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Regioselective and stereoselective hydroxylation is of vital importance in the functionalization of steroids, which remains challenging in organic synthesis. In particular, the C12-hydroxy steroids play a significant role in the synthesis of many important steroidal drugs.

We review the recent progress of synthesis routes for four α,ω-C6 bifunctional compounds (6-hydroxyhexanoic acid, 6-aminocaproic acid, 1,6-hexanediol and 1,6-hexanediamine), focusing on routes involving sustainable catalysts or renewable feedstocks.

Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering. Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g. Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.

Aliphatic α,ω-diamines (DAs) are important monomer precursors in polyamide plastic manufacturing. However, the dominant industrial process for DA synthesis involves energy-intensive, multistage chemical reactions that are harmful to the environment. For instance, 1,6-hexanediamine (HMD), one of most prominent monomers in nylon-66 synthesis, is mainly synthesized with currently high technological control by butadiene hydrocyanation, which suffers from the use of highly toxic hydrogen cyanide, unsatisfactory selectivity and a complex separation process. Thus, the development of sustainable green DA synthetic routes is highly desired. Herein, we report an efficient one-potin vivobiocatalytic cascade for the transformation of cycloalkanes into DAs with the aid of advanced techniques, including the RetroBioCat tool for biocatalytic route design, enzyme mining for finding appropriate enzymes and microbial consortia construction for efficient pathway assembly. As a result, DAs are successfully produced by the developed microbial consortia-based biocatalytic system, especially HMD, and product concentrations as high as 16.5 mM and 7.6 mM are achieved when using cyclohexanol (CHOL) or cyclohexane (CH) as substrates, respectively. This also represents the highest HMD biosynthesis productivity to date. Other cycloalkanes also serve as substrates, indicating the generality of our approach.

Artificial biorefinery of oleic acid into 1,10-decanedioic acid represents a revolutionizing route to the sustainable production of chemically difficult-to-make bifunctional chemicals. However, the carbon atom economy is extremely low (56%) due to the formation of unifunctional

This study presents an in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants or site-saturation mutagenesis libraries in a PCR-independent manner. The plasmid DNA is double digested with Cas9 bearing specific single guide RNAs to remove the target nucleotides. Next, T5 exonuclease excises both 5'-ends of the linearized plasmid to generate homologous regions of approximately 15 nt. Subsequently, a short dsDNA of approximately 30-50 bp containing the desired mutation cyclizes the plasmid through base pairing and introduces the mutation into the plasmid. The gaps are repaired in Escherichia coli host cells after transformation. This method is highly efficient and accurate. Both single and multiple site-directed mutagenesis can be successfully performed, especially to large sized plasmids. This method demonstrates the great potential for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.

Cytochrome P450 monooxygenases (P450s) play crucial roles in the oxyfunctionalization of non-activated hydrocarbons, thus bridging the gap between simple molecules and high value-added fine chemicals. The introduction of P450s into artificially designed cascade reactions provides an exciting opportunity to accomplish challenging reactions and access organic compounds that cannot be achieved by traditional chemical catalysts or by natural metabolic pathways. The main objective of this review is to provide an overview of different types of artificially designed multi-step cascades in which P450s are involved as key catalysts in the biosynthesis of various organic molecules. The different efforts include in vitro multi-enzymatic biocatalytic cascades, in vivo biocatalytic cascades as well as chemo-enzymatic hybrid cascades. Overall, this work provides an overview of cascade reactions involving P450s with various potential applications for the industrial production of food, cosmetics, polymers and pharmaceuticals.

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity., One mutant, six targets: The regio‐ and stereoselective C−H activation for hydroxylation of six different steroids with formation of C7β alcohols was accomplished using a single P450 mutant evolved by protein library construction based on a special DNA assembly and cloning procedure. The C7β steroidal alcohols, not readily accessible by synthetic reagents or catalysts, are of intense interest as therapeutic drugs.

A PCR-independent in vitro site-directed mutagenesis method was established. Cas12a from Francisella novicida (FnCas12a) linearizes the plasmid with single digestion. T5 exonuclease removes the target nucleotide. A short single- or double-stranded mutagenic oligonucleotide introduces the mutation. This rapid and simple mutagenesis method is referred to as FnCas12a and T5 exonuclease mediated site-directed mutagenesis system (CT5-SDM). The platform is also suitable for the mutagenesis of plasmids larger than 10 kb. KEY POINTS: Site-directed mutagenesis mediated by single-stranded DNA. Removing target site with T5 exonuclease. Highly efficient cleavage of target DNA with FnCas12a.

