF hosts engineered for chemical production

This project developed and implemented a modular CRISPR activation and interference (CRISPRa/i) platform to accelerate strain optimization and pathway development for industrially relevant microbial hosts. By integrating multiplexed transcriptional perturbation tools with data-driven Design–Build–Test–Learn (DBTL) workflows, the team achieved reductions in cycle time and enhanced production of industrial aromatics, particularly 4-aminocinnamic acid (4-ACA), in Pseudomonas putida. Key accomplishments included: ● Development of a robust, tunable CRISPRa/i system in P. putida that enabled efficient multi-target gene regulation via guide RNA (gRNA) programs ● Completion of two full DBTL cycles, guided by machine learning (ML) models trained on transcriptomic and performance data, reducing engineering time by over 30% ● Optimization of multi-gene regulatory programs to balance expression of host and pathway modules, improve 4-ACA titers, and resolve metabolic bottlenecks ● Demonstration of system portability through a limited proof-of-concept extension in Acinetobacter baylyi, underscoring the generalizability of the approach ● Evaluation of strain performance on lignocellulosic biomass-derived substrates, demonstrating the feasibility of converting renewable carbon into aromatic building blocks These results illustrate the feasibility of applying ML-guided CRISPRa/i perturbation strategies to accelerate strain development in complex microbial systems. The resulting tools and datasets contribute to DOE objectives by improving platform predictability, reducing development costs, and enabling broader access to sustainable, economically viable bioproduction technologies.

09 BIOMASS FUELS↗

NASA NTRS2019

Bionutrients-1, On-Demand Production of Nutrients in Space

Future long-duration missions face significant challenges maintaining crew health. A critical area is supplying adequate nutrition, as certain vitamins and nutrients in supplied foods and supplements demonstrate substantial degradation during extended storage. To address this issue, we are developing and flight-testing a platform technology that demonstrates in situ microbial production of targeted nutrients over extended mission durations. This 5-year experiment, known as BioNutrients-1, was started on the International Space Station in May 2019. It involves two components: an on-orbit hydration and production experiment; and the development of space-compatible, key bio-manufacturing microorganisms. On-orbit testing utilizes a small “production pack” system that encloses sterile edible growth substrate and desiccated Saccharomyces cerevisiae strains genetically engineered to produce the nutrients beta-carotene or zeaxanthin. On hydration and mixing of the production pack, the organisms revive and grow until limited by the depletion of growth media, hypothetically leading to consistent amounts of biomass and nutrients. In eventual mission applications, the packet contents would be heat treated to inactivate the microorganisms prior to consumption. For these flight experiments, the packet will not be heat treated, but will instead be frozen for return to Earth for analyses. In addition to the production pack trials, 14 different microorganisms/treatments were also delivered to ISS for long-duration storage. These samples will be intermittently returned to Earth and analyzed to determine survival rates and genomics. For this presentation, initial data from returned samples and ground controls will be discussed.

Hindupur, Aditya↗

NASA NTRS2019

BioNutrients-1: On-Demand Production of Nutrients in Space

Future long-duration missions face significant challenges maintaining crew health. A critical area is supplying adequate nutrition, as certain vitamins and nutrients in supplied foods and supplements demonstrate substantial degradation during extended storage. To address this issue, we are developing and flight-testing a platform technology that demonstrates in situ microbial production of targeted nutrients over extended mission durations. This 5-year experiment, known as BioNutrients-1, was started on the International Space Station in May 2019. It involves two components: an on-orbit hydration and production experiment; and the development of space-compatible, key bio-manufacturing microorganisms. On-orbit testing utilizes a small "production pack" system that encloses sterile edible growth substrate and desiccated Saccharomyces cerevisiae strains genetically engineered to produce the nutrients beta-carotene or zeaxanthin. On hydration and mixing of the production pack, the organisms revive and grow until limited by the depletion of growth media, hypothetically leading to consistent amounts of biomass and nutrients. In eventual mission applications, the packet contents would be heat treated to inactivate the microorganisms prior to consumption. For these flight experiments, the packet will not be heat treated, but will instead be frozen for return to Earth for analyses. In addition to the production pack trials, 14 different microorganisms/treatments were also delivered to ISS for long-duration storage. These samples will be intermittently returned to Earth and analyzed to determine survival rates and genomics. For this presentation, initial data from returned samples and ground controls will be discussed.

