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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth

Summary The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl‐CoA carboxylase (ACCase). As the rate‐limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α‐carboxyltransferase (Crα‐CT) subunit of the ACCase by yeast two‐hybrid protein–protein interaction assay. Three independent CRISPR‐Cas9 mediated knockout mutants for CrCTI1 each produced an ‘enhanced oil’ phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα‐CT. However, mutating all six predicted phosphorylation sites within Crα‐CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β‐oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.

Li, Zhongze [Aix‐Marseille Université, CEA, CNRS, ↗
DOE OSTI2020

A review of methods for the reconstruction and analysis of integrated genome-scale models of metabolism and regulation

The current survey aims to describe the main methodologies for extending the reconstruction and analysis of genome-scale metabolic models and phenotype simulation with Flux Balance Analysis mathematical frameworks, via the integration of Transcriptional Regulatory Networks and/or gene expression data. Although the surveyed methods are aimed at improving phenotype simulations obtained from these models, the perspective of reconstructing integrated genome-scale models of metabolism and gene expression for diverse prokaryotes is still an open challenge.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Flux balance analysis of the ammonia-oxidizing bacterium Nitrosomonas europaea ATCC19718 unravels specific metabolic activities while degrading toxic compounds

The ammonia-oxidizing bacterium Nitrosomonas europaea has been widely recognized as an important player in the nitrogen cycle as well as one of the most abundant members in microbial communities for the treatment of industrial or sewage wastewater. Its natural metabolic versatility and extraordinary ability to degrade environmental pollutants (e.g., aromatic hydrocarbons such as benzene and toluene) enable it to thrive under various harsh environmental conditions. Constraint-based metabolic models constructed from genome sequences enable quantitative insight into the central and specialized metabolism within a target organism. These genome-scale models have been utilized to understand, optimize, and design new strategies for improved bioprocesses. Reduced modeling approaches have been used to elucidate Nitrosomonas europaea metabolism at a pathway level. However, genome-scale knowledge about the simultaneous oxidation of ammonia and pollutant metabolism of N . europaea remains limited. Here, we describe the reconstruction, manual curation, and validation of the genome-scale metabolic model for N . europaea , i GC535. This reconstruction is the most accurate metabolic model for a nitrifying organism to date, reaching an average prediction accuracy of over 90% under several growth conditions. The manually curated model can predict phenotypes under chemolithotrophic and chemolithoorganotrophic conditions while oxidating methane and wastewater pollutants. Calculated flux distributions under different trophic conditions show that several key pathways are affected by the type of carbon source available, including central carbon metabolism and energy production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Towards oilcane: Engineering hyperaccumulation of triacylglycerol into sugarcane stems

Abstract Metabolic engineering to divert carbon flux from sucrose to oil in high biomass crop like sugarcane is an emerging strategy to boost lipid yields per hectare for biodiesel production. Sugarcane stems comprise more than 70% of the crops' biomass and can accumulate sucrose in excess of 20% of their extracted juice. The energy content of oils in the form of triacylglycerol (TAG) is more than twofold that of carbohydrates. Here, we report a step change in TAG accumulation in sugarcane stem tissues achieving an average of 4.3% of their dry weight (DW) in replicated greenhouse experiments by multigene engineering. The metabolic engineering included constitutive co‐expression of wrinkled1; diacylglycerol acyltransferase1‐2; cysteine‐oleosin; and ribonucleic acid interference‐suppression of sugar‐dependent1. The TAG content in leaf tissue was also elevated by more than 400‐fold compared to non‐engineered sugarcane to an average of 8.0% of the DW and the amount of total fatty acids reached about 13% of the DW. With increasing TAG accumulation an increase of 18:1 unsaturated fatty acids was observed at the expense of 16:0 and 18:0 saturated fatty acids. Total biomass accumulation, soluble lignin, Brix and juice content were significantly reduced in the TAG hyperaccumulating sugarcane lines. Overcoming this yield drag by engineering lipid accumulation into late stem development will be critical to exceed lipid yields of current oilseed crops.

