The article we tweeted today was published in "B." on Biotechnol Lett
yproduct-free geraniol glycosylation by whole-cell biotransformation with recombinant E
scherichia coli", written by Xenia Priebe et al. at the Institute of Chemical Engineering of Industrial Students in Munich, Germany.
Geraniol is a very important fragrance in the consumer goods industry and can be glucosized by the UDP-glucose-dependent glucose-dependent glucose transferase VvGT14a glucosamine in grapes to obtain more stable geran glycosides. E. coli expressing VvGT14a is a convenient whole-cell biocatalyst for this biotransformation due to its inherent UDP-glucose regenerative capacity. The low water solubility and high cytotoxicity of geraniol can be overcome in two-phase systems, where the non-aqueous phase is used as an in situ substrate reservoir. However, the effects of different process variables on bipolar whole-cell biotransformation are unclear. Therefore, the goal of the study was to identify potential bottlenecks in the biotransformation process by providing geraniol in situ by isopropyl myristate as a second non-aqueous phase. ResultsInstately, insufficient glucose supply can be ruled out by measuring intracellular glucose concentration. Instead, the oxygen supply is identified as a bottleneck. In addition, chloramphenicol acetyltransferase-generating by-product coumeryl acetate is considered a limiting factor for high yield. The use of a whole-cell biocatalyst lacking CAT prevents the formation of coumyl acetate, and 100% selectively obtains geranin during L-scale biotransformation.
Supply of intracellular UDP-glucose during geraniol biotransformation
In order to better understand the supply of UDP-glucose during the bulk bioconversion of geraniol in E. coli pLysS, in the process of biotransformation on the mL scale, UDP-glucose and its metabolite UDP are quantified intracellularly, without the intermittent process of adding geraniol as a reference, the conclusion must be expressed carefully due to the rather low amount of data. However, the authors believe that certain trends can be observed: for both the biotransformation and reference processes, the qualitative and quantitative processes of UDP-glucose are similar: it falls within the first 10 hours of the process, followed by a fairly constant process. For UDP-glucose as substrates, all measured concentrations were well above 16uM. During the first 10 hours of biotransformation, UDP levels increase and subsequently decrease. A reduction in the reference process can also be observed; however, there is no data at the first sample point. The UDP concentrations of all assays are well known to be lower than the semi-maximum inhibitory UDP concentration of VvGT14a of 600uM. During biotransformation, the concentration of geranyl glucoside increases linearly over the first 12 h and remains unchanged thereafter. However, based on the data provided here, it can be ruled out that insufficient supply of UDP-glucose is the cause of the stagnation of gesporin concentrations. As a result, a different bottleneck seems to exist.
The role of oxygen supply in the biotransformation of geraniol
This bottleneck manifests itself in the availability of oxygen during biotransformation. Oxygen-limiting conditions 3 h before bioconversion result in an increase in the conversion rate of glinideol, forming up to 4 g · Strong acetate of L-1 and a pH decreases from pH 6.5 to pH 5.5. By increasing the agitator speed from 2200 rpm to 4000 rpm on the milliliter level, an oxygen-rich reaction environment can be generated, resulting in 0.8 g · Complete conversion of L-1 geraniol, complete inhibition of acetate formation and less pronounced reduction of pH. In addition, cell death can be avoided: although cells with a process with a lower stirring rate enter the death phase near the end of the process, the biological catalysts of experiments with higher stirring speed transition to a stable phase after apparent cell growth within the first 8 h. However, despite the complete conversion of geraniol, the yield of geraniol glucoside reached only 49.6% (based on the range from 0.8 g L-1 geraniol to 1.64 g · L-1 maximum available product concentration), indicating the formation of by-products. In fact, as speculated in the introduction, the formation of geranimate can be confirmed. Geranimate accumulates only in isopropyl myristate, not in the aqueous phase.
Eliminates the formation of by-products during geraniol biotransformation
It is well known that in the biocatalyst used here, CAT encoded on the plasmid pLysS can catalyze acetylation of geraniol. Thus, the modified strain E. coli pLysSA, which replaced the CAT gene with the alpha β-lactamase gene, was studied for their respective by-product formations. The strain did not produce any geroxylate, proving that CAT is responsible for the formation of geroxylate. Both the original and improved strains showed 37 mg · Rate of formation of the same initial volume of coriain at L-1h-1. However, in order to obtain this rate with modified strains, the expression of VvGT14a must be performed with 1 mm IPTG instead of 0.1 mm IPTG, which is used for the original strain during high cell density culture. The authors further tested the results of applying different medium additives during biotransformation with improved strains, particularly due to the weaker expression of VvGT14a compared to the original strain. One of them was the addition of kanamycin to a final concentration of 0.03 g·h-1, however this did not lead to higher product formation. This indicates that there is no problem with plasmid loss during biotransformation.
The confounding of CAT was further verified by comparing the acetylation capacity of different alcohol substrates of the two E. coli strains BL21 (DE3) and BL21 (DE3) strains. Chloramphenicol is used as the reference substrate, and benzyl alcohol, 2-phenethyl alcohol, vanillin, geraniol and linalool are acetylated substrates. BL21(DE3)pLysS has the highest activity against chloramphenicol. In addition, the strain also acetylated all substrates except linalool. BL21 (DE3) is either completely inactive to the substrate or has little activity against benzyl alcohol and geraniol, which is significantly lower than BL21(DE3)pLysS.
0.4 L scale non-byproduct-free geranin production
Since E. coli pLysSA is a suitable strain for geraniol-free glucosylation, the authors investigated whether the concentration of geranyl glucoside could be further increased during biotransformation in a 0.4 L scale stirred tank reactor. Instead of batched glucose, glucose feed is applied, as if the feed shows a favorable effect on reducing acetate formation. In addition, the biotransformation temperature ranged from 30 °C to 37 °C and 16 g · Increased biocatalyst concentration of L-1. Throughout the process, linear geranyl glucoside is formed, with a geraniol glycosylation selectivity of 100%. Due to insufficient power inputs to the reactor system used, dissolved oxygen cannot maintain an air saturation of more than 30% throughout the feed batching process. However, by operating under glucose-restricted conditions, pronounced acetate formation can be avoided. The initial high concentration of acetate degrades rapidly within 10 hours. The biocatalyst shows slight cell growth and after 48 h the final concentration of 930 mg l-1 is obtained with a final concentration of 930 mg l-1 of geranyl glucoside. The conversion rate of geraniol is 30%, and the yield of geranilin is 28%. Still, because geraniol remains and products form a linear trend, biotransformation seems to be able to continue beyond the application process time. In addition, experiments have shown that carbon-derived glucose does not have to be supplied in excess batches as described earlier, but can be added to the reactor. However, the biocatalyst should not be transferred to a quiescent state, as this can negatively affect the bioconversion of geraniol. Typically, about 70-80% of the glucose provided is used to maintain cellular processes, 15-20% is used for the formation of biomass, and 2% is required for the formation of geranyl glucoside.
conclusion
The study was the first to analyze in detail the whole-cell biotransformation of geraniol by Escherichia coli expressing UDP-glucose-dependent glucose transferase, which can serve as the best starting point for the design of other glycosylation processes.
Finishing: High
Article link https://doi.org/10.1007/s10529-020-02993-z