Micrococcus luteus naturally produces alkenes, unsaturated aliphatic hydrocarbons, and represents a promising host to produce hydrocarbons as constituents of biofuels and lubricants. In this work, we identify the genes for key enzymes of the branched-chain amino acid catabolism in M. luteus, whose first metabolic steps lead also to the formation of primer molecules for branched-chain fatty acid and olefin biosynthesis, and demonstrate how these genes can be used to manipulate the production of specific olefins in this organism. We constructed mutants of several gene candidates involved in the branched-chain amino acid metabolism or its regulation and investigated the resulting changes in the cellular fatty acid and olefin profiles by GC/MS. The gene cluster encoding the components of the branched-chain α-keto acid dehydrogenase (BCKD) complex was identified by deletion and promoter exchange mutagenesis. Overexpression of the BCKD gene cluster resulted in about threefold increased olefin production whereas deletion of the cluster led to a drastic reduction in branched-chain fatty acid content and a complete loss of olefin production. The specificities of the acyl-CoA dehydrogenases of the branched amino acid degradation pathways were deduced from the fatty acid and olefin profiles of the respective deletion mutant strains. In addition, growth experiments with branched amino acids as the only nitrogen source were carried out with the mutants in order to confirm our annotations. Both the deletion mutant of the BCKD complex, responsible for the further degradation of all three branched-chain amino acids, as well as the deletion mutant of the proposed isovaleryl-CoA dehydrogenase (specific for leucine degradation) were not able to grow on leucine in contrast to the parental strain. In conclusion, our experiments allow the unambigous assignment of specific functions to the genes for key enzymes of the branched-chain amino acid metabolism of M. luteus. We also show how this knowledge can be used to engineer the isomeric composition and the chain lengths of the olefins produced by this organism.
In the food and pharmaceutical industries, evaluating the sterilization performance preceding aseptic production processes is of central importance. In the case of hydrogen peroxide sterilization of solid surfaces, bioindicators (BI) consisting of spores of Bacillus atrophaeus or Geobacillus stearothermophilus are used to validate the effectiveness and efficiency of the inactivation procedure. Commercial production of G. stearothermophilus is commonly performed on agar plates, where cultivation and sporulation conditions are not well-defined. Therefore, the produced BI can vary in their resistance, which in turn creates unacceptable uncertainties in the evaluation of aseptic processes. Submerged production in the bioreactor would allow more control over sporulation conditions, while reducing production time, resistance variability, and avoidance of false-positive or false-negative test results. In addition, submerged production of G. stearothermophilus so far was a challenge to achieve sufficiently high spore concentrations for BI production. This study reports on the development of a method for submerged production of G. stearothermophilus spores (pH 7.0, 57 °C, 30% pO2) that can achieve 1.6 × 107 spores/mL with a resistance against 35% H2O2 at 25 °C of D25°C,35% H2O2 = 73 s. This resistance ranks within the range of commercially available BI, making the results directly transferable to industrial applications.
The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two‐step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one‐step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one‐step BI production is technically possible but at the expense of spore yield. The two‐step manufacturing process can realize almost 10‐fold higher spore yields.
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