Ji-Won Song

Multistep Enzymatic Synthesis of Long-Chain α,ω-Dicarboxylic and ω-Hydroxycarboxylic Acids from Renewable Fatty Acids and Plant Oils†

A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid.

Genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium Thermus thermophilus HB27

BackgroundThermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. However, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. Thus, it is highly required to consider systems biological approaches where T. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological characteristics under such harsh conditions.ResultsWe reconstructed a genome-scale metabolic model of T. thermophilus, i TT548, the first ever large-scale network of a thermophilic bacterium, accounting for 548 unique genes, 796 reactions and 635 unique metabolites. Our initial comparative analysis of the model with Escherichia coli has revealed several distinctive metabolic reactions, mainly in amino acid metabolism and carotenoid biosynthesis, producing relevant compounds to retain the cellular membrane for withstanding high temperature. Constraints-based flux analysis was, then, applied to simulate the metabolic state in glucose minimal and amino acid rich media. Remarkably, resulting growth predictions were highly consistent with the experimental observations. The subsequent comparative flux analysis under different environmental conditions highlighted that the cells consumed branched chain amino acids preferably and utilized them directly in the relevant anabolic pathways for the fatty acid synthesis. Finally, gene essentiality study was also conducted via single gene deletion analysis, to identify the conditional essential genes in glucose minimal and complex media.ConclusionsThe reconstructed genome-scale metabolic model elucidates the phenotypes of T. thermophilus, thus allowing us to gain valuable insights into its cellular metabolism through in silico simulations. The information obtained from such analysis would not only shed light on the understanding of physiology of thermophiles but also helps us to devise metabolic engineering strategies to develop T. thermophilus as a thermostable microbial cell factory.

3′-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli

3′-Untranslated region (3′UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3′UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3′UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3′UTR. In summary, 3′UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells.

Activation of the Glutamic Acid-Dependent Acid Resistance System in Escherichia coli BL21(DE3) Leads to Increase of the Fatty Acid Biotransformation Activity.

The biosynthesis of carboxylic acids including fatty acids from biomass is central in envisaged biorefinery concepts. The productivities are often, however, low due to product toxicity that hamper whole-cell biocatalyst performance. Here, we have investigated factors that influence the tolerance of Escherichia coli to medium chain carboxylic acid (i.e., n-heptanoic acid)-induced stress. The metabolic and genomic responses of E. coli BL21(DE3) and MG1655 grown in the presence of n-heptanoic acid indicated that the GadA/B-based glutamic acid-dependent acid resistance (GDAR) system might be critical for cellular tolerance. The GDAR system, which is responsible for scavenging intracellular protons by catalyzing decarboxylation of glutamic acid, was inactive in E. coli BL21(DE3). Activation of the GDAR system in this strain by overexpressing the rcsB and dsrA genes, of which the gene products are involved in the activation of GadE and RpoS, respectively, resulted in acid tolerance not only to HCl but also to n-heptanoic acid. Furthermore, activation of the GDAR system allowed the recombinant E. coli BL21(DE3) expressing the alcohol dehydrogenase of Micrococcus luteus and the Baeyer-Villiger monooxygenase of Pseudomonas putida to reach 60% greater product concentration in the biotransformation of ricinoleic acid (i.e., 12-hydroxyoctadec-9-enoic acid (1)) into n-heptanoic acid (5) and 11-hydroxyundec-9-enoic acid (4). This study may contribute to engineering E. coli-based biocatalysts for the production of carboxylic acids from renewable biomass.

Cloning, expression, and characterization of P450 monooxygenase CYP102H1 from Nocardia farcinica

To isolate a new P450 monooxygenase belonging to the CYP102 family, CYP102H1 of Nocardia farcinica IFM 10152 (i.e., pnf11580) was cloned, expressed, and partially characterized. CYP102H1 gene was amplified from pNF1 of N. farcinica and cloned into expression vectors (i.e., pTrc99A, pET28a(+)). When Escherichia coli BL21(DE3) codon+ strain was transformed with pET28a-CYP102H1 and the culture was induced with 1.0 mM isopropyl-β-d-thio-galactoside in a complex medium at 30°C, CYP102H1 could be expressed in soluble form even though soluble form was not dominant. The enzyme showed typical features of heme proteins; a spectrum of reduced CO bound form showed typical maximum at 450 nm. When the biotransformation of linoleic acid was carried out in a reconstituted system consisting of CYP102H1 and redox partner proteins of Pseudomonas putida (i.e., CamAB), ω1-hydroxylinoleic acid was detected with gas chromatography/mass spectrometry analysis, suggesting that CYP102H1 catalyzes oxygenation of linoleic acid at ω-1 position, which is typical in CYP102A subfamily members.

Intracellular transformation rates of fatty acids are influenced by expression of the fatty acid transporter FadL in Escherichia coli cell membrane

Fatty acids have a low permeability through the cell membrane. Therefore, the intracellular biotransformation of fatty acids can be slow due to supply limitations. The effects of expression level of the fatty acid transporter FadL in Escherichia coli on the biotransformations were investigated. The enhanced expression of FadL led to 5.5-fold increase of the maximum reaction rate Vmax (i.e., 200 μmol/min per g dry cells (200 U/g dry cells)) of the recombinant E. coli expressing a hydratase of Stenotrophomonas maltophilia in the periplasm with respect to hydration of oleic acid. The FadL expression level was also critical for oxidation of 12- and 10- hydroxyoctadecanoic acid by the recombinant E. coli expressing an alcohol dehydrogenase (ADH) of Micrococcus luteus. In addition, the multistep biotransformation of ricinoleic acid into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) by the recombinant E. coli expressing the ADH of M. luteus and a Baeyer-Villiger monooxygenase of Pseudomonas putida KT2440 was 2-fold increased to 40 U/g dry cells with expression of FadL to an appropriate level. The FadL expression level is one of the critical factors to determine whole-cell biotransformation rates of not only long chain fatty acids but also hydroxy fatty acids. This study may contribute to whole-cell biocatalyst engineering for biotransformation of hydrophobic substances.