1,6-Hexanediol (HDO) is an important precursor in the polymer industry. The current industrial route to produce HDO involves energy intensive and hazardous multistage (four-pot–four-step) chemical reactions using cyclohexane (CH) as the starting material, which leads to serious environmental problems. Here, we report the development of a biocatalytic cascade process for the biotransformation of CH to HDO under mild conditions in a one-pot–one-step manner. This cascade biocatalysis operates by using a microbial consortium composed of three E. coli cell modules, each containing the necessary enzymes. The cell modules with assigned functions were engineered in parallel, followed by combination to construct E. coli consortia for use in biotransformations. The engineered E. coli consortia, which contained the corresponding cell modules, efficiently converted not only CH or cyclohexanol to HDO, but also other cycloalkanes or cycloalkanols to related dihydric alcohols. In conclusion, the newly developed biocatalytic process provides a promising alternative to the current industrial process for manufacturing HDO and related dihydric alcohols.

Terpenoids, formed by cyclization and/or permutation of isoprenes, are the most diverse and abundant class of natural products with a broad range of significant functions. One family of the critical enzymes involved in terpenoid biosynthesis is terpene cyclases (TCs), also known as terpene synthases (TSs), which are responsible for forming the ring structure as a backbone of functionally diverse terpenoids. With the recent advances in biotechnology, the researches on terpene cyclases have gradually shifted from the genomic mining of novel enzyme resources to the analysis of their structures and mechanisms. In this review, we summarize both the new methods for genomic mining and the structural mechanisms of some typical terpene cyclases, which are helpful for the discovery, engineering and application of more and new TCs.

Directed evolution of stereo-, regio-, and chemoselective enzymes has enriched the toolbox of synthetic organic chemistry. Among the different gene mutagenesis techniques, saturation mutagenesis (S...

In the present study, we upgraded Pyrococcus furiosus Argonaute (PfAgo) mediated nucleic acid detection method and established a highly sensitive and accurate molecular diagnosis platform for the large-scale screening of COVID-19 infection. Briefly, RT-PCR was performed with the viral RNA extracted from nasopharyngeal or oropharyngeal swabs as template to amplify conserved regions in the viral genome. Next, PfAgo, guide DNAs and molecular beacons in appropriate buffer were added to the PCR products, followed by incubating at 95 °C for 20–30 min. Subsequently, the fluorescence signal was detected. This method was named as SARS-CoV-2 PAND. The whole procedure is accomplished in approximately an hour with the using time of the Real-time fluorescence quantitative PCR instrument shortened from >1 h to only 3–5 min per batch in comparison with RT-qPCR, hence the shortage of the expensive Real-time PCR instrument is alleviated. Moreover, this platform was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The diagnostic results of clinic samples with SARS-CoV-2 PAND displayed 100% consistence with RT-qPCR test., Graphical abstract Image 1

Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s., Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

Cytochrome P450 enzyme CYP109B1 is a versatile biocatalyst exhibiting hydroxylation activities toward various substrates. However, the regio- and stereoselective steroid hydroxylation by CYP109B1 is far less explored. In this study, the oxidizing activity of CYP109B1 is reconstituted by coupling redox pairs from different sources, or by fusing it to the reductase domain of two self-sufficient P450 enzymes P450RhF and P450BM3 to generate the fused enzyme. The recombinant Escherichia coli expressing necessary proteins are individually constructed and compared in steroid hydroxylation. The ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499) from Synechococcus elongates is found to be the best redox pair for CYP109B1, which gives above 99% conversion with 73% 15β selectivity for testosterone. By contrast, the rest ones and the fused enzymes show much less or negligible activity. With the aid of redox pair of Fdr_0978/Fdx_1499, CYP109B1 is used for hydroxylating different steroids. The results show that CYP109B1 displayed good to excellent activity and selectivity toward four testosterone derivatives, giving all 15β-hydroxylated steroids as main products except for 9 (10)-dehydronandrolone, for which the selectivity is shifted to 16β. While for substrates bearing bulky substitutions at C17 position, the activity is essentially lost. Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is revealed by computational analysis, thus providing theoretical basis for directed evolution to further improve its catalytic properties.

Steroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio- and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.

