Synthetic biology toolkit of Ralstonia eutropha (Cupriavidus necator).

Synthetic biology encompasses many kinds of ideas and techniques with the common theme of creating something novel. The industrially relevant microorganism, Ralstonia eutropha (also known as Cupriavidus necator), has long been a subject of metabolic engineering efforts to either enhance a product it naturally makes (polyhydroxyalkanoate) or produce novel bioproducts (e.g., biofuels and other small molecule compounds). Given the metabolic versatility of R. eutropha and the existence of multiple molecular genetic tools and techniques for the organism, development of a synthetic biology toolkit is underway. This toolkit will allow for novel, user-friendly design that can impart new capabilities to R. eutropha strains to be used for novel application. This article reviews the different synthetic biology techniques currently available for modifying and enhancing bioproduction in R. eutropha. KEY POINTS: • R. eutropha (C. necator) is a versatile organism that has been examined for many applications. • Synthetic biology is being used to design more powerful strains for bioproduction. • A diverse synthetic biology toolkit is being developed to enhance R. eutropha's capabilities.

Elucidating regulation of polyhydroxyalkanoate metabolism in Ralstonia eutropha: Identification of transcriptional regulators from phasin and depolymerase genes.

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by liquid chromatography with tandem mass spectrometry, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using ß-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli, and their interaction with the promoters investigated in vitro by electrophoretic mobility shift assays. We could show that R. eutropha DNA-binding xenobiotic response element-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.

High-cell-density fed-batch strategy to manufacture tailor-made P(HB-co-HHx) by engineered Ralstonia eutropha at laboratory scale and pilot scale.

The transition towards a sustainable bioeconomy requires the development of highly efficient bioprocesses that enable the production of bulk materials at a competitive price. This is particularly crucial for driving the commercialization of polyhydroxyalkanoates (PHAs) as biobased and biodegradable plastic substitutes. Among these, the copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB-co-HHx)) shows excellent material properties that can be tuned by regulating its monomer composition. In this study, we developed a high-cell-density fed-batch strategy using mixtures of fructose and canola oil to modulate the molar composition of P(HB-co-HHx) produced by Ralstonia eutropha Re2058/pCB113 at 1-L laboratory scale up to 150-L pilot scale. With cell densities >100 g L-1 containing 70-80 wt% of PHA with tunable HHx contents in the range of 9.0-14.6 mol% and productivities of up to 1.5 g L-1 h-1, we demonstrate the tailor-made production of P(HB-co-HHx) at an industrially relevant scale. Ultimately, this strategy enables the production of PHA bioplastics with defined material properties on the kilogram scale, which is often required for testing and adapting manufacturing processes to target diverse applications.

Microbially synthesized poly(hydroxybutyrate-co-hydroxyhexanoate) with low to moderate hydroxyhexanoate content: Properties and applications.

Plastic pollution is the biggest environmental concern of our time. Breakdown products like micro- and nano-plastics inevitably enter the food chain and pose unprecedented health risks. In this scenario, bio-based and biodegradable plastic alternatives have been given a momentum aiming to bridge a transition towards a more sustainable future. Polyhydroxyalkanoates (PHAs) are one of the few thermoplastic polymers synthesized 100 % via biotechnological routes which fully biodegrade in common natural environments. Poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] is a PHA copolymer with great potential for the commodity polymers industry, as its mechanical properties can be tailored through fine-tuning of its molar HHx content. We have recently developed a strategy that enables for reliable tailoring of the monomer content of P(HB-co-HHx). Nevertheless, there is often a lack of comprehensive investigation of the material properties of PHAs to evaluate whether they actually mimic the functionalities of conventional plastics. We present a detailed study of P(HB-co-HHx) copolymers with low to moderate hydroxyhexanoate content to understand how the HHx monomer content influences the thermal and mechanical properties and to link those to their abiotic degradation. By increasing the HHx fractions in the range of 2 - 14 mol%, we impart an extension of the processing window and application range as the melting temperature (Tm) and glass temperature (Tg) of the copolymers decrease from Tm 165 °C to 126 °C, Tg 4 °C to -5.9 °C, accompanied by reduced crystallinity from 54 % to 20 %. Elongation at break was increased from 5.7 % up to 703 % at 14 mol% HHx content, confirming that the range examined was sufficiently large to obtain ductile and brittle copolymers, while tensile strength was maintained throughout the studied range. Finally, accelerated abiotic degradation was shown to be slowed down with an increasing HHx fraction decreasing from 70 % to 55 % in 12 h.

Workflow for shake flask and plate cultivations with fats for polyhydroxyalkanoate bioproduction.

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy.

