Anaerobic glucose uptake in Pseudomonas putida KT2440 in a bioelectrochemical system.

Providing an anodic potential in a bio-electrochemical system to the obligate aerobe Pseudomonas putida enables anaerobic survival and allows the cells to overcome redox imbalances. In this setup, the bacteria could be exploited to produce chemicals via oxidative pathways at high yield. However, the absence of anaerobic growth and low carbon turnover rates remain as obstacles for the application of such an electro-fermentation technology. Growth and carbon turnover start with carbon uptake into the periplasm and cytosol. P. putida KT2440 has three native transporting systems for glucose, each differing in energy and redox demand. This architecture previously led to the hypothesis that internal redox and energy constraints ultimately limit cytoplasmic carbon utilization in a bio-electrochemical system. However, it remains largely unclear which uptake route is predominantly used by P. putida under electro-fermentative conditions. To elucidate this, we created three gene deletion mutants of P. putida KT2440, forcing the cells to exclusively utilize one of the routes. When grown in a bio-electrochemical system, the pathway mutants were heavily affected in terms of sugar consumption, current output and product formation. Surprisingly, however, we found that about half of the acetate formed in the cytoplasm originated from carbon that was put into the system via the inoculation biomass, while the other half came from the consumption of substrate. The deletion of individual sugar uptake routes did not alter significantly the secreted acetate concentrations among different strains even with different carbon sources. This means that the stoichiometry of the sugar uptake routes is not a limiting factor during electro-fermentation and that the low rates might be caused by other reasons, for example energy limitations or a yet-to-be-identified oxygen-dependent regulatory mechanism.

A Coculture of Photoautotrophs and Hydrolytic Heterotrophs Enables Efficient Upcycling of Starch from Wastewater toward Biomass-Derived Products: Synergistic Interactions Impacting Metabolism of the Consortium.

Even with particular interest in sustainable development, due to the limited types of bioavailable carbon sources that could support heterotrophic/mixotrophic growth, microalgae-derived products still suffer from inconsistent yield and high costs. This study demonstrates a successful cocultivation of the photoautotroph Chlorella vulgaris with a hydrolytic-enzyme-abundant heterotroph, Saccharomycopsis fibuligera, enabling efficient starch upcycling from water/wastewater toward enhancing microalgae-dominant biomass and lipid production. The enzymatic activities of S. fibuligera contributed to the hydrolysis of starch into glucose, generating a 7-fold higher biomass through mixotrophic/heterotrophic growth of C. vulgaris. Further, scanning transmission electron microscopy (STEM) and quantitative analysis suggested a significantly induced accumulation of lipids in C. vulgaris. Results of meta-transcriptomics revealed the critical regulatory role of illumination in interaction shifting. Gene expression for glycolysis and lipid biosynthesis of C. vulgaris were highly activated during dark periods. Meanwhile, during illumination periods, genes coding for glucoamylase and the sulfur-related activities in S. fibuligera were significantly upregulated, leading to induced starch hydrolysis and potential increased competition for sulfur utilization, respectively. This study indicates that hydrolytic organisms could collaborate to make starch bioavailable for nonhydrolytic microalgae, thus broadening the substrate spectrum and making starch a novel biotechnological feedstock for microalgae-derived products, e.g., biofuels or single-cell protein.

Utilizing Cyanobacteria in Biophotovoltaics: An Emerging Field in Bioelectrochemistry.

Anthropogenic global warming is driven by the increasing energy demand and the still dominant use of fossil energy carriers to meet these needs. New carbon-neutral energy sources are urgently needed to solve this problem. Biophotovoltaics, a member of the so-called bioelectrochemical systems family, will provide an important piece of the energy puzzle. It aims to harvest the electrons from sunlight-driven water splitting using the natural oxygenic photosystem (e.g., of cyanobacteria) and utilize them in the form of, e.g., electricity or hydrogen. Several key aspects of biophotovoltaics have been intensively studied in recent years like physicochemical properties of electrodes or efficient wiring of microorganisms to electrodes. Yet, the exact mechanisms of electron transfer between the biocatalyst and the electrode remain unresolved today. Most research is conducted on microscale reactors generating small currents over short time-scales, but multiple experiments have shown biophotovoltaics great potential with lab-scale reactors producing currents over weeks to months. Although biophotovoltaics is still in its infancy with many open research questions to be addressed, new promising results from various labs around the world suggest an important opportunity for biophotovoltaics in the decades to come. In this chapter, we will introduce the concept of biophotovoltaics, summarize its recent key progress, and finally critically discuss the potentials and challenges for future rational development of biophotovoltaics.

