Enantioselective Hydrolysis of Racemic and <i>Meso</i>-Epoxides with Recombinant <i>Escherichia coli</i> Expressing Epoxide Hydrolase from <i>Sphingomonas</i> sp. HXN-200: Preparation of Epoxides and Vicinal Diols in High <i>ee</i> and High Concentration

A unique epoxide hydrolase (SpEH) from <i>Sphingomonas</i> sp. HXN-200 was identified and cloned based on genome sequencing and expressed in <i>Escherichia coli</i>. The engineered <i>E. coli</i> (SpEH) showed the same selectivity and substrate specificity as the wild type strain and 172 times higher activity than <i>Sphingomonas</i> sp. HXN-200 for the hydrolysis of styrene oxide <b>1</b>. Hydrolysis of racemic styrene oxide <b>1</b>, substituted styrene oxides <b>3</b>, <b>5</b>–<b>7</b>, and <i>N</i>-phenoxycarbonyl-3,4-epoxypiperidine <b>8</b> (200–100 mM) with resting cells of <i>E. coli</i> (SpEH) gave (<i>S</i>)-epoxides <b>1</b>, <b>3</b>, <b>5</b>–<b>7</b> and (−)-<b>8</b> in 98.0–99.5% enantiomeric excess (<i>ee</i>) and 37.6–46.5% yield. Hydrolysis of cyclopentene oxide <b>9</b>, cyclohexene oxide <b>10</b>, and <i>N</i>-benzyloxycarbonyl-3,4-epoxypyrrolidine <b>11</b> (100 mM) afforded the corresponding (<i>R</i>, <i>R</i>)-vicinal <i>trans</i>-diols <b>12</b>–<b>14</b> in 86–93% <i>ee</i> and 90–99% yield. The <i>ee</i> of (1<i>R</i>, 2<i>R</i>)-cyclohexane-1,2-diol <b>13</b> was improved to 99% by simple crystallization. These biotransformations showed high specific activity (0.28–4.3 U/mg cdw), product concentration, product/cells ratio, and cell-based productivity. Hydrolysis at even higher substrate concentration was also achieved: (<i>S</i>)-<b>1</b> was obtained in 430 mM (51 g/L_org) and 43% yield; (1<i>R</i>, 2<i>R</i>)-<b>13</b> was obtained in 500 mM (58 g/L) and >99% yield. Gram-scale preparation of epoxides (<i>S</i>)-<b>1</b>, (<i>S</i>)-<b>3</b>, (<i>S</i>)-<b>6</b> and diols (1<i>R</i>, 2<i>R</i>)-<b>12</b>, (1<i>R</i>, 2<i>R</i>)-<b>13</b>, (3<i>R</i>, 4<i>R</i>)-<b>14</b> were also demonstrated. <i>E. coli</i> (SpEH) cells showed the highest enantioselectivity to produce (<i>S</i>)-<b>1</b> (<i>E</i> of 39) among all known EHs in the form of whole cells or free enzymes and the highest enantioselectivities to produce (<i>S</i>)-<b>3</b>, <b>5</b>, <b>6</b>, <b>7</b>, (−)-<b>8</b>, and (<i>R</i>, <i>R</i>)-<b>14</b> (<i>E</i> of 36, 35, 28, 57, 22, and 28) among all known EHs. The easily available and highly active <i>E. coli</i> (SpEH) cells are the best biocatalysts known thus far for the practical preparation of these useful and valuable enantiopure epoxides and vicinal diols via hydrolysis

Enhancing Gastrodin Production in <i>Yarrowia lipolytica</i> by Metabolic Engineering

Gastrodin, 4-hydroxybenzyl alcohol-4-O-β-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica

Enhancing Enantioselectivity and Productivity of P450-Catalyzed Asymmetric Sulfoxidation with an Aqueous/Ionic Liquid Biphasic System

