Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-d-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways. These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.Singapore-MIT Alliance for Research and Technology (SMART

Effects of lipids with different oxidation levels on protein degradation and biogenic amines formation in Sichuan-style sausages

ABS T R A C T We evaluated the effects of different oxidation levels of lipids on protein degradation and biogenic amines (BAs) formation during Sichuan-style sausages processing. Lipids with varying degrees of oxidation were obtained through storage at different temperatures and added as raw materials of Sichuan-style sausages, followed by the analyses of lipid oxidation, protein degradation, biogenic amine content, and other indicators. During the pro-cessing, with increasing degree of lipid oxidation, the contents of peroxide value (POV), thiobarbituric acid reactive substances (TBARs), protein degradation index (PI), amino acid nitrogen (AAN), free amino acids (FAAs), and BAs increased. Based on the protein electrophoresis results, the higher the oxidation degree of pig backfat, the higher degree of sarcoplasmic protein oxidation, and the greater myofibril protein degradation. Pearson correlation revealed that lipid oxidation, protein degradation, and BAs content correlated significantly (P < 0.05).Peer reviewe

Identification and optimization of critical media composition for the production of lycopene in PTS01 strain.

<p>(A) Half-normal probability plot of the results of minimal resolution IV experiment design (The details of the experiment design were shown in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075164#pone.0075164.s001" target="_blank">File S1</a>). In the half-normal probability plot, the line was called the near zero line. The estimated effect of an unimportant factor will typically be on or close to a near-zero line, while the estimated effect of an important factor will typically be displaced well off the line. (B) RSM plot of lycopene production versus concentrations of glycerol and KH₂PO₄ of PTS01.</p

Relationship of the central metabolic and DXP pathways.

<p>The abbreviations for metabolites in the figure were as follows: glycerol (Gly), glucose (Glu), glucose-6-phosphate (G6P), glycerol-3-phosphate (G3P), dihydroxyacetone phosphate (DHAP), glyceraldehyde 3-phosphate (GAP), 1,3-biphospho-glycerate (G1,3P), 3-phospho-glycerate (3PG), 2-phospho-glycerate (2PG), phosphoenolpyruvate (PEP), the phosphotransferase system (PTS), pyruvate (PYR), oxaloacetate (OAA), tricarboxylic acid cycle (TCA), 1-deoxy-D-xylulose 5-phosphate (DXP), 2C-methyl-D-erythritol 4-phosphate (MEP), 4-diphosphocytidyl-2C-methyl D-erythritol (CDP-ME), 4-diphosphocytidyl-2C-methyl D-erythritol 2-phosphate (CDP-MEP), 2C-methyl-D-erythritol 2,4-diphosphate (MEC), hydroxylmethylbutenyl diphosphate (HMBPP), isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), phosphate (PPi), carbohydrate phosphotransferase system (PTS). The abbreviations for enzyme-coding genes in the figure are as follows: PTS enzyme IIBC (<i>ptsG</i>), histidine protein (<i>ptsH</i>), PTS enzyme I (<i>ptsI</i>), PTS enzyme IIA(<i>crr</i>), glycerol facilitator (<i>glpF</i>), glycerol kinase (<i>glpK</i>), glycerol-3-phosphate dehydrogenase (<i>glpK</i>), triose phosphate isomerase (<i>tpi</i>), glyceraldehyde-3-phosphate dehydrogenase A (<i>gapA</i>), phosphoglycerate kinase (<i>pgk</i>), phosphoglycero mutase III (<i>gpmM</i>), phosphoglyceromutase I (<i>gpmA</i>), enolase (<i>eno</i>), PEP carboxylase (<i>ppc</i>), PEP carboxykinase (<i>pck</i>), phosphoenolpyruvate synthetase (<i>ppsA</i>), pyruvate kinase type I and II (<i>pykFA</i>), DXP synthase (<i>dxs</i>), DXP reductase (<i>dxr</i>), CDPME synthase (<i>ispD</i>), CDPME kinase (<i>ispE</i>), CDPMEP synthase (<i>ispF</i>), HMBPP synthase (<i>ispG</i>), HMBPP reductase (<i>ispH</i>), IPP isomerase (<i>idi</i>), farnesyl pyrophosphate (<i>FPP</i>) synthase (<i>ispA</i>), GGPP synthase (<i>crtE</i>), phytoene synthase (<i>crtB</i>) and phytoene desaturase (<i>crtI</i>) and amorpha-1,4-diene synthase (<i>ADS</i>).</p

