Recombinant Production and Biochemical Characterization of Thermostable Arabinofuranosidase from Acidothermophilic Alicyclobacillus Acidocaldarius

The complete enzymatic degradation of lignocellulosic biomass requires the cooperative action of cellulosic, hemicellulosic, and lignolytic enzymes such as cellulase, xylanase, laccase, galactosidase, and arabinofuranosidase. Arabinofuranosidases (E.C 3.2.1.55), which belong to the glycoside hydrolase family of enzymes, hydrolyze the 1,3- and 1,5-α-arabinosyl bonds in L-arabinose- containing molecules. L-arabinoses are present in hemicellulosic part of lignocellulosic biomass. Arabinofuranosidases also play an important role in the complete hydrolysis of arabinoxylans. Analysis of the genome project and CAZY database revealed two putative arabinofuranosidase genes in the A. acidocaldarius genome. The aim of the study was cloning, heterologous expression, purification and biochemical characterization of the arabinofuranosidase enzyme encoded in A. acidocaldarius genome. For this purpose, the AbfA gene of the arabinofuranosidase protein was cloned into the pQE-40 vector, heterologously expressed in E. coli BL21 GOLD (DE3) and successfully purified using His-Tag. Biochemical characterization of the purified enzyme revealed that A. acidocaldarius arabinofuranosidase exhibited activity over a wide pH and temperature range with optimum activity at 45 ºC and pH 6.5 in phosphate buffer towards 4-nitrophenyl-α-L-arabinofuranoside as the substrate. In addition, the enzyme is highly stable over wide range of temperature and maintaining 60% of its activity after 90 min of incubation at 80 ºC. Through the bioinformatics studies, the homology model of A. acidocaldarius arabinofuranosidase was generated and the substrate binding site and residues located in this site were identified. Further molecular docking analysis revealed that the substrate located in the catalytically active pose and, residues N174, E175, and E294 have direct interaction with 4-nitrophenyl-α-L-arabinofuranoside. Moreover, based on phylogenetic analysis, A. acidocaldarius arabinofuranosidase exists in the sub-group of intracellular arabinofuranosidases, and G. stearothermophilus and B.subtilis arabinofuranosidases are close relatives of A. acidocaldarius arabinofuranosidase. This is the first study to report the gene cloning, recombinant expression and biochemical and bioinformatic characterization of an auxiliary GH51 arabinofuranosidase from an acidothermophilic bacterium A. acidocaldarius. © 2023, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature

Synthesis of oxo-fatty acid esters in a whole cell cascade reaction with engineered monooxygenase (P450 BM3) and dehydrogenase (cpADH5) variants

Hydroxyl or ketone functionalized fatty acid methyl esters are important compounds for production of pharmaceuticals, cosmetics or dietary supplements. For instance, 3-hydroxyhexanoate or its methyl ester is used in the synthesis of laulimalide which is an anticancer drug. Biocatalysis through enzymatic cascades has drawn attention for the development of more efficient, sustainable, and greener synthetic processes to produce complex valuable chemical compounds. Furthermore, whole cell catalysts which employs enzyme cascade offers important advantageous over classical chemical catalysis and enzyme based biotransformation such as; cofactor regeneration by the cell metabolism, omission of protein purification steps and increased enzymes. In this thesis, a whole cell catalyst which includes a P450 BM3 and cpADH5 coupled cascade reaction was developed for the synthesis of hydroxy- or keto- fatty acid methyl esters, which is a greener route for production. P450 BM3 monooxygenase from Bacillus subtilis and alcohol dehydrogenase from Candida parapsilosis (cpADH5) are powerful enzymes and have been used for the synthesis of many industrially relevant compounds. However, P450 BM3 and cpADH5 poorly accept fatty acid methyl esters and hydroxy-fatty acid methyl esters as substrate respectively. P450 BM3 and cpADH5 enzymes were engineered in order to enable the conversion of methyl hexanoate by P450 BM3 and methyl 3-hydroxyhexanoate by cpADH5. In chapter I, the directed evolution of P450 BM3 was performed by employing the KnowVolution approach. The P450 BM3 YE_M1_2 variant exhibited boosted performance toward methyl hexanoate. Initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 32 fold higher when compared to WT enzyme. Furthermore, 1.5 fold increased methyl 3-hydroxyhexanoate production was obtained with YE_M1_2 variant (YE_M1_2; 2.75 mM and WT; 1.8 mM). In addition, a second improved P450 BM3 variant named as YE_M3_1 showed 5.4 fold improved initial oxidation rate (YE_M3_1; 1085 min-1 and WT; 146 min-1) in comparison to WT and 1.6 fold increased total product formation when compared to P450 BM3 F87A/A328I parental variant (YE_M3_1; 1.3 mM and F87A/A328I; 0.8 mM). cpADH5 enzyme was engineered through computational assisted approach for conversion of methyl 3-hydroxyhexanoate into corresponding ketone. Molecular docking studies revealed that W286 is located in the small binding pocket and limits the access to substrates that contain aliphatic chains longer than ethyl substituent. In addition to W286, L119 residue was mutated as well through site saturation mutagenesis. Experimental findings showed that, L119 and W286 are key residues to boost oxidation of medium chain methyl 3-hydroxy fatty acids. Kinetic characterization of W286A showed a 5.5 fold increase of Vmax and a 9.6 fold decrease of Km values toward methyl 3-hydroxyhexanoate (Vmax: 2.48 U/mg and Km: 4.76 mM). Simultaneous saturation at positions 119 and 286 yielded a double mutant (L119M/W286S) with more than 30 fold improved activity toward methyl 3-hydroxyoctanoate (WT: no conversion; L119M/W286S: 30 %). Interestingly, L119M/W286S variant exhibited inverted enantiopreference (S-enantiomer ≥ 99% activity decrease and R-enantiomer > 20 fold activity improvement) toward methyl 3-hydroxybutyrate. Additionally, a comparison of the mutagenesis methods Iterative Saturation Mutagenesis (ISM) and OmniChange was performed. cpADH5 enzyme was engineered through multiple site saturation mutagenesis for oxidation of methyl 3-hydroxyhexanoate into corresponding ketone. The residues; C57, W116, L119, and W286 which lie in substrate binding pocket of cpADH5 were selected through computational analysis and from literature. Selected positions were mutated through ISM and OmniChange. Furthermore the employed methods as well as the obtained activity improvements were compared. The best variant (C57V/W286S) obtained from OmniChange showed 108 fold improved activity while the best variant (W286A) from ISM showed 82 fold improved methyl 3-hydroxyhexanoate oxidation in comparison to cpADH5 WT. Moreover, molecular docking analysis also supported the superiority of OmniChange variant. The cpADH5 C57V/W286S variant had 0.77 kcal/mol less binding energy (W286A: -4.74 kcal/mol and C57V/W286S: -5.51 kcal/mol) and decrease in catalytically important distances. Finally, the whole cell catalyst for synthesis of methyl 3-hydroxyhexanoate or methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. Under the optimized process conditions, the whole cell catalyst produced up to 2 mM total product after 48 h., Biotransformation with crude cell lysate yielded 3.8 mM total product after 48 h with almost 40% conversion rate. A whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate or methyl 3-oxohexanoate was successfully established by co-expressing P450 BM3 and cpADH5 engineered variants