Biocatalytic asymmetric sulfoxidation represents a green method to prepare the useful and valuable enantiopure sulfoxides, but this method sometimes suffers from unsatisfied enantioselectivity and low productivity due to substrate and product inhibitions. Here we developed an aqueous/ionic liquid (IL) biphasic system for simultaneously enhancing the enantioselectivity and productivity of P450 monooxygenase-catalyzed asymmetric sulfoxidations of sulfides <b>1</b>, <b>3</b>, <b>5</b>, <b>7</b>, and <b>9</b>, as the first example of this kind for a biooxidation. Escherichia coli (P450pyrI83H-GDH) coexpressing P450pyrI83H monooxygenase and glucose dehydrogenase was engineered for the asymmetric sulfoxidations with cofactor recycling, giving higher <i>R</i>-enantioselectivity than any other known P450 monooxygenases and showing high specific activities. The inhibition to the reactions and the toxicity to the cells of the substrates and products were investigated and mostly avoided by using a KP buffer/[P_6,6,6,14]­[NTf₂] biphasic reaction system, in which the IL showed excellent biocompatibility to the cells and high solubility to the substrates and products. Sulfoxidations of <b>1</b>, <b>3</b>, <b>5</b>, <b>7</b>, and <b>9</b> with the resting E. coli cells in the biphasic system increased the product concentration from 9.4 to 20 mM for (<i>R</i>)-phenyl methyl sulfoxide <b>2</b>, from 1.9 to 9.9 mM for (<i>R</i>)-4-fluorophenyl methyl sulfoxide <b>4</b>, from 5.4 to 16 mM for (<i>R</i>)-ethyl phenyl sulfoxide <b>6</b>, from 4.2 to 22 mM for (<i>R</i>)-methyl <i>p</i>-tolyl sulfoxide <b>8</b>, and from 5.7 to 24 mM for (<i>R</i>)-methyl <i>p</i>-methoxyphenyl sulfoxide <b>10</b>, respectively, and improved the product ee from 85 to 99% for (<i>R</i>)-<b>2</b>, from 80 to 98% for (<i>R</i>)-<b>4</b>, from 88 to 96% for (<i>R</i>)-<b>6</b>, from 35 to 62% for (<i>R</i>)-<b>8</b>, and from 53 to 67% for (<i>R</i>)-<b>10</b>, respectively. The enhancements in enantioselectivity are possibly caused by the low substrate concentrations in the aqueous phase of the biphasic system. Preparative sulfoxidations to produce the useful and valuable sulfoxides (<i>R</i>)-<b>2</b>, (<i>R</i>)-<b>4</b>, and (<i>R</i>)-<b>6</b> in 99%, 98%, and 96% ee, respectively, were demonstrated

A Designed Chemoenzymatic Route for Efficient Synthesis of 6‑Dehydronandrolone Acetate: A Key Precursor in the Synthesis of C7-Functionalized Steroidal Drugs

Dehydronandrolone acetate (3) is a crucial precursor for the synthesis of C7-functionalized steroidal drugs. However, the current production method involves a laborious and environmentally unfriendly five-step chemical process, resulting in a low efficiency. To overcome this, we report a chemoenzymatic strategy, involving a one-pot biocatalytic C7β-hydroxylation/C17β-ketoreduction of 19-norandrostenedione (1) by combination of P450 monooxygenase and 17-ketosteroid reductase to generate C7β-hydroxynandrolone (2) as an intermediate, followed by a one-pot chemical dehydration and esterification to form 3. Impressively, the gram-scale synthesis of 3 was achieved with 93% isolated yield, which outperforms the traditional chemical approach (68% yield), thereby signaling great potential for industrial applications

Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

Unspecific peroxygenases (UPOs), secreted by fungi, demonstrate versatility in catalyzing challenging selective oxyfunctionalizations. However, the number of peroxygenases and corresponding variants with tailored selectivity for a broader substrate scope is still limited due to the lack of efficient engineering strategies. In this study, a new unspecific peroxygenase from Coprinopsis marcescibilis (CmaUPO) is identified and characterized. To enhance or reverse the enantioselectivity of wildtype (WT) CmaUPO catalyzed asymmetric hydroxylation of ethylbenzene, CmaUPO was engineered using an efficient superfolder-green-fluorescent-protein (sfGFP)-mediated secretion system in Escherichia coli. Iterative saturation mutagenesis (ISM) was used to target the residual sites lining the substrate tunnel, resulting in two variants: T125A/A129G and T125A/A129V/A247H/T244A/F243G. The two variants greatly improved the enantioselectivities [21% ee (R) for WT], generating the (R)-1-phenylethanol or (S)-1-phenylethanol as the main product with 99% ee (R) and 84% ee (S), respectively. The sfGFP-mediated secretion system in E. coli demonstrates applicability for different UPOs (AaeUPO, CciUPO, and PabUPO-I). Therefore, this developed system provides a robust platform for heterologous expression and enzyme engineering of UPOs, indicating great potential for their sustainable and efficient applications in various chemical transformations

