Co-feeding enhances the yield of methyl ketones.

The biotechnological production of methyl ketones is a sustainable alternative to fossil-derived chemical production. To date, the best host for microbial production of methyl ketones is a genetically engineered Pseudomonas taiwanensis VLB120 ∆6 pProd strain, achieving yields of 101 mgg-1 on glucose in batch cultivations. For competitiveness with the petrochemical production pathway, however, higher yields are necessary. Co-feeding can improve the yield by fitting the carbon-to-energy ratio to the organism and the target product. In this work, we developed co-feeding strategies for P. taiwanensis VLB120 ∆6 pProd by combined metabolic modeling and experimental work. In a first step, we conducted flux balance analysis with an expanded genome-scale metabolic model of iJN1463 and found ethanol as the most promising among five cosubstrates. Next, we performed cultivations with ethanol and found the highest reported yield in batch production of methyl ketones with P. taiwanensis VLB120 to date, namely, 154 mg g-1 methyl ketones. However, ethanol is toxic to the cell, which reflects in a lower substrate consumption and lower product concentrations when compared to production on glucose. Hence, we propose cofeeding ethanol with glucose and find that, indeed, higher concentrations than in ethanol-fed cultivation (0.84 g Laq-1 with glucose and ethanol as opposed to 0.48 g Laq-1 with only ethanol) were achieved, with a yield of 85 mg g-1. In a last step, comparing experimental with computational results suggested the potential for improving the methyl ketone yield by fed-batch cultivation, in which cell growth and methyl ketone production are separated into two phases employing optimal ethanol to glucose ratios. ONE-SENTENCE SUMMARY: By combining computational and experimental work, we demonstrate that feeding ethanol in addition to glucose improves the yield of biotechnologically produced methyl ketones.

Determination of double bond positions in methyl ketones by gas chromatography-mass spectrometry using dimethyl disulfide derivatives.

RATIONALE: Methyl ketones are of interest for the application as biofuels. The fatty acid metabolism of different microbes has been rearranged to achieve a sustainable production of methyl ketones. The biofuel properties and possible further chemical modifications of these methyl ketones are influenced by their chain length, as well as their degree of saturation and the corresponding double bond position. METHODS: A method based on gas chromatography-electron ionization; mass spectrometry (GC-EI-MS) was used to determine the double bond position of methyl ketones. Derivatization using dimethyl disulfide (DMDS) and an iodine catalyst enabled the activation of the double bonds for selective fragmentation during electron ionization. The cleavage led to key fragments in the Orbitrap high-resolution mass spectrum and allowed the unequivocal localization of the double bond position of the respective monounsaturated methyl ketone. RESULTS: The double bond position of several medium chain length methyl ketones originating from the gram-negative bacterium Pseudomonas taiwanensis (P. taiwanensis) VLB120 was determined. The DMDS derivatives of methyl ketones can yield isobaric fragment ions for different possible double bond positions, which can be distinguished only using high-resolution MS. The double bond position of all methyl ketones deriving from P. taiwanensis VLB120 was the same, counting from the end of the aliphatic chain, and was determined as ω-7. CONCLUSIONS: The derivatization of medium chain length monounsaturated methyl ketones with DMDS allowed the determination of the corresponding double bond position via GC-EI-MS. High-resolution MS is needed to differentiate possible double bond positions that yield isobaric fragment ions.

Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH.

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced Khalf at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).