Oleic acid based experimental evolution of Bacillus megaterium yielding an enhanced P450 BM3 variant

Abstract

Background Unlike most other P450 cytochrome monooxygenases, CYP102A1 from Bacillus megaterium (BM3) is both soluble and fused to its redox partner forming a single polypeptide chain. Like other monooxygenases, it can catalyze the insertion of oxygen unto the carbon-hydrogen bond which can result in a wide variety of commercially relevant products for pharmaceutical and fine chemical industries. However, the instability of the enzyme holds back the implementation of a BM3-based biocatalytic industrial processes due to the important enzyme cost it would prompt. Results In this work, we sought to enhance BM3’s total specific product output by using experimental evolution, an approach not yet reported to improve this enzyme. By exploiting B. megaterium’s own oleic acid metabolism, we pressed the evolution of a new variant of BM3, harbouring 34 new amino acid substitutions. The resulting variant, dubbed DE, increased the conversion of the substrate 10-pNCA to its product p-nitrophenolate 1.23 and 1.76-fold when using respectively NADPH or NADH as a cofactor, compared to wild type BM3. Conclusions This new DE variant, showed increased organic cosolvent tolerance, increased product output and increased versatility in the use of either nicotinamide cofactors NADPH and NADH. Experimental evolution can be used to evolve or to create libraries of evolved BM3 variants with increased productivity and cosolvent tolerance. Such libraries could in turn be used in bioinformatics to further evolve BM3 more precisely. The experimental evolution results also supports the hypothesis which surmises that one of the roles of BM3 in Bacillus megaterium is to protect it from exogenous unsaturated fatty acids by breaking them down.

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward large alkyl hydroxylation are shown (light green spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed, with refs: 1 (), 21 (), 25 (), 26 ( , , , , , ), 27 (), 28 (), 30 (), 47 ( , , , , , , , , , , , , , , , − ), 51 ( , , , , , , , ), 52 ( , , , , ), 55 (), 58 ( , , , ), 62 (), 67 (), 69 (), 70 (), 72 ( , , ), 73 ( , ), 74 ( , , , , , , , , , , , , , , , , ), 75 ( , , , , , , , , − ), 78 (…”

“… All positions of the P450 BM3 heme domain that have been mutated and show improvement toward large alkyl hydroxylation are shown (light green spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed, with refs: 1 ( 320 ), 21 ( 325 ), 25 ( 446 ), 26 ( 2 , 287 , 320 , 429 , 446 , 447 ), 27 ( 325 ), 28 ( 320 ), 30 ( 325 ), 47 ( 2 , 195 , 217 , 238 , 241 , 244 , 245 , 287 , 288 , 293 , 348 , 426 , 429 , 433 , 438 , 446 − 454 ), 51 ( 2 , 238 , 241 , 245 , 270 , 325 , 446 , 451 ), 52 ( 217 , 244 , 290 , 348 , 433 ), 55 ( 325 ), 58 ( 290 , 359 , 455 , 456 ), 62 ( 381 ), 67 ( 325 ), 69 ( 24 ), 70 ( 325 ), 72 ( 2 , 24 , …”

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward large alkyl hydroxylation are shown (light green spheres). Mutated positions in the heme domain (residues 1−471) and in the reductase domain (472−1049) are listed, with refs: 1 (320), 21 (325), 25 (446), 26(2,287,320,429,446,447), 27 (325), 28 (320), 30 (325), 47(2, 195, 217, 238, 241, 244, 245, 287, 288, 293, 348, 426, 429, 433, 438, 446−454), 51(2,238,241,245,270,325,446,451), 52(217,244,290,348,433), 55 (325), 58(290,359,455,456), 62(381), 67(325), 69(24), 70 (325), 72 (2, 24, 296), 73(195,313), 74(2,157,238,241,244,287,288,306,429,431,441,442,446,447,453,457,458), 75 (2, 12, 24, 166, 219, 309, 319, 452, 458−461), 78 (2, 12, 24, 217, 220, 244, 245, 296, 309, 319, 348,...…”

“…431, 441, 442, 446, 447, 451−468), 88(458), 94(217,244,245,348,426,433,438,452,454), 96 (278), 97(381,469,470), 100(290,359,455,456), 106 (290), 107(290,359,455,456), 108(195), 109(325,470), 119(325), 127 (320), 135(290,320,359,455,456), 138(220,244,245,424), 142(12, 217, 244, 245, 348, 426, 433, 438, 442, 452−454, 461, 462), 143(195), 145 (290, 325, 359, 455, 456), 148 (325), 156 (381), 171 (2, 238), 309, 325, 446), 267 (2, 195, 473), 268 (24, 166, 254, 291, 294), 274 (290, 359, 455, 456), 276 (2, 168, 238), 278 (325), 286 (166), 290(12,217,220,244,245,348,424,426,433,438,442,452,…”