09 BIOMASS FUELS↗
DOE OSTI2026

Mobile genetic elements shape microbial diversity and functions in thawing permafrost soils

Ecosystems are shaped by communities of microorganisms whose niches and impacts depend on functional profiles influenced by gene gains and losses. Culture-based experiments demonstrate that mobile genetic elements (MGEs) can mediate gene flux, but quantitative understanding of these dynamics in natural systems remains limited. Here we develop and apply a systematic, meta-omic framework to investigate MGEs in a complex natural system using an 8-year soil time series collected at Stordalen Mire, in Sweden’s thawing permafrost margin. In this climate-critical peatland, we identify ~2.1 million MGE recombinases across 89 microbial phyla and assess ecological distributions, affected functions, past mobility and current activity. This revealed an active mobilome that shapes natural genetic diversity via differential impacts on major phyla and affects a wide range of functions, including metabolic genes involved in carbon flux and nutrient cycling. These findings and this analytic framework suggest avenues towards a better understanding of MGE diversity, activity, mobility and impacts across ecosystems.

Biological and medical sciences↗
DOE OSTI2022

Predicting stress response and improved protein overproduction in Bacillus subtilis

Abstract Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis , i JT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed i JT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like i JT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolic Pathway Engineering

Modern microbial and enzyme engineering and their advancement are increasingly dependent on the marriage of a wide range of sophisticated technologies. For students entering the field of biotechnology, the outlook is indeed daunting. Expertise at levels beyond that of simple familiarity will be needed to conduct competitive research. It goes without saying that to be competitive, all new researchers in this field will need basic preparation in molecular biology, biochemistry, and genetics. Further, experience working with concepts and experimental tools in enzyme biochemistry and kinetics, gene editing, computational metabolic pathway modeling, experimental pathway flux analysis, and computational clustering tools to process complex data sets will be vital for success. We speculate that most biotechnology researchers in early career at this time will build teams of collaborators to address these disparate science fields rather than attempt to become experts in one lab.

BASIC BIOLOGICAL SCIENCES,BIOMASS FUELS↗
DOE OSTI2021

Microbial drivers of methane emissions from unrestored industrial salt ponds

Wetlands are important carbon (C) sinks, yet many have been destroyed and converted to other uses over the past few centuries, including industrial salt making. A renewed focus on wetland ecosystem services (e.g., flood control, and habitat) has resulted in numerous restoration efforts whose effect on microbial communities is largely unexplored. We investigated the impact of restoration on microbial community composition, metabolic functional potential, and methane flux by analyzing sediment cores from two unrestored former industrial salt ponds, a restored former industrial salt pond, and a reference wetland. We observed elevated methane emissions from unrestored salt ponds compared to the restored and reference wetlands, which was positively correlated with salinity and sulfate across all samples. 16S rRNA gene amplicon and shotgun metagenomic data revealed that the restored salt pond harbored communities more phylogenetically and functionally similar to the reference wetland than to unrestored ponds. Archaeal methanogenesis genes were positively correlated with methane flux, as were genes encoding enzymes for bacterial methylphosphonate degradation, suggesting methane is generated both from bacterial methylphosphonate degradation and archaeal methanogenesis in these sites. These observations demonstrate that restoration effectively converted industrial salt pond microbial communities back to compositions more similar to reference wetlands and lowered salinities, sulfate concentrations, and methane emissions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cyanobacterial circadian regulation enhances bioproduction under subjective nighttime through rewiring of carbon partitioning dynamics, redox balance orchestration, and cell cycle modulation

