Metabolic engineering of Shewanella oneidensis to produce glutamate and itaconic acid.

Shewanella oneidensis is a gram-negative bacterium known for its unique respiratory capabilities, which allow it to utilize a wide range of electron acceptors, including solid substrates such as electrodes. For a future combination of chemical production and electro-fermentation, the goal of this study was to expand its product spectrum. S. oneidensis was metabolically engineered to optimize its glutamate production and to enable production of itaconic acid. By deleting the glutamate importer gltS for a reduced glutamate uptake and pckA/ptA to redirect the carbon flux towards the TCA cycle, a ∆3 mutant was created. In combination with the plasmid pG2 carrying the glutamate dehydrogenase gdhA and a specific glutamate exporter NCgl1221 A111V, a 72-fold increase in glutamate concentration compared to the wild type was achieved. Along with overexpression of gdhA and NCgl1221 A111V, the deletion of gltS and pckA/ptA as well as the deletion of all three genes (∆3) was examined for their impact on growth and lactate consumption. This showed that the redirection of the carbon flux towards the TCA cycle is possible. Furthermore, we were able to produce itaconic acid for the first time with a S. oneidensis strain. A titer of 7 mM was achieved after 48 h. This suggests that genetic optimization with an expression vector carrying a cis-aconitate decarboxylase (cadA) and a aconitate hydratase (acnB) along with the proven redirection of the carbon flux to the TCA cycle enabled the production of itaconic acid, a valuable platform chemical used in the production of a variety of products. KEY POINTS: •Heterologous expression of gdhA and NCgl1221_A111V leads to higher glutamate production. •Deletion of ackA/pta redirects carbon flux towards TCA cycle. •Heterologous expression of cadA and acnB enables itaconic acid production.

New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum.

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured. KEY POINTS: • Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum • Cultivation conditions of the pre-culture are a reason for the acid crash • Inoculum from cells in stationary growth phase ensures shift to solventogenesis.

Application of gas diffusion electrodes in bioeconomy: An update.

The transition of today's fossil fuel based chemical industry toward sustainable production requires improvement of established production processes as well as development of new sustainable and bio-based synthesis routes within a circular economy. Thereby, the combination of electrochemical and biotechnological advantages in such routes represents one important keystone. For the electrochemical generation of reactants from gaseous substrates such as O2 or CO2 , gas diffusion electrodes (GDE) represent the electrodes of choice since they overcome solubility-based mass transport limitations. Within this article, we illustrate the architecture, function principle and fabrication of GDE. We highlight the application of GDE for conversion of CO2 using abiotic catalysts for subsequent biosynthesis as well as the application of microbial catalysts at GDE for CO2 conversion. The reduction of oxygen at GDE is summarized for the application of oxygen depolarized cathodes in microbial fuel cells and generation of H2 O2 to drive enzymatic reactions. Finally, engineering aspects such as scale-up and the modeling of GDE-based processes are described. This review presents an update on the application of GDE in bio-based production systems and emphasizes their large potential for sustainable development of new pathways in bioeconomy.

Methylotrophic bacteria with cobalamin-dependent mutases in primary metabolism as potential strains for vitamin B12 production.

Several bacterial species are known for their ability to synthesize vitamin B12 but biotechnological vitamin B12 production today is restricted to Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the rising popularity of veganism leads to a growing demand for vitamin B12 and thereby interest in alternative strains which can be used as efficient vitamin B12 sources. In this work, we demonstrate that methylotrophic microorganisms which utilize the ethylmalonyl-CoA pathway containing B12-dependent enzymes are capable of active vitamin B12 production. Several bacteria with an essential function of the pathway were tested for vitamin B12 synthesis. Among the identified strains, Hyphomicrobium sp. DSM3646 demonstrated the highest vitamin B12 levels reaching up to 17.9 ± 5.05 µg per g dry cell weight. These relatively high vitamin B12 concentrations achieved in simple cultivation experiments were performed in a mineral methanol medium, which makes Hyphomicrobium sp. DSM3646 a new promising cobalamin-producing strain.

Identification of vitamin B12 producing bacteria based on the presence of bluB/cobT2 homologues.

OBJECTIVES: The objective of the study was to develop a strategy for the identification of new vitamin B12-producing species and to characterize their production capability using a fast and sensitive LC-MS/MS method developed in this study. RESULTS: Searching for homologues of the bluB/cobT2 fusion gene known to be responsible for the production of the active vitamin B12 form in P. freudenreichii was shown to be a successful strategy for the identification of new vitamin B12-producing strains. The analysis of the identified strains via LC-MS/MS showed the ability of Terrabacter sp. DSM102553, Yimella lutea DSM19828 and Calidifontibacter indicus DSM22967 to produce the active form of vitamin B12. Further analysis of vitamin B12 production capability of Terrabacter sp. DSM102553 in M9 minimal medium and peptone-based media revealed that the highest yield of 2.65 µg of vitamin B12 per g dry cell weight was obtained in M9 medium. CONCLUSIONS: The proposed strategy enabled identification of Terrabacter sp. DSM102553, whose relatively high yields obtained in the minimal medium open new perspectives for the possible application of the strain for biotechnological vitamin B12 production.

