Biotransformation Of Phytosterols To Androstenedione In Systems Containing Deep Eutectic Solvents

As the second-largest class of drugs on the world market,steroid drugs are of great medical and economic value.The whole-cell side-chain cleavage of phytosterols by microorganisms such as Mycobacterium sp.is known as an environmentally friendly method for the production of androstenedione(AD),which is an important intermediate to produce steroids.However,AD yield was limited by factors such as low water solubility of phytosterols,product inhibition and degradation,which was difficult to be applied in large-scale industrial production.In recent years,deep eutectic solvents(DESs)have been employed as a new kind of green solvent,which presented good performance in biotransformation.Actually,the whole-cell bioconversion involving multi-enzyme catalytic reactions is more challenging because a closed intracellular environment is not always available in the presence of DESs.In this work,biotransformation systems containing DESs were constructed for the production of AD via side-chain cleavage of phytosterols by Mycobacterium sp.MB 3683 resting cells.Meanwhile the mechanisms of action of DESs were also discussed in detail to enhance the biotransformation efficiency and promote the development of green process engineering.First,38 kinds of DESs with different hydrogen bond donors and hydrogen bond acceptors were synthesized and their physicochemical properties were evaluated relevant to phytosterols biotransformation by Mycobacterium sp.The stabilities,hydrophilicities,phytosterols solubilities of DESs and buffering performances of buffers containing DESs were determined.The organic acid components in DESs caused a complete loss of cellular biocatalytic activity by destroying the buffering capacity.In terms of hydrogen bond acceptors species,it was found that significantly higher biotransformation efficiencies were achieved by betaine-based DESs than those of choline chloride-based DESs.And in terms of hydrogen bond donors species,higher biotransformation efficiencies were achieved by non-glucose sugars as the hydrogen bond donors of DESs than others.Moreover,the trend towards higher biotransformation efficiency was linked to higher hydrophilicity of DESs.Ultimately,the results indicated that two kinds of DESs,i.e.Bet/Mal(betaine/maltose,with the molar ratio of 4:1)and Bet/Gal(betaine/galactose,with the molar ratio of 5:2),presented more significant enhancements on AD yield,which were available for further biocatalytic researches.Second,the effects of DESs types and their concentrations on AD yield during phytosterols biotransformation by Mycobacterium sp.resting cells were investigated,taking into account of the cell membrane integrity,cell morphology,cell viability,and AD accumulation and degradation in systems containing DESs.The results indicated the damage to cell membrane and cell viability by the choline chloride-based DESs was more severe than that by betaine-based DESs,which further led to the decreases in biotransformation efficiency.For betaine-based DESs,in systems with 5-10%(volume ratio,the same below)Bet/Mal or Bet/Gal,the cell membrane integrities were well maintained to provide a stable intracellular catalytic environment.The concentration of androsta-1,4-diene-3,17-dione(ADD)as the AD degradation intermediate decreased significantly during the biotransformation process,implying that the AD degradation pathway was significantly inhibited.The AD accumulation phase of the biotransformation could be extended to 48 h,thus the long-term and efficient accumulation of AD was ensured.Ultimately,a maximum AD yield enhancement of 38.9%was obtained in the 10%Bet/Gal system with the AD production of 0.670 g/L.Finally,the AD production by immobilized Mycobacterium sp.resting cells in systems with DESs were preliminarily investigated.The cryogel beads polymerized with 2-hydroxyethyl methacrylate and butyl methacrylate were selected as the immobilization carriers.By alleviating the loss of cell catalytic activity due to cell aggregation,a 6.8-fold higher AD yield per unit of cells than that of the suspension cultured resting cells biotransformation system was achieved.The ability of cryogel beads to mitigate cell membrane damage by DESs was observed,and DESs could also improve AD production by inhibiting product degradation as previously.While in the semi-continuous biotransformation process,the total AD yield per unit of cells of 401.4 mg/g DCW was achieved in the system containing 10%Bet/Mal after 3 cycles biotransformation,which was significantly higher compared to both the semi-continuous buffer system(266.7 mg/g DCW)or the suspension cultured resting cells buffer system(33.4 mg/g DCW).DESs are now generally acknowledged as a new class of green solvents similar to ILs in properties and designability,which have also the advantages of inexpensive raw materials and simple preparation.A significant positive effect on the biotransformation was exhibited by DESs in the process of side-chain cleavage of phytosterols by Mycobacterium sp.cells,which implied the potential application of DESs in the biocatalytic process for the production of steroid drug intermediates.