Analysing intracellular isoprenoid metabolites in diverse prokaryotic and eukaryotic microbes.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

The anoxic electrode-driven fructose catabolism of Pseudomonas putida KT2440.

Pseudomonas putida (P. putida) is a microorganism of interest for various industrial processes, yet its strictly aerobic nature limits application. Despite previous attempts to adapt P. putida to anoxic conditions via genetic engineering or the use of a bioelectrochemical system (BES), the problem of energy shortage and internal redox imbalance persists. In this work, we aimed to provide the cytoplasmic metabolism with different monosaccharides, other than glucose, and explored the physiological response in P. putida KT2440 during bioelectrochemical cultivation. The periplasmic oxidation cascade was found to be able to oxidize a wide range of aldoses to their corresponding (keto-)aldonates. Unexpectedly, isomerization of the ketose fructose to mannose also enabled oxidation by glucose dehydrogenase, a new pathway uncovered for fructose metabolism in P. putida KT2440 in BES. Besides the isomerization, the remainder of fructose was imported into the cytoplasm and metabolized. This resulted in a higher NADPH/NADP+ ratio, compared to glucose. Comparative proteomics further revealed the upregulation of proteins in the lower central carbon metabolism during the experiment. These findings highlight that the choice of a substrate in BES can target cytosolic and periplasmic oxidation pathways, and that electrode-driven redox balancing can drive these pathways in P. putida under anaerobic conditions.

Technical-scale biophotovoltaics for long-term photo-current generation from Synechocystis sp. PCC6803.

A carbon-free energy supply is essential to sustain our future. Biophotovoltaics (BPV) provides a promising solution for hydrogen supply by directly coupling light-driven water splitting to hydrogen formation using oxygenic photoautotrophic cyanobacteria. However, BPV is currently limited by its low photon-to-current efficiency, and current experimental setups at a miniaturized scale hinder the rational investigation of the process and thus system optimization. In this article, we developed and optimized a new technical-scale (~250 ml working volume) BPV platform with defined and controllable operating parameters. Factors that interfered with reproducible and stable current output signals were identified and adapted. We found that the classical BG11 medium, used for the cultivation of cyanobacteria and also in many BPV studies, caused severe interferences in the bioelectrochemical experiments. An optimized nBG11 medium guaranteed a low and stable background current in the BPV reactor, regardless of the presence of light and/or mediators. As proof-of-principle, a very high long-term light-dependent current output (peak current of over 20 µA) was demonstrated in the new set-up over 12 days with living Synechocystis sp. PCC6803 cells and validated with appropriate controls. These results report the first reliable BPV platform generating reproducible photocurrent while still allowing quantitative investigation, rational optimization, and scale-up of BPV processes.

Trans-4-hydroxy-L-proline production by the cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria play an important role in photobiotechnology. Yet, one of their key central metabolic pathways, the tricarboxylic acid (TCA) cycle, has a unique architecture compared to most heterotrophs and still remains largely unexploited. The conversion of 2-oxoglutarate to succinate via succinyl-CoA is absent but is by-passed by several other reactions. Overall, fluxes under photoautotrophic growth conditions through the TCA cycle are low, which has implications for the production of chemicals. In this study, we investigate the capacity of the TCA cycle of Synechocystis sp PCC 6803 for the production of trans-4-hydroxy-L-proline (Hyp), a valuable chiral building block for the pharmaceutical and cosmetic industries. For the first time, photoautotrophic Hyp production was achieved in a cyanobacterium expressing the gene for the L-proline-4-hydroxylase (P4H) from Dactylosporangium sp. strain RH1. Interestingly, while elevated intracellular Hyp concentrations could be detected in the recombinant Synechocystis strains under all tested conditions, detectable Hyp secretion into the medium was only observed when the pH of the medium exceeded 9.5 and mostly in the late phases of the cultivation. We compared the rates obtained for autotrophic Hyp production with published sugar-based production rates in E. coli. The land-use efficiency (space-time yield) of the phototrophic process is already in the same order of magnitude as the heterotrophic process considering sugar farming as well. But, the remarkable plasticity of the cyanobacterial TCA cycle promises the potential for a 23-55 fold increase in space-time yield when using Synechocystis. Altogether, these findings contribute to a better understanding of bioproduction from the TCA cycle in photoautotrophs and broaden the spectrum of chemicals produced in metabolically engineered cyanobacteria.