Abstract Background The industrial feasibility of photosynthetic bioproduction using cyanobacterial platforms remains challenging due to insufficient yields, particularly due to competition between product formation and cellular carbon demands across different temporal phases of growth. This study investigates how circadian clock regulation impacts carbon partitioning between storage, growth, and product synthesis in Synechococcus elongatus PCC 7942, and provides insights that suggest potential strategies for enhanced bioproduction. Results After entrainment to light-dark cycles, PCC 7942 cultures transitioned to constant light revealed distinct temporal patterns in sucrose production, exhibiting three-fold higher productivity during subjective night compared to subjective day despite moderate down-regulation of genes from the photosynthetic apparatus. This enhanced productivity coincided with reduced glycogen accumulation and halted cell division at subjective night time, suggesting temporal separation of competing processes. Transcriptome analysis revealed coordinated circadian clock-driven adjustment of the cell cycle and rewiring of energy and carbon metabolism, with over 300 genes showing differential expression across four time points. The subjective night was characterized by altered expression of cell division-related genes and reduced expression of genes involved in glycogen synthesis, while showing upregulation of glycogen degradation pathways, alternative electron flow components, the pentose phosphate pathway, and oxidative decarboxylation of pyruvate. These molecular changes created favorable conditions for product formation through enhanced availability of major sucrose precursors (glucose-1-phosphate and fructose-6-phosphate) and maintained redox balance through multiple mechanisms. Conclusions Our analysis of circadian regulatory rewiring of carbon metabolism and redox balancing suggests two potential approaches that could be developed for improving cyanobacterial bioproduction: leveraging natural circadian rhythms for optimizing cultivation conditions and timing of pathway induction, and engineering strains that mimic circadian-driven metabolic shifts through controlled carbon flux redistribution and redox rebalancing. While these strategies remain to be tested, they could theoretically improve the efficiency of photosynthetic bioproduction by enabling better temporal separation between cell growth, carbon storage accumulation, and product synthesis phases.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Microbial production of high octane and high sensitivity olefinic ester biofuels

Abstract Background Advanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety. Results Optimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose. Conclusion Our study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.

09 BIOMASS FUELS↗
NASA NTRS1973

On the physical basis of a theory of human thermoregulation.

Theoretical study of the physical factors which are responsible for thermoregulation in nude resting humans in a physical steady state. The behavior of oxidative metabolism, evaporative and convective thermal fluxes, fluid heat transfer, internal and surface temperatures, and evaporative phase transitions is studied by physiological/physical modeling techniques. The modeling is based on the theories that the body has a vital core with autothermoregulation, that the vital core contracts longitudinally, that the temperature of peripheral regions and extremities decreases towards the ambient, and that a significant portion of the evaporative heat may be lost underneath the skin. A theoretical basis is derived for a consistent modeling of steady-state thermoregulation on the basis of these theories.

Iberall, A. S.↗
DOE OSTI2025

A dynamic protein interactome drives energy conservation and electron flux in Thermococcus kodakarensis

ABSTRACT Life is supported by energy gains fueled by catabolism of a wide range of substrates, each reliant on the selective partitioning of electrons through redox ( red uction and ox idation) reactions. Electron flux through tunable and regulated protein interactions provides dynamic routes for energy conservation, but how electron flux is regulated in vivo , particularly for archaeal metabolisms that support rapid growth at the thermodynamic limits of life, is poorly understood. Identification of bona fide in vivo protein assemblies and how such assemblies dictate the totality of electron flux is critical to our understanding of the regulation imposed on metabolism, energy production, and energy conservation. Here, 25 key proteins in central metabolic redox pathways in the model, genetically accessible, hyperthermophilic archaeon Thermococcus kodakarensis , were purified to reveal an extensive, dynamic, and tightly interconnected network of protein interactions that responds to environmental cues (such as the availability of various reductive sinks) to direct electron flux to maximize energetic gains. Interactions connecting disparate functions suggest many catabolic and anabolic activities occur in spatial proximity in vivo , and while protein complexes have been historically defined under optimal conditions, many of these complexes appear to maintain alternative partnerships in changing conditions. The totality of the results obtained redefines our understanding of in vivo assemblies driving ancient metabolic strategies supporting the growth of modern Archaea. IMPORTANCE Given the potential for rational genetic manipulations of biofuel- and biotech-promising archaea to yield transformative results for major markets, it is a priority to define how the metabolisms of such species are controlled, at least in part, by in vivo protein assemblies, and from such, define routes of energy flux that can be most efficiently altered toward biofuel or biotechnological gains. Proteinaceous electron carriers (PECs, such as ferredoxins) offer the potential for specific protein–protein interactions to coordinate selective reductive flow. Employing the model, genetically accessible, hyperthermophilic archaeon, Thermococcus kodakarensis , we establish the metabolic protein interactome of 25 key redox proteins, revealing that each redox active protein has a dynamic partnership profile, suggesting catabolic and anabolic activities may occur in concert and in temporal and spatial proximity in vivo . These results reveal critical importance in evaluating the newly identified partnerships and their role and utility in providing regulated redox flux in T. kodakarensis .

