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The number of genes encoding β-oxidation enzymes in Cupriavidus necator H16 (synonym, Ralstonia eutropha H16) is high, but only the operons A0459-A0464 and A1526-A1531, each encoding four genes for β-oxidation enzymes, were expressed during growth with long-chain-length fatty acids (LCFAs). However, we observed that C. necator ΔA0459-A0464 ΔA1526-A1531 and C. necator H16 showed the same growth behavior during growth with decanoic acid and shorter FAs. The negative effect of the deletion of these two operons increased with an increasing chain length of the utilized FAs. Transcriptome sequencing (RNA-Seq) revealed the expression profiles of genes involved in the catabolism of medium-chain-length fatty acids (MCFAs) in C. necator H16. Operon A0459-A0464 was expressed only during growth with nonanoic acid, whereas operon A1526-A1531 was highly expressed during growth with octanoic and nonanoic acid. The gene clusters B1187-B1192 and B0751-B0759 showed a log2 fold change in expression of up to 4.29 and 4.02, respectively, during growth with octanoic acid and up to 8.82 and 5.50, respectively, with nonanoic acid compared to sodium gluconate-grown cells. Several acyl-CoA ligases catalyze the activation of MCFAs with coenzyme A (CoA), but fadD3 (A3288), involved in activation of LCFAs, was not detected. The expression profiles of C. necator strain ΔA0459-A0464 ΔA1526-A1531 showed that the growth with nonanoic acid resulted in the expression of further β-oxidation enzyme-encoding genes. Additional insights into the transport of FAs in C. necator H16 revealed the complexity and putative involvement of the DegV-like protein encoded by A0463 in the transport of odd-chain-length FAs and of siderophore biosynthesis in the transport mechanism. IMPORTANCE Although Cupriavidus necator H16 has been used in several studies to produce polyhydroxyalkanoates from various lipids, the fatty acid metabolism is poorly understood. The β-oxidation of long-chain-length FAs has been investigated, but the tremendous number of homologous genes encoding β-oxidation enzymes hides the potential for variances in the expressed genes for catabolism of shorter FAs. The catabolism of medium-chain-length FAs and connected pathways has not been investigated yet. As more sustainable substrates such as lipids and the production of fatty acids and fatty acid derivates become more critical with the dependency on fossil-based substances, understanding the complex metabolism in this highly diverse workhorse for biotechnology, C. necator, is inevitable. For further metabolic engineering and construction of production strains, we investigated the metabolism during growth on medium-chain-length FAs by RNA-Seq.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO 2 ) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO 2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO 2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO 2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Many homologous genes encoding β-oxidation enzymes have been 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 from 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. C. necator ΔB0198-B0200 strain 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 two-dimensional (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 has been thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (A. Pohlmann, W. F. Fricke, F. Reinecke, B. Kusian, et al., Nat Biotechnol 24:1257-1262, 2006, https://doi.org/10.1038/nbt1244). 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.

The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator ), which is used for biopolymer production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells and the minimal biotin demand of a biotin-auxotrophic R. eutropha strain were determined. Three consecutive cultivations in biotin-free medium were necessary to prevent growth of the auxotrophic mutant, and 40 ng/ml biotin was sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin was necessary to ensure growth comparable to that of the wild type, which is similar to the demand of biotin-auxotrophic mutants among other prokaryotic and eukaryotic microbes. A phenotypic complementation of the R. eutropha ΔbioF mutant was only achieved by homologous expression of bioF of R. eutropha or heterologous expression of bioF of Bacillus subtilis but not by bioF of Escherichia coli Together with the results from bioinformatic analysis of BioFs, this leads to the assumption that the intermediate of biotin synthesis in R. eutropha is pimeloyl-CoA instead of pimeloyl-acyl carrier protein (ACP) like in the Gram-positive B. subtilis Internal biotin content was enhanced by homologous expression of accB , whereas homologous expression of accB and accC2 in combination led to decreased biotin concentrations in the cells. Although a DNA-binding domain of the regulator protein BirA is missing, biotin synthesis seemed to be influenced by the amount of acceptor protein present. IMPORTANCE Ralstonia eutropha is applied in industry for the production of biopolymers and serves as a research platform for the production of diverse fine chemicals. Due to its ability to grow on hydrogen and carbon dioxide as the sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert inexpensive resources into value-added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bioengineering of the strain and for the development of new strategies for biotechnological production., (Copyright © 2020 American Society for Microbiology.)

Unlabelled: Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli The purified enzyme AtfG25 showed acyltransferase activity with C12- or C16-acyl-CoA, C12 to C18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of AtfG25 on lipid accumulation, the respective gene, atfG25, was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that AtfG25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study., Importance: A novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)