Team:Evry Paris-Saclay/HP/Annexe/Antonin
Rare Sugar Bioproduction - State of the Art
Bioproduction is the synthesis of molecules of interest based on living organisms. This can be achieved in macroorganisms such as mice with the production of antibodies, growth factors or even vaccines. On the other hand, industrials typically use microorganisms and exploit the fermentative capacities of particular species like Saccharomyces cerevisiae or Escherichia coli. Throughout the years, industrialists managed to develop the knowledge to obtain compounds in a rational manner thanks to microorganisms.
For our project, we focused our work on the bioproduction of a rare sugar, the D-Psicose or D-Allulose, a C3 epimer of D-Fructose. Its chemical production is complex (Click here to find more) but it could be easily bioproduced with fructose as a substrate. The reaction requires a D-Psicose 3-epimerase (EC: 5.1.3.30) or D-tagatose 3-epimerase (EC: 5.1.3.31) to convert fructose into psicose [1]. Today, psicose can be found at $1000 per gram form Sigma Aldrich and between $35 and $60 per kilogram form All-u-Lose®, a food grade product.
As of today, it appears that no industrialist is currently producing psicose using bioproduction. However, there have been many reports on D-psicose production using D-psicose 3-epimerase (Dpe) from few microbial organisms, one of the first being the D-tagatose 3-epimerase from Pseudomonas cichorii [2]. Psicose bioproduction could be made in whole cell cultures by adding the substrate and using the optimal conditions for cell growth. But most of the organisms expressing these Dpe don’t correspond to the FDA’s standards. For instance, Gram-negative bacteria such as E. coli have at their surface some LPS, which have a strong immunogenicity. Some strains could also excrete toxins that could initiate food poisoning. Therefore, industrialists have to insert the epimerase from these strains into Generally Recognized As Safe (GRAS) strains such as Bacillus subtilis, which have been authorized by the FDA for food production [3]
There are many culture methods, with three main types: Batch, Fed Batch and Chemostat, depending on how the substrate is added to the culture. The problem with whole cell, is that most D-psicose 3-epimerases have an optimal work temperature of 60 to 80°C [1], which is deadly for any bioproduction microorganism. This technique doesn’t provide the best yields but requires little material and allows a strong resistance against environmental perturbations. It also limits the need of purification steps by centrifuging the bacteria, therefore removing a great portion of byproducts. However, we have found no publication describing a whole cell culture production of psicose where the enzyme and the sugar are produced in living cells.
To enhance the yields of this production, it is possible to lysate the cells to release all the enzymes for further bioconversion [4, 5, 6]. After a first step where the bacteria are grown to express the recombinant protein, it can be purified thanks to multiple extraction methods. Membranes can be broken by mechanical force with an Amico-French press, by sonication or even with glass beads inside a bead beater. The goal here is to recover the enzyme and purify it. Therefore, it is better to avoid using lytic enzymes which could be unsuitable for consumption. After this purification, the conversion can start with the optimal conditions for the enzyme to ensure greater yields, like 23% at 70°C [5]. The D-psicose 3-epimerase can be fixed to maximize the interaction with the substrate, which allowed an incredible yield of 70% at 45°C with a mutated Dpe [6].
To keep the advantages of whole cell production, stability, resistance to environmental perturbations, avoiding purification steps, and still enhance the production, cells can be permeabilized [7]. The permeabilization can be performed with detergents, solvents (acetone, chloroform, ethanol, methanol, toluene), salts and chemicals such as EDTA. By piercing the membrane, the substrate and product are allowed to transfer through the cell, therefore maximizing the reaction. The conversion rate of fructose into psicose could be doubled with this technique, for instance a yield of 31% at 65°C [7].
Another method is called cell free bioproduction [8]. This technique does not rely on living organisms and allows fast and controlled protein synthesis at a low price. This can be achieved by adding a plasmid coding for a recombinant protein to a cell lysate or purified enzymes and coenzymes. The transcription and translation machineries have their activity without the cell structure. Therefore, there is no formation of byproducts and the substrate cannot be used for biomass synthesis, increasing the production yields.
References
- [1] Van Overtveldt S, Verhaeghe T, Joosten HJ, van den Bergh T, Beerens K, Desmet T. A structural classification of carbohydrate epimerases: From mechanistic insights to practical applications. Biotechnol Adv (2015) 33, 1814-1828.
- [2] Itoh H, Okaya H, Khan AR, Tajima S, Hayakawa S, Izumori K. Purification and characterization of D-tagatose 3-epimerase from Pseudomonas sp. ST-244. Biosci Biotechnol Biochem (1994) 58, 2168-2171.
- [3] Chen J, Jin Z, Gai Y, Sun J, Zhang D. A food-grade expression system for D-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker. Bioresour Bioprocess (2017) 4, 9.
- [4] Zhu Y, Men Y, Bai W, Li X, Zhang L, Sun Y, Ma Y. Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production. Biotechnol Lett (2012) 34, 1901-1906.
- [5] Zhang W, Li H, Zhang T, Jiang B, Zhou L, Mu W. Characterization of a D-psicose 3-epimerase from Dorea sp. CAG317 with an acidic pH optimum and a high specific activity. J Mol Catal B: Enzymatic (2015) 120, 68-74.
- [6] Choi JG, Ju YH, Yeom SJ, Oh DK. Improvement in the thermostability of D-psicose 3-epimerase from Agrobacterium tumefaciens by random and site-directed mutagenesis. Appl Environ Microbiol (2011) 77, 7316-7320.
- [7] Park CS, Kim T, Hong SH, Shin KC, Kim KR, Oh DK. D-Allulose production from D-fructose by permeabilized recombinant cells of Corynebacterium glutamicum cells expressing D-allulose 3-epimerase Flavonifractor plautii. PLoS One (2016) 11, e0160044.
- [8] Rollin JA, Kin Tamb T, Zhang YHP. New biotechnology paradigm: cell-free biosystems for biomanufacturing. Green Chem (2013) 15, 1708-1719.
