Modular pathway rewiring of Saccharomyces cerevisiae enables high-level production of L-ornithine

Baker's yeast Saccharomyces cerevisiae is an attractive cell factory for production of chemicals and biofuels. Many different products have been produced in this cell factory by reconstruction of heterologous biosynthetic pathways; however, endogenous metabolism by itself involves many metabolites of industrial interest, and de-regulation of endogenous pathways to ensure efficient carbon channelling to such metabolites is therefore of high interest. Furthermore, many of these may serve as precursors for the biosynthesis of complex natural products, and hence strains overproducing certain pathway intermediates can serve as platform cell factories for production of such products. Here we implement a modular pathway rewiring (MPR) strategy and demonstrate its use for pathway optimization resulting in high-level production of L-ornithine, an intermediate of L-arginine biosynthesis and a precursor metabolite for a range of different natural products. The MPR strategy involves rewiring of the urea cycle, subcellular trafficking engineering and pathway re-localization, and improving precursor supply either through attenuation of the Crabtree effect or through the use of controlled fed-batch fermentations, leading to an L-ornithine titre of 1,041±47 mg l−1 with a yield of 67 mg (g glucose)−1 in shake-flask cultures and a titre of 5.1 g l−1 in fed-batch cultivations. Our study represents the first comprehensive study on overproducing an amino-acid intermediate in yeast, and our results demonstrate the potential to use yeast more extensively for low-cost production of many high-value amino-acid-derived chemicals.


Saccharomyces cerevisiae
This study

Saccharomyces cerevisiae
This study

ORN-X(KanMX)
MATa SUC2 MAL2-8c ura3-52 his3-∆1 P ARG3 ::P KEX2 car2∆::LoxP-T CTC1 -AGC1-P tHXT1 -P TPI -ORT1-T pYX212 ura3::      The uptake and generation rates are given in mg/g dry cell weight per h; b The uptake and generation rates are given in g/g dry cell weight per hour; c Yield from glucose, g/g; d Yield from glucose, mg/g; Supplementary The uptake and generation rates are given in mg/g dry cell weight per h; b The uptake and generation rates are given in g/g dry cell weight per hour; c Yield from glucose, g/g; d Yield from glucose, mg/g; e ND: not detected. Supplementary

Plasmid construction
All the plasmids used in this study can be found in Supplementary  Table 2).
Defined minimal medium (Delft medium) as described before was used for both batch cultivations and fed-batch fermentations of L-ornithine producing strains 10 . Luria Bertani (LB) broth with 80 mg l −1 ampicillin was used for maintenance of E. coli DH5α harboring appropriate plasmids.

Shake flask cultivation for L-ornithine production
Shake flask cultivation was used to evaluate the L-ornithine producing strains. 20 ml cultures were started in 100 ml unbaffled cotton-stopped flasks by inoculating an amount of pre-culture that resulted in a final optical density of 0.05 at 600 nm (OD600). The strains were grown at 30 °C with 200 rpm. orbital shaking in defined minimal medium (Delft medium) with 20 g l −1 glucose 10 . Samples were taken periodically to measure the cell mass, L-ornithine titre, residual glucose and other metabolites.

Fed-batch cultivation of L-ornithine producing strains
For fed-batch cultivations 10 , strains were first grown in a batch culture with Delft medium and then an initial volume of 900 ml Delft media in a 3 l bioreactor was inoculated to a cell density of 0.05. Cells were cultivated at 30 ºC, 600 rpm agitation, 1 vvm air flow, dissolved oxygen above 30% (controlled by adjusting the air flow and agitation) and pH 5.5 (controlled by 2 M KOH).
After the glucose and part of the ethanol were consumed, the exponential feed was started.
The temperature, agitation, gassing, pH and composition of the off-gas were monitored and controlled using the DasGip monitoring and control system. The effluent gas from the fermentation was analyzed for real-time determination of oxygen and CO 2 concentration by DasGip fed-batch pro® gas analysis systems with the off gas analyzer GA4 based on zirconium dioxide and two-beam infrared sensor.
A feed strategy was designed keeping the volumetric growth rate constant. An exponential feed rate ν(t) (l h -1 ) was calculated according to: where x 0 , s 0 and V 0 were the biomass density (g DCW l -1 ), the substrate concentration (g l -1 ) and the reactor volume (l) at the start of the fed-batch process, Yxs was the respiratory yield coefficient (g glucose gDCW -1 ); s f was the concentration of the growth limiting substrate (g glucose l -1 ) in the reservoir; μ 0 the was the specific growth rate (h -1 ) during the feed phase and t the feeding time.