The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, among other things, amino acid bias, which means that the development of efficient PCR-free methods is desirable. Here, we present a highly efficient in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants in a PCR-free manner. First, it involves plasmid digestion by utilizing a complex of Cas9 with specific single guide RNA (sgRNA) followed by degradation with T5 exonuclease to generate a 15 nt homologous region. Second, primers containing the desired mutations are annealed to form the double-stranded DNA fragments, which are then ligated into the linearized plasmid. In theory, neither the size of the target plasmid nor the unavailable restriction enzyme site poses any problems that may arise in traditional techniques. In this study, single and multiple site-directed mutagenesis were successfully performed even for a large size plasmid (up to 9.0 kb). Moreover, a PCR-free site-saturation mutagenesis library on single site and two adjacent sites of a green fluorescent protein was also generated with promising results. This demonstrates the great potential of the ICM system for creating high-quality mutant libraries in directed evolution as an alternative to PCR-based saturation mutagenesis, thus facilitating research on synthetic biology.

Biocatalytic C–H amination is one of the most challenging tasks. C-H amination reaction can hardly be driven efficiently by solely one enzyme so far. Thus, enzymatic synergy represents an alternative strategy. Herein, we report an “Artificially Bioamination Pathway” for C–H amination of cyclohexane as a model substrate. Three enzymes, a monooxygenase P450BM3 mutant, an alcohol dehydrogenase ScCR from Streptomyces coelicolor and an amine dehydrogenase EsLeuDH from Exiguobacterium sibiricum, constituted a clean cascade reaction system with easy product isolation. Two independent cofactor regeneration systems were optimized to avoid interference from the endogenous NADH oxidases in the host E. coli cells. Based on a stepwise pH adjustment and ammonium supplement strategy, and using an in vitro mixture of cell-free extracts of the three enzymes, cyclohexylamine was produced in a titer of 14.9 mM, with a product content of up to 92.5%. Furthermore, designer cells coexpressing the three required enzymes were constructed and their capability of alkane bio-amination was examined. This artificially designed bioamination paves an attractive approach for enzymatic synthesis of amines from accessible and cheap alkanes.

The P450-mediated asymmetric hydroxylation of inert C–H bonds is a chemically challenging reaction. Self-sufficient P450LaMO from the CYP116B subfamily could catalyze the transformation of 1,2,3,4-tetrahydronaphthalene to (S)-tetralol, despite its poor enantioselectivity (er 66 : 34) and product selectivity (the ratio of alcohol and ketone, ak, 76 : 24). To improve the selectivity, phenylalanine scanning and further protein engineering were performed to reshape the active pocket of P450LaMO, resulting in a mutant (T121V/Y385F/M391L) with not only improved (S)-enantioselectivity (er 98 : 2) but also excellent product selectivity (ak 99 : 1), in contrast to another mutant L97F/T121F/E282V/T283Y with complementary (R)-enantioselectivity (er 23 : 77). Moreover, the enantiopure (S)-alcohols formed by the P450LaMO-catalyzed oxidation of a series of alkylbenzenes are potentially important building blocks in the pharmaceutical industry. This Phe-based enantioselectivity engineering used for reshaping the active pocket of P450s could provide a guide to the protein evolution of other CYP116B members.

Ketoreductases are tools for the synthesis of chiral alcohols in industry. However, the low activity of natural enzymes often restricts their use in industrial applications. On the basis of computational analysis and previous reports, two residues (F92 and F94) probably affecting the activity of ketoreductase CgKR1 were identified. By tuning these two residues, the CgKR1-F92C/F94W variant was obtained that exhibited higher activity toward all 28 structurally diverse substrates examined than the wild-type enzyme. Among them, 13 substrates have a specific activity over 50 U mg–1 (54–775 U mg–1). Using CgKR1-F92C/F94W as a catalyst, five substrates at high loading (>100 g–1 L–1) were reduced completely in gram-scale preparative reactions. This approach provides accesses to pharmaceutically relevant chiral alcohols with high enantioselectivity (up to 99.0% ee) and high space-time yield (up to 583 g–1 L–1 day–1). Molecular dynamics simulations highlighted the crucial role of residues 92 and 94 in activity impr...