The Taguchi orthogonal array design results.

<p>To examine the influence of each enzyme on amopha-4,11-diene (AD) yield, the average effects analysis was determined. The five enzymes can be classified into two main groups. A: Average values of each level of factors Erg12, Erg8 and Idi on AD yield. The group of enzymes has a positive correlation with AD yield. B: Average values of each level of factors Erg19 and IspA on AD yield. The group of enzymes has little or no effect on AD yield. C: The half-normal plot indicates the significant factors on AD yield. Factor A, B, D represent Erg12, Erg8 and Idi respectively. The abbreviations are as follows. Erg12: mevalonate kinase, Erg8: phosphomevalonate kinase, Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase.</p

Inhibitory effect of IspA and analysis of the precipitates.

<p>A set of separate experiments was conducted to validate the inhibitory effect of IspA. This was attributed to the precipitation of FPP. A: Fold change in amorpha-4,11-diene (AD) yield when increasing IspA and Idi concentrations while keeping other enzymes at reference level. Fold change in AD yield was calculated by normalizing against AD yield obtained by reference enzyme levels, as indicated by the arrows. Presented data were average of triplicates and standard errors were drawn on the plot. B: UPLC-(TOF)MS analysis of the intermediates in the precipitates. Presented data were average of triplicates and standard errors were drawn on the plot. C: SDS-PAGE analysis of enzymes in the precipitates. The molecular weight of the each band present in the protein marker is indicated. The abbreviations are as follows. Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase, MVA: mevalonic acid, FPP: farnesyl pyrophosphate.</p

Transcriptional analysis of <i>dxs</i>, <i>idi</i>, <i>ispD ispF</i>, <i>crtE</i>, <i>crtB</i> and <i>crtI</i>.

<p>PTS02 was grown in 2xPY and induced with different concentrations of L-arabinose (0.1, 1 and 10 mM). Columns labelled “C” represent the expression levels of PTS01 grown in OPT1 in the absence of the inducer (L-arabinose). Fold changes of transcriptional levels of these genes were normalized to the expression levels of corresponding genes of PTS01 grown in 2xPY in the absence of the inducer (L-arabinose).</p

Coded level combinations for a five-level, two factor response surface methodology with central composite design.

*<p>Refer to table S3B for the actual enzyme concentrations corresponding to the coded levels. A: farnesyl pyrophosphate synthase (IspA), B: amorpha-4,11-diene synthase (Ads).</p

Taguchi L16 (4⁵) orthogonal arraray design and results.

*<p>Refer to table S3A for the actual enzyme concentrations corresponding to the coded levels. A: mevalonate kinase (Erg12), B: phosphomevalonate kinase (Erg8), C: diphosphomevalonate decarboxylase (Erg19), D: isopentenyl pyrophosphate isomerase (Idi), E: farnesyl pyrophosphate synthase (IspA).</p

Comparison of lycopene production, growth curve and PEP levels of MG01 and PTS01.

<p>(A) Growth curve and lycopene productions of PTS01 and MG01 in optimal glycerol medium (OPT1) at 37°C, with a shaking speed of 300 rpm. (B) Comparison of PEP levels in MG01 and PTS01 at different growth stages (early-log phase, pre-induction, 9 h; mid-log phase, 16 h post-induction; stationary phase, 36 h post-induction). Both of the strains were grown in OPT1 at 37°C, with a shaking speed of 300 rpm).</p