Contribution of Enzyme Catalysis to the Achievement of the United Nations' Sustainable Development Goals.

In September 2015, the United Nations General Assembly established the 2030 Agenda for Sustainable Development, which includes 17 Sustainable Development Goals (SDGs) [...].

Microbial electrosynthesis of methane and acetate-comparison of pure and mixed cultures.

The electrochemical process of microbial electrosynthesis (MES) is used to drive the metabolism of electroactive microorganisms for the production of valuable chemicals and fuels. MES combines the advantages of electrochemistry, engineering, and microbiology and offers alternative production processes based on renewable raw materials and regenerative energies. In addition to the reactor concept and electrode design, the biocatalysts used have a significant influence on the performance of MES. Thus, pure and mixed cultures can be used as biocatalysts. By using mixed cultures, interactions between organisms, such as the direct interspecies electron transfer (DIET) or syntrophic interactions, influence the performance in terms of productivity and the product range of MES. This review focuses on the comparison of pure and mixed cultures in microbial electrosynthesis. The performance indicators, such as productivities and coulombic efficiencies (CEs), for both procedural methods are discussed. Typical products in MES are methane and acetate, therefore these processes are the focus of this review. In general, most studies used mixed cultures as biocatalyst, as more advanced performance of mixed cultures has been seen for both products. When comparing pure and mixed cultures in equivalent experimental setups a 3-fold higher methane and a nearly 2-fold higher acetate production rate can be achieved in mixed cultures. However, studies of pure culture MES for methane production have shown some improvement through reactor optimization and operational mode reaching similar performance indicators as mixed culture MES. Overall, the review gives an overview of the advantages and disadvantages of using pure or mixed cultures in MES. KEY POINTS: • Undefined mixed cultures dominate as inoculums for the MES of methane and acetate, which comprise a high potential of improvement • Under similar conditions, mixed cultures outperform pure cultures in MES • Understanding the role of single species in mixed culture MES is essential for future industrial applications.

Fermentative α-Humulene Production from Homogenized Grass Clippings as a Growth Medium.

Green waste, e.g., grass clippings, is currently insufficiently recycled and has untapped potential as a valuable resource. Our aim was to use juice from grass clippings as a growth medium for microorganisms. Herein, we demonstrate the production of the sesquiterpene α-humulene with the versatile organism Cupriavidus necator pKR-hum on a growth medium from grass clippings. The medium was compared with established media in terms of microbial growth and terpene production. C. necator pKR-hum shows a maximum growth rate of 0.43 h-1 in the grass medium and 0.50 h-1 in a lysogeny broth (LB) medium. With the grass medium, 2 mg/L of α-humulene were produced compared to 10 mg/L with the LB medium. By concentrating the grass medium and using a controlled bioreactor in combination with an optimized in situ product removal, comparable product concentrations could likely be achieved. To the best of our knowledge, this is the first time that juice from grass clippings has been used as a growth medium without any further additives for microbial product synthesis. This use of green waste as a material represents a new bioeconomic utilization option of waste materials and could contribute to improving the economics of grass biorefineries.

Upgrading Kolbe Electrolysis-Highly Efficient Production of Green Fuels and Solvents by Coupling Biosynthesis and Electrosynthesis.

The chemical industry is transitioning to more sustainable and biobased processes. One key element of this transition is coupling energy fluxes and feedstock utilization for optimizing processes, routes and efficiencies. Here, we show for the first time the coupling of the Kolbe electrolysis at the anode with a subsequent microbial conversion of the cathodically produced co-product hydrogen. Kolbe electrolysis of valeric acid yields the liquid drop-in fuel additive n-octane. Subsequently, the solvent isopropanol is produced by resting Cupriavidus necator cells using gaseous electrolysis products (esp. CO2 and H2 ). The resting microbial cells show carbon efficiencies of up to 41 % and Coulombic/Faradaic efficiencies of 60 % and 80 % for anodic and cathodic reactions, respectively. The implementation of a paired electrolyser resulted in superior process performances with overall efficiencies of up to 64.4 %.

Modeling and simulation-based design of electroenzymatic batch processes catalyzed by unspecific peroxygenase from A. aegerita.