An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µs ' with CDW was observed in the range of 5-69 g L-1 (R2 = 0.98). The growth rates calculated based on µs ' were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µs ' as a function of the absorption coefficient µa allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.

Tailoring the HHx monomer content of P(HB-co-HHx) by flexible substrate compositions: scale-up from deep-well-plates to laboratory bioreactor cultivations.

The enhanced material properties exhibited by the microbially synthetized polyhydroxyalkanoate (PHA) copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] evidence that this naturally biodegrading biopolymer could replace various functionalities of established petrochemical plastics. In fact, the thermal processability, toughness and degradation rate of P(HB-co-HHx) can be tuned by modulating its HHx molar content enabling to manufacture polymers à-la-carte. We have developed a simple batch strategy to precisely control the HHx content of P(HB-co-HHx) to obtain tailor-made PHAs with defined properties. By adjusting the ratio of fructose to canola oil as substrates for the cultivation of recombinant Ralstonia eutropha Re2058/pCB113, the molar fraction of HHx in P(HB-co-HHx) could be adjusted within a range of 2-17 mol% without compromising polymer yields. The chosen strategy proved to be robust from the mL-scale in deep-well-plates to 1-L batch bioreactor cultivations.

Native feedstock options for the polyhydroxyalkanoate industry in Europe: A review.

The United Nations defined 17 Sustainable Development Goals (SDGs) in 2016 and agreed on fighting to confront the climate change and protecting the oceans and forests. Subsequently, the sustainable production of bioplastics is gradually gaining reputation and significance. With the usage of bioplastics such as biodegradable polyhydroxyalkanoates (PHAs) various SDGs would be tackled, but costs remain a crucial factor for competing against fossil-based plastics. Appropriate local feedstock selection can help to reduce the production costs and minimize transportation routes. In this work, four feedstock generations are introduced and respective conversion strategies to PHA are presented. Whilst the focus is on mapping the abundances of feedstocks and potential PHA production capacities in Europe, utilization of animal by-product streams is also highlighted as a rather unconventional but highly abundant feedstock for PHA production.

Substrate-Flexible Two-Stage Fed-Batch Cultivations for the Production of the PHA Copolymer P(HB-co-HHx) With Cupriavidus necator Re2058/pCB113.

Recent studies of the impact and dimension of plastic pollution have drawn the attention to finding more sustainable alternatives to fossil-based plastics. Microbially produced polyhydroxyalkanoates (PHAs) biopolymers are strong candidates to replace conventional plastic materials, due to their true biodegradability and versatile properties. However, widespread use of these polymers is still hindered by their high cost of production. In the present study, we target high yields of the PHA copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)] using a substrate-flexible two-stage fed-batch approach for the cultivation of the recombinant Cupriavidus necator strain Re2058/pCB113. A more substrate-flexible process allows to cope with constant price fluctuations and discontinuous supply of feedstocks on the market. Utilizing fructose for biomass accumulation and rapeseed oil for polymer production resulted in a final biomass concentration of 124 g L-1 with a polymer content of 86 wt% holding 17 mol% of HHx. Productivities were further optimized by operating the biomass accumulation stage in a "drain and fill" modus where 10% of the culture broth was recycled for semi-continuous biomass accumulation, after transferring 90% to a second bioreactor for PHA production. This strategy succeeded in shortening process times rising productivity yields to ∼1.45 g L-1 h-1.

Bioprocess Development for Lantibiotic Ruminococcin-A Production in Escherichia coli and Kinetic Insights Into LanM Enzymes Catalysis.

Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (<1 µg L-1) and difficulties to cultivate the obligately anaerobic organism. We recently reported the reconstruction of the RumA biosynthesis machinery in Escherichia coli where the fully modified and active peptide was expressed as a fusion protein together with GFP. In the current study we developed a scale-up strategy for the biotechnologically relevant heterologous production of RumA, aimed at overproducing the peptide under conditions comparable with those in industrial production settings. To this end, glucose-limited fed-batch cultivation was used. Firstly, parallel cultivations were performed in 24-microwell plates using the enzyme-based automated glucose-delivery cultivation system EnPresso® B to determine optimal conditions for IPTG induction. We combined the bioprocess development with ESI-MS and tandem ESI-MS to monitor modification of the precursor peptide (preRumA) during bioreactor cultivation. Dehydration of threonine and serine residues in the core peptide, catalyzed by RumM, occurs within 1 h after IPTG induction while formation of thioether cross-bridges occur around 2.5 h after induction. Our data also supplies important information on modification kinetics especially with respect to the fluctuations observed in the various dehydrated precursor peptide versions or intermediates produced at different time points during bioreactor cultivation. Overall, protein yields obtained from the bioreactor cultivations were >120 mg L-1 for the chimeric construct and >150 mg L-1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.