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.

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.

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.

Autotrophic Production of the Sesquiterpene α-Humulene with Cupriavidus necator in a Controlled Bioreactor.

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L-1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

Growth medium and electrolyte-How to combine the different requirements on the reaction solution in bioelectrochemical systems using Cupriavidus necator.

Microbial electrosynthesis is a relatively new research field where microbial carbon dioxide fixation based on the energy supplied by a cathode is investigated. Reaction media used in such bioelectrochemical systems have to fulfill requirements of classical biotechnology as well as electrochemistry. The design and characterization of a medium that enables fast electroautotrophic growth of Cupriavidus necator in microbial electrosynthesis was investigated in detail. The identified chloride-free medium mainly consists of low buffer concentration and is supplied with trace elements. Biotechnologically relevant parameters, such as high-specific growth rates and short lag phases, were determined for growth characterization. Fast growth under all conditions tested, i.e. heterotrophic, autotrophic and electroautotrophic was achieved. The lag phase was shortened by increasing the FeSO4 concentration. Additionally, electrochemical robustness of the reaction media was proven. Under reductive conditions, no deposits on electrodes or precipitations in the media were observed and no detectable hydrogen peroxide evolved. In the bioelectrochemical system, no lag phase occurred and specific growth rate of C. necator was 0.09 h⁻¹. Using this medium shortens seed train drastically and enables fast electrobiotechnological production processes based on C. necator.

Expanding the genetic tool box for Cupriavidus necator by a stabilized L-rhamnose inducible plasmid system.

The Gram negative bacterium Cupriavidus necator is well known for the accumulation of poly(3-hydroxybutyrate) and its fast lithoautotrophic growth, leading in high cell densities. Although the host was engineered for the heterologous production of diverse chemicals and biopolymers in recent years, tool box of stabilized inducible expression systems is still limited. To avoid plasmid loss during fermentation processes and to allow expression of complex proteins, a tunable L-rhamnose inducible system was established and characterized using enhanced green fluorescent protein (eGFP). The construct was stabilized by a previously established partitioning system. An increase of fluorescence signal intensity in different media was shown with inducer concentrations up to 11mM L-rhamnose. The strongest effects were measured at quite low concentrations - high tunability was observed between 0 and 0.4-1mM (depending on the medium used). Expression is tightly regulated and could be increased over 140-fold in complex medium and approximately 60-fold in minimal medium due to induction with 11mM L-rhamnose. Varying induction times were characterized regarding growth behavior and expression pattern, taking into consideration problems that may arise during expression of toxic proteins. The novel plasmid expands the tool box for engineering the highly flexible production host C. necator.

Investigation of plasmid-induced growth defect in Pseudomonas putida.

Genetic engineering in bacteria mainly relies on the use of plasmids. But despite their pervasive use for physiological studies as well as for the design and optimization of industrially used production strains, only limited information about plasmid induced growth defects is available for different replicons and organisms. Here, we present the identification and characterization of such a phenomenon for Pseudomonas putida transformants carrying the pBBR1-derived plasmid pMiS1. We identified the kanamycin resistance gene and the transcription factor encoding rhaR gene to be causal for the growth defect in P. putida. In contrast, this effect was not observed in Escherichia coli. The plasmid-induced growth defect was eliminated after introduction of a mutation in the plasmid-encoded rep gene, thus enabling construction of the non-toxic variant pMiS4. GFP reporters construct analyses and qPCR experiments revealed a distinctly lowered plasmid copy number for pMiS4, which is probably the reason for alleviation of the growth defect by this mutation. Our work expands the knowledge about plasmid-induced growth defects and provides a useful low-copy pBBR1 replicon variant.

Reactor concepts for bioelectrochemical syntheses and energy conversion.

In bioelectrochemical systems (BESs) at least one electrode reaction is catalyzed by microorganisms or isolated enzymes. One of the existing challenges for BESs is shifting the technology towards industrial use and engineering reactor systems at adequate scales. Due to the fact that most BESs are usually deployed in the production of large-volume but low-value products (e.g., energy, fuels, and bulk chemicals), investment and operating costs must be minimized. Recent advances in reactor concepts for different BESs, in particular biofuel cells and electrosynthesis, are summarized in this review including electrode development and first applications on a technical scale. A better understanding of the impact of reactor components on the performance of the reaction system is an important step towards commercialization of BESs.