RESUMEN
Owing to the nondeterministic and nonlinear nature of gene expression, the steady-state intracellular protein abundance of a clonal population forms a distribution. The characteristics of this distribution, including expression strength and noise, are closely related to cellular behavior. However, quantitative description of these characteristics has so far relied on arrayed methods, which are time-consuming and labor-intensive. To address this issue, we propose a deep-learning-assisted Sort-Seq approach (dSort-Seq) in this work, enabling high-throughput profiling of expression properties with high precision. We demonstrated the validity of dSort-Seq for large-scale assaying of the dose-response relationships of biosensors. In addition, we comprehensively investigated the contribution of transcription and translation to noise production in Escherichia coli, from which we found that the expression noise is strongly coupled with the mean expression level. We also found that the transcriptional interference caused by overlapping RpoD-binding sites contributes to noise production, which suggested the existence of a simple and feasible noise control strategy in E. coli.
Asunto(s)
Aprendizaje Profundo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
Syngas fermentation processes with acetogens represent a promising process for the reduction of CO2 emissions alongside bulk chemical production. However, to fully realize this potential the thermodynamic limits of acetogens need to be considered when designing a fermentation process. An adjustable supply of H2 as electron donor plays a key role in autotrophic product formation. In this study an anaerobic laboratory scale continuously stirred tank reactor was equipped with an All-in-One electrode allowing for in-situ H2 generation via electrolysis. Furthermore, this system was coupled to online lactate measurements to control the co-culture of a recombinant lactate-producing Acetobacterium woodii strain and a lactate-consuming Clostridium drakei strain to produce caproate. When C. drakei was grown in batch cultivations with lactate as substrate, 1.6 g·L-1 caproate were produced. Furthermore, lactate production of the A. woodii mutant strain could manually be stopped and reinitiated by controlling the electrolysis. Applying this automated process control, lactate production of the A. woodii mutant strain could be halted to achieve a steady lactate concentration. In a co-culture experiment with the A. woodii mutant strain and the C. drakei strain, the automated process control was able to dynamically react to changing lactate concentrations and adjust H2 formation respectively. This study confirms the potential of C. drakei as medium chain fatty acid producer in a lactate-mediated, autotrophic co-cultivation with an engineered A. woodii strain. Moreover, the monitoring and control strategy presented in this study reinforces the case for autotrophically produced lactate as a transfer metabolite in defined co-cultivations for value-added chemical production.
RESUMEN
The use of gaseous and air-captured CO2 for technical biosynthesis is highly desired, but elusive so far due to several obstacles including high energy (ATP, NADPH) demand, low thermodynamic driving force and limited biosynthesis rate. Here, we present an ATP and NAD(P)H-free chemoenzymatic system for amino acid and pyruvate biosynthesis by coupling methanol with CO2. It relies on a re-engineered glycine cleavage system with the NAD(P)H-dependent L protein replaced by biocompatible chemical reduction of protein H with dithiothreitol. The latter provides a higher thermodynamic driving force, determines the reaction direction, and avoids protein polymerization of the rate-limiting enzyme carboxylase. Engineering of H protein to effectively release the lipoamide arm from a protected state further enhanced the system performance, achieving the synthesis of glycine, serine and pyruvate at g/L level from methanol and air-captured CO2. This work opens up the door for biosynthesis of amino acids and derived products from air.
Asunto(s)
NAD , Ácido Pirúvico , Ácido Pirúvico/metabolismo , NAD/metabolismo , Aminoácidos , Dióxido de Carbono , Metanol , Adenosina TrifosfatoRESUMEN
In this study, we show how electrochemically mediated bioconversion can greatly increase the co-production of 1,3-propanediol and organic acids from glycerol in an industrial bioprocess using a Clostridum pasteurianum mutant. Remarkably, an enhanced butyrate formation was observed due to a weakened butanol pathway of the mutant. This allowed the strain to have a higher ATP generation for an enhanced growth, higher glycerol consumption and PDO production. The PDO titer reached as high as 120.67 g/L at a cathodic current of -400 mA, which is 33% higher than that without electricity, with a concurrent increase of butyric acid by 80%. To fully recover the increased PDO and organic acids, a novel downstream process combining thin film evaporation of PDO and esterification of organic acids with ethanol was developed. This enables the efficient co-production of PDO, ethyl acetate and ethyl butyrate with a high overall carbon use of 87%.