Unspecific peroxygenases have attracted interest due to their ability to catalyze the oxygenation of various types of C-H bonds using only hydrogen peroxide as a cosubstrate. Due to the instability of these enzymes at even low hydrogen peroxide concentrations, careful fed-batch addition of the cosubstrate or ideally in situ production is required. While various approaches for hydrogen peroxide addition have been qualitatively assessed, only limited kinetic data concerning enzyme inactivation and peroxide accumulation has been reported so far. To obtain quantitative insights into the kinetics of such a process, a detailed data set for a peroxygenase-catalyzed benzylic hydroxylation coupled with electrochemical hydrogen peroxide production is presented. Based on this data set, we set out to model such an electroenzymatic process. For this, initial velocity data for the benzylic hydroxylation is collected and an extended Ping-Pong-Bi-Bi type rate equation is established, which sufficiently describes the enzyme kinetic. Moreover, we propose an empirical inactivation term based on the collected data set. Finally, we show that the full model does not only describe the process with sufficient accuracy, but can also be used predictively to control hydrogen peroxide feeding rates To limit the concentration of this critical cosubstrate in the system.

Gram-scale production of the sesquiterpene α-humulene with Cupriavidus necator.

Terpenoids have an impressive structural diversity and provide valuable substances for a variety of industrial applications. Among terpenes, the sesquiterpenes (C15 ) are the largest subclass with bioactivities ranging from aroma to health promotion. In this article, we show a gram-scale production of the sesquiterpene α-humulene in final aqueous concentrations of 2 g L-1 with the recombinant strain Cupriavidus necator pKR-hum in a fed-batch mode on fructose as carbon source and n-dodecane as an extracting organic phase for in situ product removal. Since C. necator is capable of both heterotrophic and autotrophic growth, we additionally modeled the theoretically possible yields of a heterotrophic versus an autotrophic process on CO2 in industrially relevant quantities. We compared the cost-effectiveness of both processes based on a production of 10 t α-humulene per year, with both processes performing equally with similar costs and gains. Furthermore, the expression and activity of 3-hydroxymethylglutaryl-CoA reductase (hmgR) from Myxococcus xanthus was identified as the main limitation of our constructed C. necator pKR-hum strain. Thus, we outlined possible solutions for further improvement of our production strain, for example, the replacement of the hmgR from M. xanthus by a plant-based variant to increase α-humulene production titers in the future.

First time ß-farnesene production by the versatile bacterium Cupriavidus necator.

BACKGROUND: Terpenes are remarkably diverse natural structures, which can be formed via two different pathways leading to two common intermediates. Among those, sesquiterpenes represent a variety of industrially relevant products. One important industrially produced product is ß-farnesene as a precursor for a jet fuel additive. So far, microbial terpene production has been mostly limited to known production hosts, which are only able to grow on heterotrophic substrates. RESULTS: In this paper, we for the first time describe ß-farnesene production by the versatile bacterial host Cupriavidus necator on fructose, which is known to grow hetero- and autotrophically and even in bioelectrochemical systems. We were able to show a growth-dependent production of ß-farnesene by expressing the ß-farnesene synthase from Artemisia annua in C. necator H16 PHB-4. Additionally, we performed a scale-up in a parallel reactor system with production titers of 26.3 ± 1.3 µM ß-farnesene with a fed-batch process. CONCLUSIONS: The ß-farnesene production titers reported in this paper are not in the same range as titers published with known heterotrophic producers E. coli or S. cerevisiae. However, this proof-of-principle study with C. necator as production host opens new synthesis routes toward a sustainable economy and leaves room for further optimizations, which have been already performed with the known production strains.

Product recovery of an enzymatically synthesized (-)-menthol ester in a deep eutectic solvent.

Deep eutectic solvents (DESs) have gained increased attention as alternative reaction media for biocatalysis in recent years. There are many investigations on biotransformations in a variety of DESs, but the purification of bioproducts from DES reaction mixtures has not yet been sufficiently addressed. The present study demonstrates a product recovery strategy from a DES reaction medium composed of (-)-menthol and dodecanoic acid. Since the DES is not formed by equimolar amounts of the substrates, but the eutectic point occurs at a 3:1 molar ratio, product isolation is an important task for effective biocatalytic process development, even if the limiting substrate is converted completely. Both DES compounds acted as substrates and reaction solvent in the lipase-catalyzed esterification to synthesize (-)-menthyl dodecanoate. The product (-)-menthyl dodecanoate ester was separated from the DES reaction mixture by a vacuum distillation step and a second esterification reaction can be performed with the recovered (-)-menthol.

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.

CO2 to Terpenes: Autotrophic and Electroautotrophic α-Humulene Production with Cupriavidus necator.