Williams, Sere A. (ORCID:0000000235509590)↗
DOE OSTI2022

Comparative Penicillium spp. Transcriptomics: Conserved Pathways and Processes Revealed in Ungerminated Conidia and during Postharvest Apple Fruit Decay

Blue mold, caused by Penicillium spp., is an impactful postharvest disease resulting in significant economic losses due to reduced pome fruit quality and mycotoxin contamination. Using two Penicillium species with different levels of aggressiveness, transcriptomics were implemented in order to identify genes expressed during apple fruit decay and loci expressed in ungerminated conidia. Total RNA was isolated from ungerminated conidia and decayed apple fruit infected with P. expansum R19 or P. polonicum RS1. There were 2442 differentially expressed genes (DEGs) between the R19 and RS1 in apple. Comparisons within species between apple and conidia revealed 4404 DEGs for R19 and 2935 for RS1, respectively. Gene ontology (GO) analysis revealed differential regulation in fungal transport and metabolism genes during decay, suggesting a flux in nutrient acquisition and detoxification strategies. In R19, the oxidoreductase GO category comprised 20% of all DEG groups in apple verses conidia. Ungerminated conidia from both species showed DEGs encoding the glyoxylate shunt and beta-oxidation, specifying the earliest metabolic requirements for germination. This is the first study to identify pre-loaded transcripts in conidia from blue mold fungi, reveal unique genes between species expressed during apple decay, and show the expression dynamics of known fungal virulence factors. These findings will enable development of targeted approaches for blue mold abatement strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

SNPeffect: identifying functional roles of SNPs using metabolic networks

Genetic sources of phenotypic variation have been a focus of plant studies aimed at improving agricultural yield and understanding adaptive processes. Genome-wide association studies identify the genetic background behind a trait by examining associations between phenotypes and single-nucleotide polymorphisms (SNPs). Although such studies are common, biological interpretation of the results remains a challenge; especially due to the confounding nature of population structure and the systematic biases thus introduced. Here, we propose a complementary analysis (SNPeffect) that offers putative genotype-to-phenotype mechanistic interpretations by integrating biochemical knowledge encoded in metabolic models. SNPeffect is used to explain differential growth rate and metabolite accumulation in A. thaliana and P. trichocarpa accessions as the outcome of SNPs in enzyme-coding genes. To this end, we also constructed a genome-scale metabolic model for Populus trichocarpa, the first for a perennial woody tree. As expected, our results indicate that growth is a complex polygenic trait governed by carbon and energy partitioning. The predicted set of functional SNPs in both species are associated with experimentally characterized growth-determining genes and also suggest putative ones. Functional SNPs were found in pathways such as amino acid metabolism, nucleotide biosynthesis, and cellulose and lignin biosynthesis, in line with breeding strategies that target pathways governing carbon and energy partition.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

Coupling flux balance analysis with reactive transport modeling through machine learning for rapid and stable simulation of microbial metabolic switching

Integrating genome-scale metabolic networks with reactive transport models (RTMs) provides a detailed description of the dynamic changes in microbial growth and metabolism. Despite promising demonstrations in the past, computational inefficiency has been pointed out as a critical issue to overcome because it requires repeated application of linear programming (LP) to obtain flux balance analysis (FBA) solutions in every time step and spatial grid. To address this challenge, we propose a new simulation method where we train and validate artificial neural networks (ANNs) using randomly sampled FBA solutions and incorporate the resulting surrogate FBA model (represented as algebraic equations) into RTMs as source/sink terms. We demonstrate the efficiency of our method via a case study of Shewanella oneidensis MR-1. During aerobic growth on lactate, S. oneidensis produces metabolic byproducts (such as pyruvate and acetate), which are subsequently consumed as alternative carbon sources when the preferred nutrients are depleted. To effectively simulate these complex dynamics, we used a cybernetic approach that models metabolic switches as the outcome of dynamic competition among multiple growth options. In both zero-dimensional batch and one-dimensional column configurations, the ANN-based surrogate models achieved substantial reduction of computational time by several orders of magnitude compared to the original LP-based FBA models. Moreover, the ANN models produced robust solutions without any special measures to prevent numerical instability. These developments significantly promote our ability to utilize genome-scale networks in complex, multi-physics, and multi-dimensional ecosystem modeling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Control of Carbon Allocation in Phenylpropanoid Metabolism

