The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mg L(-1) homogentisate and 84 microg L(-1) geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 microg g(-1)). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound-delta-tocotrienol (15 microg g(-1))-in E. coli.

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography-mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector.

Applying systems biology tools to study n-butanol degradation in Pseudomonas putida KT2440To smoothen the process of n-butanol formation in Pseudomonas putida KT2440, detailed knowledge of the impact of this organic solvent on cell physiology and regulation is of outmost importance. Here, we conducted a detailed systems biology study to elucidate cellular responses at the metabolic, proteomic, and transcriptional level. Pseudomonas putida KT2440 was cultivated in multiple chemostat fermentations using n-butanol either as sole carbon source or together with glucose. Pseudomonas putida KT2440 revealed maximum growth rates (μ) of 0.3 h −1 with n-butanol as sole carbon source and of 0.4 h −1 using equal C-molar amounts of glucose and nbutanol. While C-mole specific substrate consumption and biomass/substrate yields appeared equal at these growth conditions, the cellular physiology was found to be substantially different: adenylate energy charge levels of 0.85 were found when n-butanol served as sole carbon source (similar to glucose as sole carbon source), but were reduced to 0.4 when n-butanol was coconsumed at stable growth conditions. Furthermore, characteristic maintenance parameters changed with increasing n-butanol consumption. 13 C flux analysis revealed that central metabolism was split into a glucose-fueled Entner-Doudoroff/pentose-phosphate pathway and an n-butanol-fueled tricarboxylic acid cycle when both substrates were coconsumed. With the help of transcriptome and proteome analysis, the degradation pathway of n-butanol could be unraveled, thus representing an important basis for rendering P. putida KT2440 from an n-butanol consumer to a producer in future metabolic engineering studies.

A two-phase biotransformation process for selective hydroxylation of n-octane to 1-octanol via Pseudomonas putida KT2440 harboring heterologously expressed P450 monooxygenase from Mycobacterium marinum is presented. Maximum cell-specific conversion rates of 12.7 mg octanol g CDW h -1 were observed not only in shaking flasks but also in 3.7-L-bioreactor studies.The bioreactor experiments were performed avoiding explosive gas mixtures by lowering volumetric power input, aeration rates and substrate concentrations. Based on a stoichiometric network of P. putida KT2440 topological studies were carried out. As a conclusion, potential limitations of NAD(P)H and/or ATP supply at production conditions can be excluded. Hence, the great potential of the host for further increasing conversion is outlined.

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