One-pot furfuralcohol (FOL) production via dehydration of corncob-derived xylose followed by bioreduction of furfural has been described. The synthesized biocompatible solid acid catalyst SO42−/SnO2-attapulgite has been characterized and used for the dehydration of xylose-rich hydrolysate, and the highest furfural yield of 44% is achieved when employing 3.6 wt% catalyst loading at 170 °C for 20 min. The recombinant Escherichia coli CCZU-A13 harboring a NADH-dependent reductase (SsCR) is found to catalyze the bioreduction of furfural to FOL, the whole-cell catalyst could tolerate as high as 300 mM furfural substrate to give 221 mM FOL after 12 h of reaction under the optimum conditions (1.0 mM glucose per mM furfural, 30 °C, pH 6.5, 0.1 g wet cells per mL). The two processes are successfully combined in a one-pot manner to transform the xylose-rich hydrolysate to furfural, and then to FOL with 44% yield based on the starting material xylose (100% FOL yield for the bioreduction step). Finally, recycling experiments for the carrageenan immobilized whole-cell and solid acid catalyst in one-pot FOL production are conducted; both catalysts show excellent recyclability and no obvious decrease in activity is detected after 5 cycles of reaction. The developed one-pot chemo-enzymatic approach is greatly useful for practical green FOL production from renewable biomass resources.

Substrate inhibition of enzymes is one of the main obstacles encountered frequently in industrial biocatalysis. Haloketone reductase SsCR was seriously inhibited by substrate 2,2′,4′-trichloroacetophenone. In this study, two essential loops were found that have a relationship with substrate binding by conducting X-ray crystal structure analysis. Three key residues were selected from the tips of the loops and substituted with amino acids with lower hydrophobicity to weaken the hydrophobic interactions that bridge the two loops, resulting in a remarkable reduction of substrate inhibition. Among these variants, L211H showed a significant attenuation of substrate inhibition, with a Ki of 16 mM, which was 16 times that of the native enzyme. The kinetic parameter kcat/Km of L211H was 3.1 × 103 s−1 mM−1, showing the comparable catalytic efficiency to that of the wild-type enzyme (WT). At the substrate loading of 100 mM, the space time yield of variant L211H in asymmetric reduction of the haloketone was 3-fold higher than that of the WT.

Cofactor regeneration using diols as "smart cosubstrates" is one of the most promising approaches, due to the thermodynamic preference and 0.5-equiv requirement. In order to establish an efficient NADPH regeneration system with 1,4-butanediol (1,4-BD), a NADP+-dependent alcohol dehydrogenase from Kluyveromyces polysporus (KpADH) was engineered to solve the kinetic imbalance. Several hotspots were identified through molecular dynamic simulation and subjected to saturation and combinatorial mutagenesis. Variant KpADHV84I/Y127M exhibited a lower KM of 15.1 mM and a higher kcat of 30.1 min-1 than WTKpADH. The oxidation of 1,4-BD to 4-hydroxybutanal was found to be the rate-limiting step, for which the kcat/KM value of double mutant KpADHV84I/Y127M was 2.00 min-1·mM-1, 11.6-fold higher than that of WTKpADH. KpADHV84I/Y127M preferred diols with a longer chain length (C5-C6). The ratio of kcat/KM toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADHV84I/Y127M was dramatically reduced by almost 100-fold compared to WTKpADH, which was advantageous for NADPH regeneration. As much as 100 mM phenylpyruvic acid could be reduced into d-phenylalanine with 99.2% conversion in 6 h using merely 0.5 equiv of 1,4-BD. Both the improved catalytic efficiency toward 1,4-BD and the balanced kcat/KM between 1,4-BD and 2-HTHF contributed to the higher NADPH regeneration efficiency. This study provides guidance for engineering alcohol dehydrogenases for cosubstrate specificity toward diols and its application in NADPH regeneration for the preparation of chiral compounds of pharmaceutical relevance.

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second-generation versions of Combinatorial Active-Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site-specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On-chip solid-phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

Biocatalytic cascade reactions using isolated stereoselective enzymes or whole cells in one-pot processes lead to value-added chiral products in a single workup. The concept has been restricted mainly to starting materials and intermediate products that are accepted by the respective wild-type enzymes. In the present study, we exploited directed evolution as a means to create E. coli whole cells for regio- and stereoselective cascade sequences that are not possible using man-made catalysts. The approach is illustrated using P450-BM3 in combination with appropriate alcohol dehydrogenases as catalysts in either two-, three-, or four-step cascade reactions starting from cyclohexane, cyclohexanol, or cyclohexanone, respectively, leading to either (R,R)-, (S,S)-, or meso-cyclohexane-1,2-diol. The one-pot conversion of cyclohexane into (R)- or (S)-2-hydroxycyclohexanone in the absence of ADH is also described.