Hindupur, Aditya↗

DOE OSTI2026

Integrating CO2 Electrolysis with Gas Fermentation to Produce Valuable Fuels and Chemicals

Many industrial activities squander CO2, decreasing process yield. We envision a future where this waste carbon is instead captured, upgraded, and valorized directly at the point of emission. Within the CO2 Reduction and Upgrading Consortium (a collaboration of seven US national laboratories and industrial partners), we are pursuing this goal by developing and de-risking new technologies for low temperature CO2 electrolysis, coupled with biological upgrading of intermediates into more valuable compounds. One such process involves electrocatalytic reduction of CO2 to generate carbon monoxide (CO). As both a carbon and energy source, CO represents an attractive feedstock for microbial upgrading by certain syngas-fermenting species, such as the autotrophic bacterium Clostridium autoethanogenum. Our team has developed new genetic tools and optimized cultivation techniques to enhance C. autoethanogenum as a platform host for the biological conversion of syngas into value-added products. For example, we have created novel CRISPR-based genetic engineering techniques to build new, genome-reduced, platform strains of C. autoethanogenum with improved growth rates. Further, we ve introduced heterologous biochemical pathways into C. autoethanogenum to enable the production of high-value compounds from syngas, such as the isoprenoid precursor mevalonic acid. With the tools of electrochemistry and synthetic biology, there is virtually no limit to the spectrum of products that could be sustainably manufactured from CO2.

09 BIOMASS FUELS↗

DOE OSTI2024

Establishing a clostridia foundry for biosystems design by integrating computational modeling, systems-level analyses, and cell-free engineering technologies (Final Report)

Rapid population growth, a rise in global living standards, and economic competitiveness have intensified the need for sustainable, low-cost biofuels and bioproducts production. Industrial biotechnology using microbial cell factories – and waste and/or renewable feedstocks – is one of the most attractive approaches for addressing this need, particularly when large-scale chemical synthesis is untenable. Unfortunately, designing, building, and optimizing biosynthetic pathways in cells remains a complex challenge. With support from the Department of Energy, we worked to address this challenge in a new interdisciplinary venture that established the world’s first clostridial Foundry for Biosystems Design (cBioFAB). Working both in vitro (cell-free) and in vivo, the goal of this project was to interweave and advance state-of-the-art computational modeling, genome editing, omics measurements, systems-biology analyses, and cell-free technologies to expand the set of platform organisms that meet DOE bioenergy goals. Specifically, we manufactured fuel and chemical intermediates via existing and de novo pathways. This report covers the outcomes of our research project.

09 BIOMASS FUELS↗

DOE OSTI2026

High yield production of 3-hydroxypropionic acid using Issatchenkia orientalis

Biomanufacturing provides a more sustainable alternative to fossil-based chemical manufacturing. 3-Hydroxypropionic acid (3HP) is a top Department of Energy value-added chemical and precursor to bioplastics, yet cost-effective microbial production remains elusive. Here, we establish the acid-tolerant yeast Issatchenkia orientalis as a robust host for low-pH 3HP biosynthesis. Genome-scale modeling identifies the β-alanine pathway as optimal, offering the highest theoretical yield and lowest oxygen requirement. Thermodynamic analysis confirms its favorability under acidic conditions. Using sequence similarity network analysis, we discover highly active aspartate 1-decarboxylase (PAND), β-alanine-pyruvate aminotransferase (BAPAT), and 3HP dehydrogenase (YDFG), which significantly improve the pathway efficiency. Next, to further elevate the production, pathway optimization through multi-copy PAND integration, byproduct elimination (knockouts of pyruvate decarboxylase and glycerol-3-phosphate dehydrogenase), and reinforcement of aspartate flux by overexpression of pyruvate carboxylase and aspartate amino transferase improves the titer to 29 g/L in shake flasks. Fed-batch fermentation at pH 4 with low-cost corn steep liquor medium further increases the production to 92 g/L with 0.7 g/g yield and 0.55 g/L/h productivity. Techno-economic analysis indicates that such performance could potentially enable a financially viable process for sustainable acrylic acid production. This work establishes I. orientalis as a next-generation platform for cost-effective 3HP production and paves the way toward industrial commercialization.

Biotechnology↗