MOESM1 of Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production

Additional file 1: Construction of plasmids. Table S1. Comparison of protoilludene synthases reported in literatures. Table S2. Cell growth of recombinant E. coli harboring MVA pathway engineered in a way of various combinations of MvUL,M,H and MvL1–6. Table S3. Cell growth of recombinant E. coli harboring MVA pathway engineered with combinations of MvUM,H and MvL2,7–13. Table S4. Strains, plasmids and primers used in this study. Figure S1. GC-FID standard curve of protoilludene. Figure S2. Residual mevalonate in culture of the strains E. coli AO/MvL1-6 with exogenous addition of mevalonate. Figure S3. Cell growth of E. coli strains harboring pBMvUL, pSMvUM and pTMvUH. Figure S4. Schematic diagram of pSMvL1–13-MvUM and pTAOMvUH

MOESM1 of Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives

Additional file 1: Table S1. Primers, plasmids and bacterial strains used in this study. Table S2. Time course analysis of oxidation of farnesol to farnesal in E. coli DH5Îą-YjgB. Figure S1. GC-FID and GC-MS profile of standard isoprenoid-based alcohols and their derivatives. Figure S2. Comparison of cell growth of the strains NA-MBF2.0, NAK-MBF2.0, NA-MBF1.0, and NAK-MBF1.0. Figure S3. Comparison of cell growth of the strains NA-MBF1.1, NA-MBF1.2, NA-MBF1.1a and NA-MBF1.2a. Figure S4. Percent composition of isoprenoid mixtures obtained from the strains NA-MBF1.1, NA-MBF1.2, NA-MBF1.1a and NA-MBF1.2a. Figure S5. GC-FID standard curves of isoprenoid alcohols and their derivatives

MOESM2 of Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives

Additional file 2: Table S3. Primers, plasmids and bacterial strains used in this study. Figure S6. GPP and FPP hydrolyzing assay of NudB

MOESM1 of Fermentative production and direct extraction of (−)-α-bisabolol in metabolically engineered Escherichia coli

Additional file 1. Figure S1. Nucleotide sequence of the E. coli codon-optimized MrBBS gene. Figure S2. Extraction efficiency of (−)-α-bisabolol using n-dodecane. Figure S3. Cell growth during optimization of (−)-α-bisabolol production in E. coli DH5α expressing MrBBS and entire MVA pathway genes

Engineering of Family-5 Glycoside Hydrolase (Cel5A) from an Uncultured Bacterium for Efficient Hydrolysis of Cellulosic Substrates

<div><p>Cel5A, an endoglucanase, was derived from the metagenomic library of vermicompost. The deduced amino acid sequence of Cel5A shows high sequence homology with family-5 glycoside hydrolases, which contain a single catalytic domain but no distinct cellulose-binding domain. Random mutagenesis and cellulose-binding module (CBM) fusion approaches were successfully applied to obtain properties required for cellulose hydrolysis. After two rounds of error-prone PCR and screening of 3,000 mutants, amino acid substitutions were identified at various positions in thermotolerant mutants. The most heat-tolerant mutant, Cel5A_2R2, showed a 7-fold increase in thermostability. To enhance the affinity and hydrolytic activity of Cel5A on cellulose substrates, the family-6 CBM from <i>Saccharophagus degradans</i> was fused to the <i>C</i>-terminus of the Cel5A_2R2 mutant using overlap PCR. The Cel5A_2R2-CBM6 fusion protein showed 7-fold higher activity than the native Cel5A on Avicel and filter paper. Cellobiose was a major product obtained from the hydrolysis of cellulosic substrates by the fusion enzyme, which was identified by using thin layer chromatography analysis.</p></div

Optimum temperature and pH for Cel5A_2R2 and Cel5A_2R2-CBM6 fusion protein.

<p>Symbols are as follows: Cel5A_2R2 (Open circles) and Cel5A_2R2-CBM6 (Closed circles). The error bars represent the standard deviation of triplicate measurements.</p

Synergistic interaction of cellobiohydrolase (CbhA) from <i>C. thermocellum</i> with Cel5A_2R2 parent protein and Cel5A_2R2-CBM6 fusion protein.

<p>The error bars represent the standard deviation of triplicate measurements.</p

Cel5A mutations covered in this work mapped onto the model of the Cel5A catalytic domain.

<p>Cellobiose at the reaction cavity is displayed as ball-and-sticks (carbon in yellow and oxygen in red). Mutated residues in each mutant are shown as sticks in different colors: D45G in orange from 1R1, V108G, and L240Q in green from 1R2, D275G in cyan from 1R3, N252D in hot pink from 1R4, D40E in purple-blue from 1R5, T195A in magenta from 2R1, F90L in blue from 2R2, the common mutation V256A in red.</p

TLC analysis of hydrolysis products of cellotriose, cellotetraose, cellopentaose, cellohexaose, CMC, PASC, filter paper, Avicel, and <i>p</i>-NPC.

<p>A: Hydrolysis (1 h) products of cellotriose and cellotetraose, B: Hydrolysis (1 h) products of cellopentaose and cellohexaose, C: Hydrolysis (5 h) products of CMC and PASC, D: Hydrolysis (16 h) products of filter paper and Avicel, and E: Hydrolysis (1 h) product of <i>p</i>-NPC. M: Standard marker, where G1 to G6 represent glucose, cellobiose, cellotriose, cellotetraose, cellopentasoe, and cellohexaose. Cello-oligosaccharides, CMC, PASC, and <i>p</i>-NPC were treated with 0.1 nmol of Cel5A_2R2-CBM6 at 55°C. The same reaction was performed using Avicel and filter paper with 1.0 nmol of Cel5A_2R2-CBM6. Reactions were performed in the absence (−) and presence (+) of the enzyme.</p

Specific enzyme activity of Cel5A_2R2 and Cel5A_2R2-CBM6 on various soluble and insoluble cellulosic substrates.

a<p>Specific activity of Cel5A_2R2-CBM6 was statistically significant from wild type Cel5A and mutant Cel5A_2R2 at two-tailed P value is less than 0.0001.</p>b<p>ND represents “Not Detected”, indicating no enzyme activity.</p