Dr. Barry Ritz, Chief Science Officer at Pure Encapsulations, adds: “Our partnership is built on a relentless effort to ensure these are the most science-backed CBD products in the world, setting the benchmark for what consumers expect and healthcare professionals demand.”
“We are extremely proud to expand our relationship with Nestlé’s healthcare professional brands into Europe after their success in the U.S. market this past year,” said Jesse Lopez, CEO and founder of Geocann.
“Their leadership team’s validation of science-backed and clinically-proven hemp products is an important milestone for our industry and provides exceptional credibility to the therapeutic benefits of cannabinoids formulated with VESIsorb.”
Pure Encapsulations – owned by Nestlé – will now sell CBD soft gel formulations which use the patented VESIsorb drug delivery system for improved absorption and bioavailability.
The first large-scale production was completed and delivered earlier this month using only European resources, including cultivation and extraction of the hemp biomass in Slovenia and drug delivery technology and soft gel capsule production in Switzerland.
The first product that Pure Encapsulations is launching in Europe is a soft gel capsule with a science-backed two-to-one ratio of CBD (cannabidiol) to BCP (beta-caryophyllene) from broad spectrum hemp extract that utilises the VESIsorb delivery system.
The CBG soft gel products utilise the VESIsorb delivery system developed by Swiss company Vesifact, a spin-off of the Federal Institute of Technology in Zurich. The delivery system is described as a self-assembling colloidal droplet delivery system, and is covered by multiple patents.
Evidence showed that some undesired byproducts could also be formed in the biosynthetic pathway of OLA 21, 22 . We hypothesized that fusing CsOLS with CsOAC may place the two enzymes in close proximity and minimize the dissipation of intermediates. In such a way, fused OLS-OAC may efficiently transfer the intermediates to form OLA with minimal byproducts. We fused the two proteins with an amino acid linker (10×Glycine) in two different orientations. The results showed that when CsOLS was fused to the N-terminus or C-terminus of the CsOAC, the obtained strains YL102 and YL103 showed a slightly decline in the production of OLA compared with the control strains YL101 (Figure 2c). It is possible that the fusion of these two proteins may negatively impact their correct folding and catalytic functions. Because the fusion of CsOLS with CsOAC could not further improve OLA production, the best production strain, YL101, was subjected to further engineering.
For performing shake flask cultivations, seed culture was carried out in the shaking tube with 2 mL seed culture medium at 30 °C and 250 r.p.m. for 48 h. Then, 0.6 mL of seed culture was inoculated into the 250 mL flask containing 30 mL of fermentation medium and grown under the conditions of 30 °C and 250 r.p.m. for 96 h. One milliliter of cell suspension was sampled every 24h for OD600 and desired metabolite measurement.
The standard protocols of Y. lipolytica transformation by the lithium acetate method were described as previously reported 16, 18 . In brief, one milliliter cells was harvested during the exponential growth phase (16-24 h) from 2 mL YPD medium (yeast extract 10 g/L, peptone 20 g/L, and glucose 20 g/L) in the 14-mL shake tube, and washed twice with 100 mM phosphate buffer (pH 7.0). Freshly cultivated yeast colony lawns picked from overnight-grown YPD plates could also be used for genetic transformation. Then, cells were resuspended in 105 μL transformation solution, containing 90 uL 50% PEG4000, 5 μL lithium acetate (2M), 5 μL boiled single stand DNA (salmon sperm, denatured) and 5 μL DNA products (including 200-500 ng of plasmids, linearized plasmids or DNA fragments), and incubated at 39 °C for 1 h, then spread on selected plates. It should be noted that the transformation mixtures needed to be vortexed for 15 seconds every 15 minutes during the process of 39 °C incubation. The selected markers, including leucine, uracil and hygromycin, were used in this study. All engineered strains after genetic transformation were undergone PCR screening using the GoTaq Green PCR kits, and the strain containing the correct gene fragment was selected to perform shaker flask cultivation. For shaking tube cultivations, 100 μL seed cultures were inoculated into 5 mL fermentation media in a 50 mL tube.
3. Results and Discussion
In this study, we have systematically investigated the enzymatic bottlenecks that restrain the efficient biosynthesis of CBD precursor olivetolic acid in the oleaginous yeast Y. lipolytica. We took a reverse engineering approach and sequentially identified that the supply of hexanoyl-CoA, malonyl-CoA, acetyl-CoA, NADPH and ATP are the rate-limiting steps. To overcome these limitations, we have screened enzymes aiming to debottleneck the pathway limitations and redirect the carbon flux toward the end-product olivetolic acid. We discovered that the use of the P. putia IvaE encoding a novel acyl-CoA synthase could efficiently convert exogenously-added hexanoic acid to hexanoyl-CoA. The co-expression of the acetyl-CoA carboxylase, the pyruvate dehydrogenase bypass, the NADPH-generating malic enzyme, as well as the activation of peroxisomal β-oxidation pathway and ATP export pathway were efficient strategies to remove the pathway bottlenecks. Collectively, these strategies have led us to construct a Y. lipolytica strain that produced olivetolic acid at a titer 83-fold higher than our initial strain (0.11 mg/L). While the current production level is low, we expect these strategies may serve as a baseline for other metabolic engineers who are interested in engineering CBD biosynthesis in oleaginous yeast species.
Engineered metabolic pathway for synthesis of olivetolic acid (OLA) in Y. lipolytica. ACC1, acetyl-CoA-carboxylase; ACS, acetic acid synthase; ANT1, adenine nucleotide transporter; CsOAC, OLA cyclase from Cannabis sativa; CsOLS, OLA synthase from C. sativa; DGA1 and DGA2, diacylglycerol acyltransferases; FAS, fatty acid synthase; MAE, malic enzyme; PDH, pyruvate dehydrogenase; PEX10, peroxisomal matrix protein.
To quantify the concentration of olivetolic acid, 0.5 mL whole cell sample with both cell pellet and liquid culture was taken. Subsequently, samples were treated with 2 U/OD zymolyase (2h, 30 °C with shaking at 1000 rpm), and then cell suspensions were added with 20% (w/v) glass beads (0.5 mm) and the cells were grinded with hand-powered electrical motor (VWR). Subsequently, the crude extracts were mixed with an equal volume of ethyl acetate (v/v), followed by vortex at room temperature for 2 hours. Organic and inorganic layers were separated by centrifugation at 12000 rpm for 10 min. Samples were extracted three times. The combined organic layers were evaporated in a vacuum oven (50 °C) and the remainders were resuspended in 0.5 mL 100% methanol. Then, 100 uL of the sample were gently transferred into a HPLC vial insert and 5 uL were injected into HPLC for OLA quantification. Under this condition, the retention time for OLA is 10.8 min.
We also observed that the pH of the fermentation media dramatically dropped to below 3.5 during the fermentation process, due to the accumulation of organic acids. This decreased pH or increased proton concentration may negatively affect membrane permeability and strain physiology 32, 36 . We next sought to control the medium pH by using either PBS buffer or CaCO3 36 . Supplementation of 20 g/L CaCO3 maintained stable pH and increased OLA titer by threefold, reaching 5.86 mg/L at 96 h, whereas PBS failed to improve OLA production (Figure 5a). We speculate that the use of PBS buffer may shift the cell metabolism, for example, the cell may increase the phospholipid or cell membrane synthesis 37 , which is competing with our goal to synthesize OLA because both pathways share the same precursor malonyl-CoA.