Team:Evry Paris-Saclay/HP/Annexe/Antonin

IGEM Evry Paris-Saclay



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 industry managed developed its knowledge to obtain compounds in a rational manner thanks to microorganism.

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 for Sigma Aldrich and between 35 and 60$ per kilogram for All-u-Lose®, a food grade product.

At the time, it appears that no industry is currently producing psicose using bioproduction. However, there have been many reports on D-psicose production within microbial organisms containing their own D-psicose 3-epimerases [2, 3, 4, 5]. But most of these organisms don’t correspond to the FDA’s standards. For instance, Gram-negative bacteria such as E. coli have at their surface some LPS, which has a strong immunogenicity. Some strains could also excrete toxins that could initiate food poisoning. Therefore, industries 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.

Psicose bioproduction can be made in whole cells by adding the substrate and using the optimal conditions for cell growth [6]. There is 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.

To enhance the yields of this production, it is possible to lysate the cells to release all the enzymes [7]. After a first step where the recombinant protein is produced inside the bacteria, 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 biocatalysis can start with the optimal conditions for the enzyme to ensure greater yields. The D-psicose-3epimerase can be fixed to maximize the interaction with the substrate.

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 [8]. 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.

Another method is called cell free bioproduction [9]. 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] Oh D. K et al. D-Psicose production from D-fructose using an isolated strain, Sinorhizobium sp. World J. Microbiol. Biotechnol. (2007) 23, 559e563
  • [3] Zhang W et al. Characterization of a d-psicose 3-epimerase from Dorea sp. CAG317 with an acidic pH optimum and a high specific activity. Journal of Molecular Catalysis. (2015) Volume 120:68-74
  • [4] Chan HC et al. Crystal structures of d-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars. Protein Cell. (2012) 3:123–131
  • [5] Choi JG et al. 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
  • [6] Chen et al. A food-grade expression system fro D-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker. Bioresour. Bioprocess. (2017) 4:9
  • [7] Zhu et al. Overexpression of D-psicose 3-epimerase from Ruminococcus sp. In Escherichia coli and its potential application D-psicose production. Biotechnol Lett (2012) 34:1901–1906
  • [8] Park C-S et al. 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(7)
  • [9] Rollin, Joseph & Kin Tam, Tsz & Zhang, Yi-Heng Percival. New Biotechnology Paradigm: Cell-Free Biosystems for Biomanufacturing. Green Chem. (2013)
  • Yoshihara A et al. Purification and characterization of D-allulose 3-epimerase derived from Arthrobacter globiformis M30, a GRAS microorganism. Journal of Bioscience and Bioengineering. (2016).

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