In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. Graphical abstract

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium

A bacterial chromosome that was naturally fused with the secondary chromosome, or “chromid,” and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae . Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family.

Biocatalytic Formation of Novel Polyesters with para-Hydroxyphenyl groups in the Backbone - Engineering Cupriavidus necator for production of high-performance materials from CO2 and electricity

Synthetic materials are integral components of consumables and durable goods and indispensable in our modern world. Polyesters are the most versatile bulk- and specialty-polymers, but their production is not sustainable, and their fate at end-of-life of great concern. Bioplastics are highly regarded alternatives but have shortcomings in material properties and commercial competitiveness with conventional synthetic plastics. These constraints have limited the success in global markets. Enabling bio-production of advanced bioplastics with superior properties from waste-derived feedstocks could change this. We have created microbial cell factories that can produce a range of aliphatic and aromatic polyesters. A DphaC1 mutant of Cupriavidus necator H16 was complemented with hydroxyacyl-CoA transferases from either Clostridium propionicum (pct540) or Clostridium difficile (hadA), respectively. These were combined with a mutant PHA synthase (phaC1437) from Pseudomonas sp. MBEL 6 19, which rescued the PHA- phenotype of the knock-out mutant and allowed polymerization of various hydroxy carboxylates, including phloretic acid. This is the first-time, incorporation of an aromatic ring in the backbone of a biological polyester was achieved. Polymers contain para-hydroxyphenyl subunits are structurally analogous to synthetic aromatic polyesters like PET and high-strength polyarylates. In a further advance, the transgenic strain was cultivated in a bio-electrochemical system under autotrophic conditions, enabling synthesis of aromatic bio-polyesters from H2 and O2 generated in situ, while assimilating CO2. Follow-up elementary flux-mode analysis established the feasibility of de novo production of twenty different polyesters from five different carbon- and energy-sources. This comprehensive study opens the door to sustainable bio-production of high-performance thermoplastics and thermosets.

Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from CO2 by a Recombinant Cupriavidus necator

The copolyester of 3-hydroxybutyrate (3HB) and 3-hydoxyhexanoate (3HHx), PHBHHx, is one of the most practical kind of bacterial polyhydroxyalkanoates due to its high flexibility and marine biodegradability. PHBHHx is usually produced from vegetable oils or fatty acids through b-oxidation, whereas biosynthesis from sugars has been achieved by recombinant strains of hydrogen-oxidizing bacterium Cupriavidus necator. This study investigated the biosynthesis of PHBHHx from CO2 as the sole carbon source by engineered C. necator strains. The recombinant strains capable of synthesizing PHBHHx from fructose were cultivated in a flask using complete mineral medium and a substrate gas mixture (H2/O2/CO2 = 8:1:1). The results of GC and 1H NMR analyses indicated that the recombinants of C. necator synthesized PHBHHx from CO2 with high cellular content. When 1.0 g/L (NH4)2SO4 was used as a nitrogen source, the 3HHx composition of PHBHHx in the strain MF01∆B1/pBBP-ccrMeJ4a-emd was 47.7 ± 6.2 mol%. Further investigation demonstrated that the PHA composition can be regulated by using (R)-enoyl-CoA hydratase (PhaJ) with different substrate specificity. The composition of 3HHx in PHBHHx was controlled to about 11 mol%, suitable for practical applications, and high cellular content was kept in the strains transformed with pBPP-ccrMeJAc-emd harboring short-chain-length-specific PhaJ.

Insights in the Degradation of Medium-Chain Length Dicarboxylic Acids in Cupriavidus necator H16 reveal Differences in β-Oxidation between Dicarboxylic Acids and Fatty Acids

Many homologous genes encoding β-oxidation enzymes were found in the genome of Cupriavidus necator H16 (synonym: Ralstonia eutropha H16). By proteome analysis, the degradation of adipic acid was investigated and showed differences to the degradation of hexanoic acid. During β-oxidation of adipic acid, activation with coenzyme A (CoA) is catalyzed by the two-subunit acyl-CoA ligase encoded by B0198 and B0199. The operon is completed by B0200 encoding a thiolase catalyzing the cleavage of acetyl-CoA at the end of the β-oxidation cycle. Strain C. necator ΔB0198-B0200 showed improved growth on adipic acid. Potential substitutes are B1239 for B0198-B0199 and A0170 as well as A1445 for B0200. A deletion mutant without all three thiolases showed diminished growth. The deletion of detected acyl-CoA dehydrogenase encoded by B2555 has an altered phenotype grown with sebacic acid but not adipic acid. With hexanoic acid, acyl-CoA dehydrogenase encoded by B0087 was detected on 2D gels. Both enzymes are active with adipoyl-CoA and hexanoyl-CoA as substrates, but specific activity indicates a higher activity of B2555 with adipoyl-CoA. 2D gels, growth experiments and enzyme assays suggest the specific expression of B2555 for the degradation of dicarboxylic acids. In C. necator H16 the degradation of carboxylic acids potentially changes with an increasing chain length. Two operons involved in growth with long-chain fatty acids seem to be replaced during growth on medium-chain carboxylic acids. Only two deletion mutants showed diminished growth. Replacement of deleted genes with one of the numerous homologous is likely. Importance The biotechnologically interesting bacterium Cupriavidus necator H16 was thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (Pohlmann et al., 2006). Nevertheless, the degradation of monocarboxylic fatty acids and dicarboxylic acids has not been elucidated completely. C. necator is used to produce value-added products from affordable substrates. One of our investigations ' primary targets is the biotechnological production of organic acids with different and specific chain lengths. The versatile metabolism of carboxylic acids recommends C. necator H16 as a candidate for producing value-added organic products. Therefore, the metabolism of these compounds is of interest, and for different applications in industry, understanding such central metabolic pathways is crucial.