Evolutionary engineering of E. coli MG1655 for tolerance against isoprenol.

BACKGROUND: Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. RESULTS: Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob. We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. CONCLUSION: Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.

Cytochrome c Reductase is a Key Enzyme Involved in the Extracellular Electron Transfer Pathway towards Transition Metal Complexes in Pseudomonas Putida.

Mediator-based extracellular electron transfer (EET) pathways can balance the redox metabolism of microbes. However, such electro-biosynthesis processes are constrained by the unknown underlying EET mechanisms. In this paper, Pseudomonas putida was studied to systematically investigate its EET pathway to transition metal complexes (i. e., [Fe(CN)6 ]3-/4- and [Co(bpy)3 ]3+/2+ ; bpy=2,2'-bipyridyl) under anaerobic conditions. Comparative proteomics showed the aerobic respiratory components were upregulated in a bioelectrochemical system without oxygen, suggesting their potential contribution to EET. Further tests found inhibiting cytochrome c oxidase activity by NaN3 and NADH dehydrogenase by rotenone did not significantly change the current output. However, the EET pathway was completely blocked, while cytochrome c reductase activity was inhibited by antimycin A. Although it cannot be excluded that cytochrome c and the periplasmic subunit of cytochrome c oxidase donate electrons to the transition metal complexes, these results strongly demonstrate that cytochrome c reductase is a key complex for the EET pathway.

Economic Process Evaluation and Environmental Life-Cycle Assessment of Bio-Aromatics Production.

The bio-based production of aromatics is experiencing a renaissance with systems and synthetic biology approaches promising to deliver bio-catalysts that will reach yields, rates, and titers comparable to already existing bulk bio-processes for the production of amino acids for instance. However, aromatic building blocks derived from petrochemical routes have a huge economic advantage, they are cheap, and very cheap in fact. In this article, we are trying to shed light on an important aspect of biocatalyst development that is frequently overlooked when working on strain development: economic and environmental impact of the production process. We estimate the production cost and environmental impact of a microbial fermentation process depending on culture pH, carbon source and process scale. As a model molecule we use para-hydroxybenzoic acid (pHBA), but the results are readily transferrable to other shikimate derived aromatics with similar carbon yields and production rates.

Characterizing the Anoxic Phenotype of Pseudomonas putida Using a Bioelectrochemical System.

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes.

Biophotovoltaics: Green Power Generation From Sunlight and Water.

Biophotovoltaics is a relatively new discipline in microbial fuel cell research. The basic idea is the conversion of light energy into electrical energy using photosynthetic microorganisms. The microbes will use their photosynthetic apparatus and the incoming light to split the water molecule. The generated protons and electrons are harvested using a bioelectrochemical system. The key challenge is the extraction of electrons from the microbial electron transport chains into a solid-state anode. On the cathode, a corresponding electrochemical counter reaction will consume the protons and electrons, e.g., through the oxygen reduction to water, or hydrogen formation. In this review, we are aiming to summarize the current state of the art and point out some limitations. We put a specific emphasis on cyanobacteria, as these microbes are considered future workhorses for photobiotechnology and are currently the most widely applied microbes in biophotovoltaics research. Current progress in biophotovoltaics is limited by very low current outputs of the devices while a lack of comparability and standardization of the experimental set-up hinders a systematic optimization of the systems. Nevertheless, the fundamental questions of redox homeostasis in photoautotrophs and the potential to directly harvest light energy from a highly efficient photosystem, rather than through oxidation of inefficiently produced biomass are highly relevant aspects of biophotovoltaics.

