Synthesis of chiral diols relevant for biofuel and fine chemical production using synthetic enzyme cascades in diverse reaction conditions
Spöring, Jan-Dirk; Rother, Dörte (Thesis advisor); Blank, Lars M. (Thesis advisor)
Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2022
Research in the field of biocatalysis has been increasing a lot in the last decades, especially due to accomplishments in genetic engineering. The motivation to use biocatalysis for the synthesis of (fine-) chemicals is the inherently high regio-, chemo-, and stereoselectivity that most enzymes have. A challenge for the application of enzyme in chemical processes was, and sometimes still is, the instability of the enzymes under process conditions, especially towards organic solvents. Most enzyme classes are evolved to be active in aqueous reaction systems and denature in organic solvents in their purified form. A change in the enzyme formulation to, e.g. lyophilized whole cells enables the enzymes to stay more stable in unconventional reaction media and opens new opportunities for the application of enzymes for organic syntheses. In this thesis, conventional and unconventional reaction media were used for the synthesis of symmetric, aliphatic, vicinal diols from biobased aldehydes. The substrates acetaldehyde, propanal, and butanal were used for a two-step enzymatic cascade employing first a carboligase and in a second step an oxidoreductase for the synthesis of 2,3 butanediol, 3,4-hexanediol, and 4,5-octanediol, respectively. In aqueous medium, several different carboligases and oxidoreductases were screened for their substrate spectrum, activity, and stereoselectivity towards the product, and in case for the oxidoreductases, also for the substrate. By combining different lyases with oxidoreductases in a modular approach, eight out of nine possible stereoisomers were successfully produced with good to excellent isomeric contents (between 68 % and >99 %) and with decent yields (between 8.2 % and 54 %) for many cases. To show that the yields and product concentrations can be improved, (4S,5S) octanediol was used as a case study for optimization of these metrics. The bottleneck of the cascade was identified to be the thermodynamic equilibrium of the reduction reaction, which lied on the substrate side. Therefore, increased cosubstrate concentrations were applied together with an increased pH, which favors the oxidation reaction of the cosubstrate. This increased the yield from 34 % to 86 % and an upscaling was performed, in which 127 mM product was produced with excellent stereoselectivity. The downstream processing proved to be rather laborious in the aqueous environment, since an organic extraction needed to be performed to separate the hydrophobic product from the hydrophilic cosubstrate, coproduct, and cell debris such as proteins from the whole cells. The latter formed an interphase during the organic extraction, which is why a protein precipitation step using HCl was performed prior to the extraction. These steps could be omitted, next to other advantages, by switching from an aqueous to a micro-aqueous reaction system with cyclopentyl methyl ether (CPME) as the main solvent. All three cascades were tested in both aqueous and micro-aqueous conditions and were found to be successful in both systems. Interestingly, the hydrophilic acetaldehyde yielded higher product concentrations in the aqueous system in comparison to the organic solvent system. This was likely due to the partitioning coefficient of the reactive and in high concentrations toxic acetaldehyde, which lied on the side of the aqueous phase and thus directly at the enzyme in the organic system. Thus, much higher concentrations of the reactive acetaldehyde were present around the enzyme and thus inactivated it. The reverse effect was observable for the more hydrophobic butanal, where the organic system resulted in higher yields than the aqueous system, probably due to the higher substrate solubility and thus availability in the organic system. Because butanal shows higher solubility in the organic system, higher substrate concentrations of up to 800 mM could be used in a scale up experiment in a 200 mL scale, which resulted in butyroin concentrations of over 250 mM and product concentrations of 79 mM meso-4,5-octanediol. The downstream processing from organic conditions proved to be simpler than from the aqueous system since the organic extraction could be omitted. Also, the specific energy demand for the product purification using distillation was lowered by 79 % by using unconventional media such as organic solvents or biphasic media. Finally, CPME was used for the connection of biocatalysis to chemocatalysis for the synthesis of cyclic acetals from simple, bio-based aldehydes. These hybrid systems can improve organic syntheses greatly by combining the benefits of both bio- and chemocatalysis such as selectivity and broad applicability, respectively. In general, the reaction conditions of both kinds of catalysts differ vastly, in this case especially due to the water susceptibility of the ruthenium catalyst and the low necessary H2 and CO2 solubility. Here, CPME was successfully shown to be a suitable solvent for the synthesis of both aliphatic diols such as 3,4-hexanediol and 4,5-octanediol from aldehydes using enzymes and 1,3-dioxolanes from these diols by a ruthenium catalyst with formate or CO2 as a C1 source and H2. Overall, enzymatic two-step cascades were used for the synthesis of aliphatic, vicinal diols in aqueous and unconventional organic reaction solvents. Both very stereospecific synthesis and high product concentrations could be reached. Organic solvents facilitated not only downstream processing, but were also used for the connection of bio- and chemocatalysis. This system can be used in the future for the connection of various enzymatic reactions with different water-susceptible chemocatalysts for the synthesis of more complex and valuable molecules.
- Department of Biology 
- Synthetic Enzyme Cascades Teaching and Research Area (FZ Jülich)