We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the ␥-cyclic C 50 carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C 40 lycopene, C 45 nonaflavuxanthin, C 50 flavuxanthin, and C 50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a ␥-cyclic C 50 carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related -cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the ␥-cyclic C 50 carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous -cyclic C 50 carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric -and ␥-cyclic C 50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C 50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

In natural and man-made ecosystems nitrifying bacteria experience frequent exposure to oxygen-limited conditions and thus have to compete for oxygen. In several reactor systems (retentostat, chemostat and sequencing batch reactors) it was possible to establish co-cultures of aerobic ammonium- and nitrite-oxidizing bacteria at very low oxygen concentrations (2-8 microM) provided that ammonium was the limiting N compound. When ammonia was in excess of oxygen, the nitrite-oxidizing bacteria were washed out of the reactors, and ammonium was converted to mainly nitrite, nitric oxide and nitrous oxide by Nitrosomonas-related bacteria. The situation could be rapidly reversed by adjusting the oxygen to ammonium ratio in the reactor. In batch and continuous tests, no inhibitory effect of ammonium, nitric oxide or nitrous oxide on nitrite-oxidizing bacteria could be detected in our studies. The recently developed oxygen microsensors may be helpful to determine the kinetic parameters of the nitrifying bacteria, which are needed to make predictive kinetic models of their competition.

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5 untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding ␤-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P antitet ), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C 50 carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.The initially used methods of deleting or overexpressing genes have been demonstrated to be inadequate for many applications in metabolic engineering (21,31,32). For example, when the goal is to optimize the expression level of a desired protein by engineering the relevant metabolic pathway, it might be necessary to change the expression of multiple enzymes simultaneously and to different levels (30,38). Also, reducing the formation of particular by-products can increase the flux of the desired product (29). In addition, low basal expression is critical for applications such as the expression of toxic genes, metabolic engineering, and control analysis (2, 14, 27, 34). This has led to an increased focus on development of genetic tools to fine-tune gene expression to the desired levels. A commonly used strategy is to make so-called promoter libraries of constitutive promoters (1,15,17). Such promoters seem to be preferred over the corresponding inducible ones for industrial scale productions because of factors such as inducer costs, sensitivity to inducer concentration, and heterogeneity of expression caused by an all-or-none effect of induction (1,19). However, the all-or-none induction effect may be eliminated if the inducer enters the cell interior by passive diffusion (20). Thus, regulatable promoter s...

The phosphometabolome is comprised of all phosphorylated metabolites including the major metabolite classes sugar phosphates and nucleoside phosphates. Phosphometabolites are invaluable in any cell as a part of primary- and energy- metabolism, and as building blocks in the biosynthesis of macromolecules. Here, we report quantitative profiling of the phosphometabolome by applying capillary ion chromatography-tandem mass spectrometry (capIC-MS/MS), ensuring improved chromatographic separation, robustness and quantitative precision. Baseline separation was achieved for six out of eight tested hexose phosphates. Quantitative precision and reproducibility was improved by introducing a fully uniformly (U) C-labeled biological extract and applying an isotope dilution (ID) correction strategy. AC-labeled biological extract does in principle contain internal standards (IS) for all metabolites, but low abundant metabolites pose a challenge, and solutions to this are discussed. The extreme reproducibility and reliability of this capIC-MS/MS method was demonstrated by running the instrumentation continuously for ten days.

Microbial culture collections are important resources for isolation of natural compounds with novel properties. In this study, a culture collection of around 1,500 pigmented heterotrophic bacteria was established. The bacteria were isolated from the sea surface microlayer at different sampling sites along the mid-part of the Norwegian coast. The bacterial isolates produced pigments of various coloration (e.g. golden, yellow, red, pink and orange). Methanol extracts of sixteen isolates were characterized with LC-Diodearray-TOF mass spectrometry analysis. The number of pigments per isolate varied considerably, and a tentative identification of the pigments was performed based on UV-absorbance profile and molecular formula assignation based on the accurate mass determination. The LC-MS analyses revealed that most of the pigments probably were carotenoids. Furthermore, we developed a high throughput LC-MS method for characterization and screening of a larger sub-fraction (300 isolates) of the culture collection. The aim was to screen and identify bacterial isolates producing carotenoids that absorb light in the UVA-Blue light. Six of the bacterial strains were selected for detailed investigation, including 16s rRNA sequencing, preparative HPLC for purification of major carotenoids and subsequent structural elucidation with NMR. Among the identified carotenoids were zeaxanthin, nostoxanthin and sarcinaxanthin, some with novel glycosylation patterns.