Food-Grade Expression of Two Laccases in Pichia pastoris and Study on Their Enzymatic Degradation Characteristics for Mycotoxins

Mycotoxin contamination poses substantial health risks to humans and animals. In this study, the two laccases PpLac1 and AoLac2 from Pleurotus pulmonarius and Aspergillus oryzae were selected and heterologously expressed in Pichia pastoris in a food-grade manner to detoxify aflatoxin B1 (AFB1), zearalenone (ZEN), and deoxynivalenol (DON). Both laccases exhibited degradation activity toward these three mycotoxins, while the efficiency of these for DON was relatively low. Therefore, molecular docking between these laccases and DON was conducted to analyze their potential interaction mechanisms. Furthermore, the degradation conditions of AFB1 and ZEN by the two laccases were optimized, and the optimal degradation rates for AFB1 and ZEN by PpLac1 reached 78.51 and 78.90%, while those for AFB1 and ZEN by AoLac2 reached 72.27 and 80.60%, respectively. The laccases PpLac1 and AoLac2 successfully transformed AFB1 and ZEN into the compounds AFQ1 and 15-OH-ZEN, which were 90 and 98% less toxic than the original compounds, respectively. Moreover, the culture supernatants demonstrated effective mycotoxin degradation results for AFB1 and ZEN in contaminated feed samples. The residual levels of AFB1 and ZEN in all samples ranged from 6.61 to 8.72 μg/kg and 3.44 to 98.15 μg/kg, respectively, and these levels were below the limit set by the European Union standards. All of the results in this study indicated that the two laccases have excellent application potential in the feed industry

Structure-Guided Triple-Code Saturation Mutagenesis: Efficient Tuning of the Stereoselectivity of an Epoxide Hydrolase

The directed evolution of enzymes promises to eliminate the long-standing limitations of biocatalysis in organic chemistry and biotechnologythe often-observed limited substrate scope, insufficient activity, and poor regioselectivity or stereoselectivity. Saturation mutagenesis at sites lining the binding pocket with formation of focused libraries has emerged as the technique of choice, but choosing the optimal size of the randomization site and reduced amino acid alphabet for minimizing the labor-determining screening effort remains a challenge. Here, we introduce structure-guided triple-code saturation mutagenesis (TCSM) by encoding three rationally chosen amino acids as building blocks in the randomization of large multiresidue sites. In contrast to conventional NNK codon degeneracy encoding all 20 canonical amino acids and requiring the screening of more than 10¹⁵ transformants for 95% library coverage, TCSM requires only small libraries not exceeding 200–800 transformants in one library. The triple code utilizes structural (X-ray) and consensus-derived sequence data, and is therefore designed to match the steric and electrostatic characteristics of the particular enzyme. Using this approach, limonene epoxide hydrolase has been successfully engineered as stereoselective catalysts in the hydrolytic desymmetrization of meso-type epoxides with formation of either (<i>R</i>,<i>R</i>)- or (<i>S</i>,<i>S</i>)-configurated diols on an optional basis and kinetic resolution of chiral substrates. Crystal structures and docking computations support the source of notably enhanced and inverted enantioselectivity

P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio- and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming trade-offs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450_BM3 mutants which enable nearly perfect regio- and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity–selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids

P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

Cytochrome P450 monooxygenases play a crucial role in the biosynthesis of many natural products and in the human metabolism of numerous pharmaceuticals. This has inspired synthetic organic and medicinal chemists to exploit them as catalysts in regio- and stereoselective CH-activating oxidation of structurally simple and complex organic compounds such as steroids. However, levels of regio- and stereoselectivity as well as activity are not routinely high enough for real applications. Protein engineering using rational design or directed evolution has helped in many respects, but simultaneous engineering of multiple catalytic traits such as activity, regioselectivity, and stereoselectivity, while overcoming trade-offs and diminishing returns, remains a challenge. Here we show that the exploitation of information derived from mutability landscapes and molecular dynamics simulations for rationally designing iterative saturation mutagenesis constitutes a viable directed evolution strategy. This combined approach is illustrated by the evolution of P450_BM3 mutants which enable nearly perfect regio- and diastereoselective hydroxylation of five different steroids specifically at the C16-position with unusually high activity, while avoiding activity–selectivity trade-offs as well as keeping the screening effort relatively low. The C16 alcohols are of practical interest as components of biologically active glucocorticoids