Microaerobic Fermentation Enables High-Titer Biosynthesis of the Rose Monoterpenes Geraniol and Geranyl Acetate in Escherichia coli

Monoterpenes are commercially important flavors and fragrances with high demand. Microbial production of monoterpenes is more sustainable than plant extraction, however, it is restricted by high product toxicity/volatility and inefficient monoterpene synthases. Hence, most reported monoterpene titers are still low for commercialization. To overcome these challenges, we utilized the rose NUDIX hydrolase instead of geraniol synthase to produce geraniol in E. coli. The supply of the monoterpene precursor, geraniol pyrophosphate (GPP), was enhanced by the mevalonate pathway optimization and screening/engineering of various GPP synthases from plants, yeasts, and bacteria. Furthermore, geraniol production was improved by deleting the competing pathway genes (tnaA, yjgB, and ackA-pta) and optimizing the bioprocess. The final strain produced 1.05 g/L monoterpenes in total including 0.91 g/L geraniol in flasks. Moreover, the geraniol strain was reprogrammed to produce geranyl acetate, reaching ∼4.1 g/L in flasks from 20 g/L glycerol (∼66% of theoretic yield). We observed that microaerobic fermentation is critical to achieve high-yield production of geraniol and geranyl acetate. By controlling the redox potential at −190 mV in 5 L bioreactors, our strain produced ∼19 g/L geranyl acetate in 100 h, with a yield of 0.12 g/g-glycerol

Additional file 3 of Mediating oxidative stress enhances α-ionone biosynthesis and strain robustness during process scaling up

Additional file 3. Oligonucleotides used for PCR amplifications

Mevalonate pathway for amorpha-4,11-diene production.

<p>The abbreviations are as follows. Erg12: mevalonate kinase, Erg8: phosphomevalonate kinase, Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase, Ads: amorpha-4,11-diene synthase, Pi: phosphate, Ppi: pyrophosphate.</p

Optimization of culture conditions for PTS01 and comparison of OPT1 with other commonly used media.

<p>(A) Lycopene production of PTS01 at different temperatures and shaking speeds. (B) Comparison of lycopene production of PTS01 in 2xPY, LB, 2xYT, 2xM9, R-media and OPT1 media. The strain was grown at 37°C, with a shaking speed of 300 rpm. (C) The growth curves of PTS01 in 2xPY, LB, 2xYT, 2xM9, R-media and OPT1 media.</p

Effects of monovalent ions.

<p>Monovalent ions were used to increase the specific activity of amorpha-4,11-diene synthase (Ads) and hence the specific amorpha-4,11-diene (AD) yield of the multienzyme synthesis reaction. A: Titration of potassium chloride concentrations, and their effects on Ads specific activity. Presented data were average of triplicates and standard errors were drawn on the plots. Student’s t-Test with paired two samples for means was used to calculate the p-value in the statistical analysis. B: Titration of different monovalent ions concentrations and their effects on AD yield by reference enzymatic levels. Fold change in AD yield was calculated by normalizing against AD yield obtained by reaction without addition of monovalent ions, as indicated by the arrow. Presented data were average of triplicates and standard errors were drawn on the plots.</p

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

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