Microbial electrosynthesis system with dual biocathode arrangement for simultaneous acetogenesis, solventogenesis and carbon chain elongation.

A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2-C4 carboxylic acids and alcohols from carbon dioxide. Each cathode chamber provides ideal pH conditions to favor acetogenesis/carbon chain elongation (pH = 6.9), and solventogenesis (pH = 4.9), respectively, without the requirement of external acid/base dosing.

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds-Present and Future Strain Construction Strategies.

The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations.

Characterization of a membrane-separated and a membrane-less electrobioreactor for bioelectrochemical syntheses.

Bioelectrochemical systems (BESs) have the potential to contribute to the energy revolution driven by the new bio-economy. Until recently, simple reactor designs with minimal process analytics have been used. In recent years, assemblies to host electrodes in bioreactors have been developed resulting in so-called "electrobioreactors." Bioreactors are scalable, well-mixed, controlled, and therefore widely used in biotechnology and adding an electrode extends the possibilities to investigate bioelectrochemical production processes in a standard system. In this work, two assemblies enabling a separated and non-separated electrochemical operation, respectively, are designed and extensively characterized. Electrochemical losses over the electrolyte and the membrane were comparable to H-cells, the bioelectrochemical standard reaction system. An effect of the electrochemical measurements on pH measurements was observed if the potential is outside the range of -1,000 to +600 mV versus Ag/AgCl. Electrobiotechnological characterization of the two assemblies was done using Shewanella oneidensis as an electroactive model organism. Current production over time was improved by a separation of anodic and cathodic chamber by a Nafion® membrane. The developed electrobioreactor was used for a scale-up of the anaerobic bioelectrochemical production of organic acids and lysine from glucose using an engineered Corynebacterium glutamicum. Comparison to a small-scale custom-made electrobioreactor indicates that anodic electro-fermentation of lysine and organic acids might not be limited by the BES setup but by the biocatalysis of the cells.

Anodic electro-fermentation: Anaerobic production of L-Lysine by recombinant Corynebacterium glutamicum.

Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidizing conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/hr. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electro-fermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

Balancing cellular redox metabolism in microbial electrosynthesis and electro fermentation - A chance for metabolic engineering.

More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering.

Toward Synthetic Biology Strategies for Adipic Acid Production: An in Silico Tool for Combined Thermodynamics and Stoichiometric Analysis of Metabolic Networks.

Adipic acid, a nylon-6,6 precursor, has recently gained popularity in synthetic biology. Here, 16 different production routes to adipic acid were evaluated using a novel tool for network-embedded thermodynamic analysis of elementary flux modes. The tool distinguishes between thermodynamically feasible and infeasible modes under determined metabolite concentrations, allowing the thermodynamic feasibility of theoretical yields to be assessed. Further, patterns that always caused infeasible flux distributions were identified, which will aid the development of tailored strain design. A review of cellular efflux mechanisms revealed that significant accumulation of extracellular product is only possible if coupled with ATP hydrolysis. A stoichiometric analysis demonstrated that the maximum theoretical product carbon yield heavily depends on the metabolic route, ranging from 32 to 99% on glucose and/or palmitate in Escherichia coli and Saccharomyces cerevisiae metabolic models. Equally important, metabolite concentrations appeared to be thermodynamically restricted in several pathways. Consequently, the number of thermodynamically feasible flux distributions was reduced, in some cases even rendering whole pathways infeasible, highlighting the importance of pathway choice. Only routes based on the shikimate pathway were thermodynamically favorable over a large concentration and pH range. The low pH capability of S. cerevisiae shifted the thermodynamic equilibrium of some pathways toward product formation. One identified infeasible-pattern revealed that the reversibility of the mitochondrial malate dehydrogenase contradicted the current state of knowledge, which imposes a major restriction on the metabolism of S. cerevisiae. Finally, the evaluation of industrially relevant constraints revealed that two shikimate pathway-based routes in E. coli were the most robust.