Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly.

Methylated polyamines as research tools.

Earlier unknown racemic ß-methylspermidine (ß-MeSpd) and γ-methylspermidine (γ-MeSpd) were -synthesized starting from crotononitrile or methacrylonitrile and putrescine. Lithium aluminum hydride reduction of the intermediate di-Boc-nitriles resulted in corresponding di-Boc-amines, which after deprotection gave target ß- and γ-MeSpd's. To prepare α-MeSpd, the starting compound, 3-amino-1-butanol, was converted into N-Cbz-3-amino-1-butyl methanesulfonate, which alkylated putrescine to give (after deprotection of amino group) the required α-MeSpd. Novel ß- and γ-MeSpd's in combination with earlier α-MeSpd are useful tools for studying enzymology and cell biology of polyamines.

Molecular breeding of advanced microorganisms for biofuel production.

Large amounts of fossil fuels are consumed every day in spite of increasing environmental problems. To preserve the environment and construct a sustainable society, the use of biofuels derived from different kinds of biomass is being practiced worldwide. Although bioethanol has been largely produced, it commonly requires food crops such as corn and sugar cane as substrates. To develop a sustainable energy supply, cellulosic biomass should be used for bioethanol production instead of grain biomass. For this purpose, cell surface engineering technology is a very promising method. In biobutanol and biodiesel production, engineered host fermentation has attracted much attention; however, this method has many limitations such as low productivity and low solvent tolerance of microorganisms. Despite these problems, biofuels such as bioethanol, biobutanol, and biodiesel are potential energy sources that can help establish a sustainable society.

Fermentation of dried distillers' grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia.

Pretreatment and hydrolysis of lignocellulosic biomass using either dilute acid, liquid hot water (LHW), or ammonium fiber expansion (AFEX) results in a complex mixture of sugars such as hexoses (glucose, galactose, mannose), and pentoses (xylose, arabinose). A detailed description of the utilization of representative mixed sugar streams (pentoses and hexoses) and their sugar preferences by the solventogenic clostridia (Clostridium beijerinckii BA101, C. acetobutylicum 260, C. acetobutylicum 824, Clostridium saccharobutylicum 262, and C. butylicum 592) is presented. In these experiments, all the sugars were utilized concurrently throughout the fermentation, although the rate of sugar utilization was sugar specific. For all clostridia tested, the rate of glucose utilization was higher than for the other sugars in the mixture. In addition, the availability of excess fermentable sugars in the bioreactor is necessary for both the onset and the maintenance of solvent production otherwise the fermentation will become acidogenic leading to premature termination of the fermentation process. During an investigation on the effect of some of the known lignocellulosic hydrolysate inhibitors on the growth and ABE production by clostridia, ferulic and p-coumaric acids were found to be potent inhibitors of growth and ABE production. Interestingly, furfural and HMF were not inhibitory to the solventogenic clostridia; rather they had a stimulatory effect on growth and ABE production at concentrations up to 2.0g/L.