Biosynthesis of 2,5-dimethylpyrazine from L-threonine by whole-cell biocatalyst of recombinant Escherichia coli / 生物工程学报

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Scaling-up batch conditions for efficient sucrose hydrolysis catalyzed by an immobilized recombinant Pichia pastoris cells in a stirrer tank reactor

Background: Invert sugar is used greatly in food and pharmaceutical industries. This paper describes scaling-up batch conditions for sucrose inversion catalyzed by the recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase entrapped in calcium alginate beads. For the first time, we describe the application of a kinetic model to predict the fractional conversion expected during sucrose hydrolysis reaction in both, a model and a prototype bioreactor with 0.5- and 5-L working volume, respectively. Results: Different scaled-up criteria used to operate the 0.5-L bioreactor were analyzed to explore the invert sugar large scale production. After model inversion studies, a 5-L scaled-up reaction system was performed in a 7-L stirred reactor. Both scaled-up criteria, immobilized biocatalyst dosage and stirring speed, were analyzed in each type of bioreactors and the collected data were used to ensure an efficient scale-up of this biocatalyst. Conclusions: To date, there is not enough information to describe the large-scale production of invert sugar using different scaled-up criteria such as dose of immobilized biocatalyst and stirring speed effect on mass transfer. The present study results constitute a valuable tool to successfully carry out this type of high-scale operation for industrial purposes.

Steroids hydroxylation catalyzed by the monooxygenase mutantfromBM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an-based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione () at 1-position, generating a hydroxylated steroid 1-OH-androstenedione (). To investigate whether C-1hydroxylation catalyzed by BM3 mutantcould be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 °C, under pH 7.0 for 4 h, with 37% transformation rate. Moreover, fourvariants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

Technologies for Surface Display of Lipase / 中国生物工程杂志

Lipase is a kind of widely used hydrolase.Surface display is an efficient method of highthroughput screening for protein engineering of lipase.Besides,lipase displaying on surface of microorganism has many advantages,such as higher stability against high temperature and organic solvent,compared with free lipase,so the host strain displaying lipase can be used as wholecell biocatalyst,which has some advantages compared with traditional immobilization of lipase.There are three kinds of host strain for displaying lipase:phage,bacteria and yeast.The surface display of lipase in the three display systems were systematically discribed,and their current uses and possible trends in the future were discussed also.

Advance on the Biotransformation of Bioactive Natural Leading Compounds / 中国生物工程杂志

Biotransformation of bioactive natural leading compounds is a kind of bioprocess in which the structure of the added bioactive natural leading compounds could be modified by biocatalysts(e.g.,enzyme,microbial,plant and animal cells) in order to produce high efficient and low toxicity compounds.The biotransformation purpose of the known bioactive natural leading compounds is to improve its efficiency,or reduce its toxicity,or improve its solubility and bioavailability.The trace and high-valued bioactive natural leading compounds also could be produced by the biotransformation,and the biotransformation of bioactive natural leading compounds is still helpful to study the mechanism of drug metabolism.The current focus of the biotransformation of bioactive natural leading compounds is on the compounds of steroid,quinine,flavone and terpene,and some important biotransformation process has been successfully screened out.Fundamental research should be done in the following fields,such as the biotransformation mechanism of bioactive natural leading compounds,biotransformation process engineering,and the efficiency evaluation of bioproducts produced by biotransformation.The latest biotechnology(e.g.,directed evolution of biocatalyst,combinatorial biotransformation,non-aqueous biotransformation,high throughput screening) should be introduced to the biotransformation of bioactive natural leading compounds,which will boost its fast development.

The Biosynthesis of ?-arbutin by Xanthomona maltophilia BT-112 / 微生物学通报

?-arbutin is biosynthesized by whole cell method with Xanthomona maltophilia BT-112.The conditions for cell biosynthesized ?-arbutin are investigated as follows:temperature,25℃;concentration of hydroquinone,30mmol/L;mol ratio of sucrose and hydroquinone,20∶1;time course of ?-arbutin biosynthesis,45 hours;rotational speed,160r/min;concentration of Xanthomona maltophilia BT-112,85g/L;concentration of K-2HPO-4-KH-2PO-4 buffer solution,25mmol/L;pH of K-2HPO-4-KH-2PO-4 buffer solution,8.0.Under the above optimal conditions,the maximum of molar conversion yield based on the amount of hydroquinone supplied reaches 86.7%.