Understanding the molecular mechanisms by which carbon flux is partitioned in plants, and how flux through the phenylpropanoid pathway in particular is regulated and allocated to different branches of the pathway is essential for the eventual rational manipulation of phenylpropanoid flux for bioenergy applications. The phenylpropanoid pathway is responsible for the biosynthesis of a diverse array of metabolites essential for structural integrity, water transport, UV protection, and defense against herbivores and pathogens. The products of the phenylpropanoid pathway, all of which are derived from phenylalanine, range from complex, insoluble polymers such as lignin and suberin, to soluble flavonoids and hydroxycinnamate esters, to volatile compounds used to attract pollinators. By far the most abundant of these is the complex heteropolymer lignin, which accounts for a significant portion of the dry weight of not only woody plants, but also of crop residues. Decreasing or altering lignin structure to provide increased cell wall digestibility could greatly increase the cost effectiveness of converting cell wall polysaccharides to ethanol or second-generation biofuels. On the other hand, it is also possible that plants engineered to accumulate higher levels of lignin could provide improved feedstock for direct combustion and electricity generation as part of a diverse bioenergy portfolio. Consistent with the enormous commitment of energy and carbon it requires, the synthesis of lignin is under tight regulatory control. In order to influence transcription, gene-specific activators and repressors must interact with basal transcription factors and the rest of the core transcriptional machinery. In eukaryotes, this interaction is bridged by the large, multi-subunit complex known as Mediator. Despite the clear importance of Mediator throughout the eukaryotes, the complex has only recently been studied in plants. We have shown that a pair of Mediator subunits, MED5a and MED5b are required for normal regulation of phenylpropanoid pathway product accumulation. Plants lacking these proteins show increased accumulation of phenylpropanoids in multiple tissues indicating that normally they directly or indirectly repress expression of phenylpropanoid pathway genes. Furthermore, we have recently shown that MED5 plays a role in dwarfing in some low lignin plants and feedback between and within metabolic pathways of secondary metabolism in Arabidopsis. Taken together with the established roles of Mediator in transcriptional regulation, and of MED5a and MED5b in the repression of phenylpropanoid metabolism, these observations suggest that MED5a and MED5b are key components of an active, transcriptional process by which phenylpropanoid homeostasis is maintained in wild-type plants. Our research focused on expanding our understanding of the role of Mediator, as well as elucidating the mechanisms by which these proteins influence phenylpropanoid biosynthesis and how metabolite accumulation within the pathway and between metabolic pathways is essential for feedback regulatory control. This research will inform future bioengineering efforts aimed at altering carbon allocation and cell wall engineering by contributing to our understanding of the role of Mediator in regulating lignin biosynthesis, a pathway of plant metabolism that is important on a global scale.

09 BIOMASS FUELS↗
DOE OSTI2026

Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous NADPH-Dependent HBD

Clostridium tyrobutyricum Δ cat 1:: adh E2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd ( Ck ) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adh E2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd ( Ck ) in Δ cat 1:: adh E2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔ cat 1:: adh E2- Pbgal-hbd ( Ck ) with a dynamic expression of hbd ( Ck ) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd ( Ck ) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd ( Ck ) expression induced at 0 or 24 h in MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) or constitutively in MΔ cat 1:: adh E2-P cat 1- hbd ( Ck ) showed significantly lower butanol yield and productivity. At 25 °C, MΔ cat 1:: adh E2-P bgal - hbd ( Ck ) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd ( Ck ) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.

Clostridium tyrobutyricum↗