Asunto(s)
Glicerol , Glicoles de Propileno , Glicerol/metabolismo , Fermentación , Glicoles de Propileno/metabolismo , PropilenglicolRESUMEN
Acetobacterium woodii is known to produce mainly acetate from CO2 and H2, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO2 and H2 via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L-1 were achieved with the AiO-electrode, whereas 8.1 g L-1 lactate were produced with direct H2 sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO2.
RESUMEN
BACKGROUND: Electro-fermentation (EF) is an emerging tool for bioprocess intensification. Benefits are especially expected for bioprocesses in which the cells are enabled to exchange electrons with electrode surfaces directly. It has also been demonstrated that the use of electrical energy in BES can increase bioprocess performance by indirect secondary effects. In this case, the electricity is used to alter process parameters and indirectly activate desired pathways. In many bioprocesses, oxidation-reduction potential (ORP) is a crucial process parameter. While C. pasteurianum fermentation of glycerol has been shown to be significantly influenced electrochemically, the underlying mechanisms are not clear. To this end, we developed a system for the electrochemical control of ORP in continuous culture to quantitatively study the effects of ORP alteration on C. pasteurianum by metabolic flux analysis (MFA), targeted metabolomics, sensitivity and regulation analysis. RESULTS: In the ORP range of -462 mV to -250 mV, the developed algorithm enabled a stable anodic electrochemical control of ORP at desired set-points and a fixed dilution rate of 0.1 h-1. An overall increase of 57% in the molar yield for 1,3-propanediol was observed by an ORP increase from -462 to -250 mV. MFA suggests that C. pasteurianum possesses and uses cellular energy generation mechanisms in addition to substrate-level phosphorylation. The sensitivity analysis showed that ORP exerted its strongest impact on the reaction of pyruvate-ferredoxin-oxidoreductase. The regulation analysis revealed that this influence is mainly of a direct nature. Hence, the observed metabolic shifts are primarily caused by direct inhibition of the enzyme upon electrochemical production of oxygen. A similar effect was observed for the enzyme pyruvate-formate-lyase at elevated ORP levels. CONCLUSIONS: The results show that electrochemical ORP alteration is a suitable tool to steer the metabolism of C. pasteurianum and increase product yield for 1,3-propanediol in continuous culture. The approach might also be useful for application with further anaerobic or anoxic bioprocesses. However, to maximize the technique's efficiency, it is essential to understand the chemistry behind the ORP change and how the microbial system responds to it by transmitted or direct effects.
Asunto(s)
Clostridium , Glicerol , Clostridium/metabolismo , Fermentación , Glicerol/metabolismo , Oxidación-Reducción , Piruvatos/metabolismoRESUMEN
Bacteria that successfully adapt to different substrates and environmental niches within the lung and overcome the immune defence can cause serious lung infections. Such infections are generally complex, and recognized as polymicrobial in nature. Both Pseudomonas aeruginosa and Streptococcus pneumoniae can cause chronic lung infections and were both detected in cystic fibrosis (CF) lung at different stages. In this study, single and dual species cultures of Pseudomonas aeruginosa and Streptococcus pneumoniae were studied under well-controlled planktonic growth conditions. Under pH-controlled conditions, both species apparently benefited from the presence of the other. In co-culture with P. aeruginosa, S. pneumoniae grew efficiently under aerobic conditions, whereas in pure S. pneumoniae culture, growth inhibition occurred in bioreactors with dissolved oxygen concentrations above the microaerobic range. Lactic acid and acetoin that are produced by S. pneumoniae were efficiently utilized by P. aeruginosa. In pH-uncontrolled co-cultures, the low pH triggered by S. pneumoniae assimilation of glucose and lactic acid production negatively affected the growth of both strains. Nevertheless, ammonia production improved significantly, and P. aeruginosa growth dominated at later growth stages. This study revealed unreported metabolic interactions of two important pathogenic microorganisms and shed new lights into pathophysiology of bacterial lung infection.