We show that CO2 can be converted by an engineered "Knallgas" bacterium (Cupriavidus necator) into the terpene α-humulene. Heterologous expression of the mevalonate pathway and α-humulene synthase resulted in the production of approximately 10 mg α-humulene per gram cell dry mass (CDW) under heterotrophic conditions. This first example of chemolithoautotrophic production of a terpene from carbon dioxide, hydrogen, and oxygen is a promising starting point for the production of different high-value terpene compounds from abundant and simple raw materials. Furthermore, the production system was used to produce 17 mg α-humulene per gram CDW from CO2 and electrical energy in microbial electrosynthesis (MES) mode. Given that the system can convert CO2 by using electrical energy from solar energy, it opens a new route to artificial photosynthetic systems.

The Oxygen Dilemma: A Severe Challenge for the Application of Monooxygenases?

Monooxygenases are promising catalysts because they in principle enable the organic chemist to perform highly selective oxyfunctionalisation reactions that are otherwise difficult to achieve. For this, monooxygenases require reducing equivalents, to allow reductive activation of molecular oxygen at the enzymes' active sites. However, these reducing equivalents are often delivered to O2 either directly or via a reduced intermediate (uncoupling), yielding hazardous reactive oxygen species and wasting valuable reducing equivalents. The oxygen dilemma arises from monooxygenases' dependency on O2 and the undesired uncoupling reaction. With this contribution we hope to generate a general awareness of the oxygen dilemma and to discuss its nature and some promising solutions.

Application of gas diffusion electrodes in bioelectrochemical syntheses and energy conversion.

Combining the advantages of biological components (e.g., reaction specificity, self-replication) and electrochemical techniques in bioelectrochemical systems offers the opportunity to develop novel efficient and sustainable processes for the production of a number of valuable products. The choice of electrode material has a great impact on the performance of bioelectrochemical systems. In addition to the redox process at the electrodes, interactions of biocatalysts with electrodes (e.g., enzyme denaturation or biofouling) need to be considered. In recent years, gas diffusion electrodes (GDEs) have proved to be very attractive electrodes for bioelectrochemical purposes. GDEs are porous electrodes, that posses a large three-phase boundary surface. At this interface, a solid catalyst supports the electrochemical reaction between gaseous and liquid phase. This mini-review discusses the application of GDEs in microbial and enzymatic fuel cells, for microbial electrolysis, in biosensors and for electroenzymatic synthesis reactions.

A simplified process design for P450 driven hydroxylation based on surface displayed enzymes.

New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc.

Efficient hydroxylation of 1,8-cineole with monoterpenoid-resistant recombinant Pseudomonas putida GS1.

In this work, monoterpenoid hydroxylation with Pseudomonas putida GS1 and KT2440 were investigated as host strains, and the cytochrome P450 monooxygenase CYP176A1 (P450cin) and its native redox partner cindoxin (CinC) from Citrobacter braakii were introduced in P. putida to catalyze the stereoselective hydroxylation of 1,8-cineole to (1R)-6ß-hydroxy-1,8-cineole. Growth experiments in the presence of 1,8-cineole confirmed pseudomonads' superior resilience compared to E. coli. Whole-cell P. putida harboring P450cin with and without CinC were capable of hydroxylating 1,8-cineole, whereas coexpression of CinC has been shown to accelerate this bioconversion. Under the same conditions, P. putida GS1 produced more than twice the amount of heterologous P450cin and bioconversion product than P. putida KT2440. A concentration of 1.1 ± 0.1 g/L (1R)-6ß-hydroxy-1,8-cineole was obtained within 55 h in shake flasks and 13.3 ± 1.9 g/L in 89 h in a bioreactor, the latter of which corresponds to a yield YP/S of 79 %. To the authors' knowledge, this is the highest product titer for a P450 based whole-cell monoterpene oxyfunctionalization reported so far. These results show that solvent-tolerant P. putida GS1 can be used as a highly efficient recombinant whole-cell biocatalyst for a P450 monooxygenase-based valorization of monoterpenoids.

Electroactive bacteria--molecular mechanisms and genetic tools.

In nature, different bacteria have evolved strategies to transfer electrons far beyond the cell surface. This electron transfer enables the use of these bacteria in bioelectrochemical systems (BES), such as microbial fuel cells (MFCs) and microbial electrosynthesis (MES). The main feature of electroactive bacteria (EAB) in these applications is the ability to transfer electrons from the microbial cell to an electrode or vice versa instead of the natural redox partner. In general, the application of electroactive organisms in BES offers the opportunity to develop efficient and sustainable processes for the production of energy as well as bulk and fine chemicals, respectively. This review describes and compares key microbiological features of different EAB. Furthermore, it focuses on achievements and future prospects of genetic manipulation for efficient strain development.