Methanol-driven andnon-natural redox cofactor mediated biocatalysis

Please click Additional Files below to see the full abstrac

Modularly engineering Rhodotorula toruloides for α-terpineol production

α-Terpineol is a monoterpenoid alcohol that has been widely used in the flavor, fragrance, and pharmaceutical industries because of its sensory and biological properties. However, few studies have focused on the microbial production of α-terpineol. The oleaginous yeast Rhodotorula toruloides is endowed with a natural mevalonate pathway and is a promising host in synthetic biology and biorefinery. The primary objective of this work was to engineer R. toruloides for the direct biosynthesis of α-terpineol. The improvement in monoterpenoid production was achieved through the implementation of modular engineering strategies, which included the enhancement of precursor supply, blocking of downstream pathways, and disruption of competing pathways. The results of these three methods showed varying degrees of favorable outcomes in enhancing α-terpineol production. The engineered strain 5L6HE5, with competitive pathway disruption and increased substrate supply, reached the highest product titer of 1.5 mg/L, indicating that reducing lipid accumulation is an efficient method in R. toruloides engineering for terpenoid synthesis. This study reveals the potential of R. toruloides as a host platform for the synthesis of α-terpineol as well as other monoterpenoid compounds

Synthesis and application of photoaffinity probe containing an intact isoprenoid chain

Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins, and proteins with molecular weight of similar to 70 KDa appeared as a major band upon Streptavidin blot analysis. (C) 2009 Elsevier Ltd. All rights reserved

PCR-based strategy for construction of multi-site-saturation mutagenic expression library

There is an increasing demand for efficient and effective methods to engineer protein variants for industrial applications, structural biology and drug development. We describe a PCR-based strategy that produces multi-site-saturation mutagenic expression library using a circular plasmid carrying the wild-type gene. This restriction digestion- and ligation-independent method involves three steps: 1) synthesis of the degenerate oligonucleotide primers, 2) incorporation of the mutations through PCR, 3) transformation into the expression host. Our strategy is demonstrated through successful construction of an E coli K12 malic enzyme expression library that contains members with simultaneous mutations on amino acid residues G311, D345 and G397. This method is in principle compatible with any circular vector that can be propagated with a dam(+) E. coli host to generate protein variant library with multiple changes, including mutation, short sequence deletion and insertion, or any mix of them. (c) 2007 Elsevier B.V All rights reserved

Conversion of fructose into 5-HMF catalyzed by GeCl4 in DMSO and [Bmim]Cl system at room temperature

Conversion of fructose into 5-hydroxymethylfurfural (HMF) catalyzed by Germanium(IV) chloride was studied in Dimethyl sulfoxide (DMSO) and [Bmim]Cl system at room temperature. The structure of ionic liquids and the ratio of ionic liquids to DMSO had a remarkable effect on this new catalytic system. The results indicated that 1-n-butyl-3-methylimidazolium chloride ([Bmim]Cl) was favorable for the dehydration of fructose to HMF. Under optimal conditions, a high HMF yield in 70% was obtained at 25 degrees C. In addition, it was also proved that there was a synergistic effect between the cation and anion of [Bmim]Cl on the dehydration of fructose to HMF by designed experiments. Finally, a possible mechanism for the dehydration of fructose to HMF was proposed. (C) 2012 Elsevier Ltd. All rights reserved

Efficient acid-catalyzed hydrolysis of cellulose in organic electrolyte solutions

A novel method for cellulose hydrolysis catalyzed by mineral acids have been developed in organic electrolyte solutions at 70 degrees C under atmosphere pressure without pretreatment. Several reaction parameters including reaction time, temperature, catalyst loading and the ratio of ionic liquids to organic solvents have been evaluated and optimized. Under optimal conditions, the maximum total reducing sugars (TRS) yield in 68.8% and glucose yield in 39.2% were obtained in 1.0 g NMP/3.0 g [Bmim]Cl system at 70 degrees C. Due to the low temperature of cellulose hydrolysis, this method shows a promising potential as an energy-efficient and cost-effective approach for the biorefinery of lignocellulosic biomass. (C) 2012 Elsevier Ltd. All rights reserved

Lipid Production from Amino Acid Wastes by the Oleaginous Yeast Rhodosporidium toruloides

Microbial lipids have been considered as promising resources for the production of renewable biofuels and oleochemicals. Various feedstocks, including sugars, crude glycerol, and volatile fatty acids, have been used as substrates for microbial lipid production, yet amino acid (AA) wastes remain to be evaluated. Here, we describe the potential to use AA wastes for lipid production with a two-stage culture mode by an oleaginous yeast strain Rhodosporidium toruloides CGMCC 2.1389. Each of the 20 proteinogenic AAs was evaluated individually as sole carbon source, with 8 showing capability to facilitate cellular lipid contents of more than 20%. It was found that L-proline was the most favored AA, with which cells accumulated lipids to a cellular lipid content of 37.3%. When blends with AA profiles corresponding to those of meat industry by-products and sheep viscera were used, the cellular lipid contents reached 27.0% and 28.7%, respectively. The fatty acid compositional analysis of these lipid products revealed similar profiles to those of vegetable oils. These results, thus, demonstrate a potential route to convert AA wastes into lipids, which is of great importance for waste management and biofuel production