Asunto(s)
Fibrosis Quística , Infecciones por Pseudomonas , Acetoína/metabolismo , Amoníaco/metabolismo , Biopelículas , Fibrosis Quística/microbiología , Cadena Alimentaria , Glucosa/metabolismo , Humanos , Ácido Láctico/metabolismo , Pulmón/microbiología , Oxígeno/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/metabolismo , Streptococcus pneumoniaeRESUMEN
CO2 in the atmosphere is a major contributor to global warming but at the same time it has the potential to be a carbon source for advanced biomanufacturing. To utilize CO2, carbonic anhydrase has been identified as a key enzyme. Furthermore, attempts have been made to accelerate the sequestration via pressure. This study aims to combine both approaches to achieve high sequestration rates. The carbonic anhydrase of the alkaliphilic cyanobacterium Coleofasciculus chthonoplastes (cahB1) and bovine carbonic anhydrase (BCA) are introduced into a high-pressure reactor to catalyze the hydration of CO2 at up to 20 bar. The reactor is filled with a CaCl2 solution. Due to the presence of Ca2+, the hydrated CO2 precipitates as CaCO3. The impact of the carbonic anhydrase is clearly visible at all pressures tested. At ambient pressure a CO2 sequestration rate of 243.68 kgCaCO3/m3 h for cahB1 was achieved compared to 150.41 kgCaCO3/m3 h without enzymes. At 20 bar the rates were 2682.88 and 2267.88 kgCaCO3/m3 h, respectively. The study shows the benefit of a combined CO2 sequestration process. To examinate the influence of the enzymes on the product formation, the precipitated CaCO3 was analyzed regarding the crystalline phase and morphology. An interchange of the crystalline phase from vaterite to calcite was observed and discussed.
RESUMEN
The glycine cleavage system (GCS) is a fundamental component of life, widely existing in microbes, plants, animals, and humans. A better understanding of the functionality and working mechanisms, and the engineering of the GCS have both scientific and practical impacts, which may lead to new knowledge and findings in life sciences, improved biomass production and human/animal health, efficient biosynthesis of chemicals, effective carbon fixation and global climate change mitigation. In this chapter, the GCS is first discussed in the context of the reductive glycine pathway (rGlyP), a recently proposed and appealing assimilation pathway of CO2 and formate, and its implementation and optimization in microorganisms for formatotrophic growth. Then, the present knowledge about the components, reactions, and working mechanisms of the GCS and related enzymes is reviewed. Particular emphasis is also placed on the conformational and structural features of the GCS proteins, especially the different forms of lipoylated H protein and its lipoylation by lipoate protein ligase (LplA). Subsequently, existing analytic methods for the components and reactions of the GCS and recent advances in quantitatively understanding and purposefully engineering the GCS are presented. Finally, perspectives of current state of the art in the GCS research are given and future research needs are highlighted.
Asunto(s)
Aminoácido Oxidorreductasas , Transferasas , Aminoácido Oxidorreductasas/metabolismo , Animales , Ingeniería Metabólica , Redes y Vías Metabólicas , Complejos Multienzimáticos/metabolismo , Transferasas/metabolismoRESUMEN
Diols encompass important bulk and fine chemicals for the chemical, pharmaceutical and cosmetic industries. During the past decades, biological production of C3-C5 diols from renewable feedstocks has received great interest. Here, we elaborate a general principle for effectively synthesizing structurally diverse diols by expanding amino acid metabolism. Specifically, we propose to combine oxidative and reductive formations of hydroxyl groups from amino acids in a thermodynamically favorable order of four reactions catalyzed by amino acid hydroxylase, L-amino acid deaminase, α-keto acid decarboxylase and aldehyde reductase consecutively. The oxidative formation of hydroxyl group from an alkyl group is energetically more attractive than the reductive pathway, which is exclusively used in the synthetic pathways of diols reported so far. We demonstrate this general route for microbial production of branched-chain diols in E. coli. Ten C3-C5 diols are synthesized. Six of them, namely isopentyldiol (IPDO), 2-methyl-1,3-butanediol (2-M-1,3-BDO), 2-methyl-1,4-butanediol (2-M-1,4-BDO), 2-methyl-1,3-propanediol (MPO), 2-ethyl-1,3-propanediol (2-E-1,3-PDO), 1,4-pentanediol (1,4-PTD), have not been biologically synthesized before. This work opens up opportunities for synthesizing structurally diverse diols and triols, especially by genome mining, rational design or directed evolution of proper enzymes.
