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

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

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

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

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

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

Purification and characterizations of individual pathway enzymes from bacterial culture.

<p>The bracket contains the specific substrate that the K_m is measured for.</p>*<p>The enzyme yield is defined as the final amount of enzymes obtained after purification from a liter bacterial culture. The results have been repeated for more than three times.</p>**<p>The molecular weight (MW) of the enzyme was calculated based on its amino acid sequence.</p