Asunto(s)
Butileno Glicoles , Escherichia coli , Alcoholes , Aminoácidos/metabolismo , Butileno Glicoles/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estrés OxidativoRESUMEN
Recent advances in biosynthesis using one-carbon (C1) compounds (e.g., CO2 and syngas) have led to first process examples of industrial demonstration for producing C1-based chemicals. In these processes, several bottlenecks such as mass-transfer limitations of substrates, limited supply of energy (ATP), and reducing equivalents and thus low cell growth and product formation rate are observed that severely hinder their technical application. As an alternative approach, C1-mixotrophy is proposed which involves co-utilization of C1 and organic substrates as complementing heterotrophic and autotrophic biosynthesis. Bulk and fine chemicals are reported to be efficiently synthetized in such a way. In this chapter, examples of C1-mixotrophy are presented and discussed to demonstrate their potential and perks. In acetogenic mixotrophy, the reductive acetyl-CoA pathway is harnessed as C1 fixation module by using native acetogens as cellular machineries. The highly adapted and efficient carbon fixation is enhanced by co-supply of reducing equivalents and energy from organic substrate. Alternatively, methanol as a highly reduced C1 compound provides carbon building blocks and reducing equivalents in methylotrophic mixotrophy, which is feasible for native and synthetic methylotrophs, broadening the range of applicable hosts. Another possibility is to make use of the anaplerotic reactions of C1 fixation naturally existing in heterotrophs. Re-wiring of carbon metabolism can lead to forced C1 fixation into the final products, thereby overcoming the inherent limitation of achievable product yield of heterotrophs. In a short to middle term, using native or synthetic pathways of C1 fixation module in a mixotrophy represents a promising and practicable bioprocess strategy. To this end, more quantitative and systematic studies regarding intracellular interactions of C1-fixation and catabolic modules are needed. Possible catabolite repression or other interfering native regulatory mechanisms in mixotrophy should be better studied. Stepwise engineering of established production strains is a necessary effort to raise the industrial relevance of C1-based biosynthesis.
Asunto(s)
Dióxido de Carbono , Carbono , Procesos Autotróficos , Dióxido de Carbono/metabolismoRESUMEN
Bioconversion of natural microorganisms generally results in a mixture of various compounds. Downstream processing (DSP) which only targets a single product often lacks economic competitiveness due to incomplete use of raw material and high cost of waste treatment for by-products. Here, we show with the efficient microbial conversion of crude glycerol by an artificially evolved strain and how a catalytic conversion strategy can improve the total products yield and process economy of the DSP. Specifically, Clostridium pasteurianum was first adapted to increased concentration of crude glycerol in a novel automatic laboratory evolution system. At m3 scale bioreactor the strain achieved a simultaneous production of 1,3-propanediol (PDO), acetic and butyric acids at 81.21, 18.72, and 11.09 g/L within only 19 h, respectively, representing the most efficient fermentation of crude glycerol to targeted products. A heterogeneous catalytic step was developed and integrated into the DSP process to obtain high-value methyl esters from acetic and butyric acids at high yields. The coproduction of the esters also greatly simplified the recovery of PDO. For example, a cosmetic grade PDO (96% PDO) was easily obtained by a simple single-stage distillation process (with an overall yield more than 77%). This integrated approach provides an industrially attractive route for the simultaneous production of three appealing products from the crude glycerol fermentation broth, which greatly improve the process economy and ecology.
Asunto(s)
Ésteres , Glicerol , Butiratos , Catálisis , Fermentación , Propilenglicol , Glicoles de PropilenoRESUMEN
A novel approach of phenotype analysis of fermentation-based bioprocesses based on unsupervised learning (clustering) is presented. As a prior identification of phenotypes and conditional interrelations is desired to control fermentation performance, an automated learning method to output reference phenotypes (defined as vector of biomass-specific rates) was developed and the necessary computing process and parameters were assessed. For its demonstration, time series data of 90 Clostridium pasteurianum cultivations were used which feature a broad spectrum of solventogenic and acidogenic phenotypes, while 14 clusters of phenotypic manifestations were identified. The analysis of reference phenotypes showed distinct differences, where potential conditionalities were exemplary isolated. Further, cluster-based balancing of carbon and ATP or the use of reference phenotypes as indicator for bioprocess monitoring were demonstrated to highlight the perks of this approach. Overall, such analysis depends strongly on the quality of the data and experimental validations will be required before conclusions. However, the automated, streamlined and abstracted approach diminishes the need of individual evaluation of all noisy dataset and showed promising results, which could be transferred to strains with comparably wide-ranging phenotypic manifestations or as indicators for repeated bioprocesses with clearly defined target.
RESUMEN
Glycine cleavage system (GCS) plays a central role in one-carbon (C1) metabolism and receives increasing interest as a core part of the recently proposed reductive glycine pathway (rGlyP) for assimilation of CO2 and formate. Despite decades of research, GCS has not yet been well understood and kinetic data are barely available. This is to a large degree because of the complexity of GCS, which is composed of four proteins (H, T, P, and L) and catalyzes reactions involving different substrates and cofactors. In vitro kinetics of reconstructed microbial multi-enzyme glycine cleavage/synthase system is desired to better implement rGlyP in microorganisms like Escherichia coli for the use of C1 resources. Here, we examined in vitro several factors that may affect the rate of glycine synthesis via the reverse GCS reaction. We found that the ratio of GCS component proteins has a direct influence on the rate of glycine synthesis, namely higher ratios of P protein and especially H protein to T and L proteins are favorable, and the carboxylation reaction catalyzed by P protein is a key step determining the glycine synthesis rate, whereas increasing the ratio of L protein to other GCS proteins does not have significant effect and the ratio of T protein to other GCS proteins should be kept low. The effect of substrate concentrations on glycine synthesis is quite complex, showing interdependence with the ratios of GCS component proteins. Furthermore, adding the reducing agent dithiothreitol to the reaction mixture not only results in great tolerance to high concentration of formaldehyde, but also increases the rate of glycine synthesis, probably due to its functions in activating P protein and taking up the role of L protein in the non-enzymatic reduction of Hox to Hred. Moreover, the presence of some monovalent and divalent metal ions can have either positive or negative effect on the rate of glycine synthesis, depending on their type and their concentration.
RESUMEN
An efficient downstream process without prior desalination was developed for recovering 1,3-propanediol (1,3-PDO) with high purity and yield from broth of a highly productive fed-batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3-PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3-PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3-PDO (< 80%). In order to improve the efficiency of final 1,3-PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3-PDO esters was achieved. This step conveniently provided free 1,3-PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3-PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3-PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high-salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3-PDO esters are crucial for the efficient separation of highly pure 1,3-PDO with acceptable yield from fermentation broth of raw glycerol.
RESUMEN
Conformational change associated with allosteric regulation in a protein is ultimately driven by energy transformation. However, little is known about the latter process. In this work, we combined steered molecular dynamics simulations and sequence conservation analysis to investigate the conformational changes and energy transformation in the allosteric enzyme aspartokinase III (AK III) from Escherichia coli. Correlation analysis of energy change at residue level indicated significant transformation between electrostatic energy and dihedral angle energy during the allosteric regulation. Key amino acid residues located in the corresponding energy transduction pathways were identified by dynamic energy correlation analysis. To verify their functions, residues with a high energy correlation in the pathways were altered and their effects on allosteric regulation of AKIII were determined. This study sheds new insights into energy transformation during allosteric regulation of AK III and proposes a strategy to identify key residues that are involved in intramolecular energy transduction and thus in driving the allosteric process.
RESUMEN
DOI: 10.1002/elsc.202000037 The cover feature visualizes our recent article about the investigation of the regulation of the Pyruvate Dehydrogenase Complex (PDC) during the lactate switch in batch cultures of Chinese Hamster Ovary cells. The relevance of this work to bioprocess engineering is highlighted in the background and the central cellular metabolic regulations are shown symbolically on the right-hand side. The regulation of PDC through phosphorylation was quantified at three regulating sites using a novel indirect flow cytometry protocol, shown as "glowing" antibodies. For details see article DOI 10.1002/elsc.202000037 on page 99.
RESUMEN
The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the "Warburg" effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation-dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody-producing CHO DP-12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl-CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate-based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.
RESUMEN
In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms.
RESUMEN
Formaldehyde (HCHO) is a reactive one-carbon compound that is interesting for biosynthesis. The assimilation of HCHO depends on the catalysis of aldolase. Here, we present a novel synthetic pathway in E. coli to convert HCHO and ethanol into 1,3-propanediol (PDO) using a deoxyribose-5-phosphate aldolase (DERA). DERA condenses HCHO and acetaldehyde to form 3-hydroxypropionaldehyde, the direct precursor of PDO formation. This new pathway opens up the possibility to synthesize an appealing C3 compound from a C1 compound and a C2 compound without carbon loss in contrast to all the other known PDO synthetic pathways where typically 30-50% of the carbons are lost as CO2 and other byproducts. The pathway is successfully demonstrated by elaborating three metabolic modules. First, DERA from Thermotoga maritima was found to be efficient for the aldol condensation and PDO production module. For the module of acetaldehyde supply from ethanol, an alcohol dehydrogenase from Hansenula polymorpha was selected. For the HCHO supply module, the control of HCHO concentration and its utilization were shown to be important for achieving the assimilation of HCHO in recombinant E. coli cells. By deleting the gene frmA for endogenous conversion of HCHO to formate and controlling HCHO at a level of about 0.6 mM, the concentration and yield of PDO were increased from initially 5.67 mM (0.43 g/L) and 0.057 mol/mol to 17.35 mM (1.32 g/L) and 0.096 mol/mol in bioconversion of ethanol and HCHO with resting E. coli cells. Further engineering of DERA and the HCHO supply module is necessary to realize the potential of this promising metabolic pathway.