ABSTRACT
Plant-derived ß-glucosidases hold promise for glycoside biosynthesis via reverse hydrolysis because of their excellent glucose tolerance and robust stability. However, their poor heterologous expression hinders the development of large-scale production and applications. In this study, we overexpressed apple seed ß-glucosidase (ASG II) in Komagataella phaffii and enhanced its production from 289 to 4322 U L-1 through expression cassette engineering and protein engineering. Upon scaling up to a 5-L high cell-density fermentation, the resultant mutant ASG IIV80A achieved a maximum protein concentration and activity in the secreted supernatant of 2.3 g L-1 and 41.4 kU L-1, respectively. The preparative biosynthesis of salidroside by ASG IIV80A exhibited a high space-time yield of 33.1 g L-1 d-1, which is so far the highest level by plant-derived ß-glucosidase. Our work addresses the long-standing challenge of the heterologous expression of plant-derived ß-glucosidase in microorganisms and presents new avenues for the efficient production of salidroside and other natural glycosides.
Subject(s)
Glucosides , Malus , Phenols , Seeds , beta-Glucosidase , Phenols/metabolism , beta-Glucosidase/genetics , beta-Glucosidase/metabolism , Glucosides/biosynthesis , Glucosides/metabolism , Glucosides/chemistry , Seeds/genetics , Seeds/metabolism , Saccharomycetales/genetics , Saccharomycetales/metabolism , Saccharomycetales/enzymology , Fermentation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Engineering/methodsABSTRACT
Invited for this issue's cover is the group of Huilei Yu at the East China University of Science and Technology. The image shows a sustainable biosynthesis route to nylon monomers from bio-based substrate α, ω-dicarboxylic acids. The Research Article itself is available at 10.1002/cssc.202301477.
Subject(s)
Diamines , Fatty Acids , Amino Acids , ChinaABSTRACT
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is a highly distinguished expression platform for the excellent synthesis of various heterologous proteins in recent years. With the advantages of high-density fermentation, P. pastoris can produce gram amounts of recombinant proteins. While not every protein of interest can be expressed to such high titers, such as Baeyer-Villiger monooxygenase (BVMO) (AcPSMO) which is responsible for pyrazole sulfide asymmetric oxidation. In this work, an excellent yeast expression system was established to facilitate efficient AcPSMO expression, which exhibited 9.5-fold enhanced secretion. Subsequently, an ultrahigh throughput screening method based on fluorescence-activated cell sorting by fusing super folder green fluorescent protein (sfGFP) in the C-terminal of AcPSMO was developed, and directed evolution was performed. The protein expression level of the superior mutant AcPSMOP1 (S58T/T252P/E336N/H456D) reached 84.6 mg/L at 100 mL shaking flask, which was 4.7 times higher than the levels obtained with the wild-type. Finally, the optimized chassis cells were used for high-density fermentation on a 5-L scale, and AcPSMOP1 protein yield of 3.4 g/L was achieved, representing approximately 85% of the total protein secreted. By directly employing the pH-adjusted supernatant as a biocatalyst, 20 g/L pyrmetazole sulfide was completely transformed into the corresponding (S)-sulfoxide, with a 78.8% isolated yield. This work confers dramatic benefits for efficient secretion of other BVMOs in P. pastoris.
Subject(s)
Mixed Function Oxygenases , Pichia , Saccharomycetales , Mixed Function Oxygenases/metabolism , Pichia/genetics , Pichia/metabolism , Recombinant Proteins/metabolism , Sulfoxides/metabolism , Sulfides/metabolismABSTRACT
Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0â mM α,ω-DAs (C6-C9) was also performed, giving 11.2â mM 1,6-HMD (56.0 % yield), 14.8â mM 1,7-heptanediamine (74.0 % yield), 17.4â mM 1,8-octanediamine (87.0 % yield), and 19.7â mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.
Subject(s)
Amino Acids , Diamines , Dicarboxylic Acids , Fatty AcidsABSTRACT
Baeyer-Villiger monooxygenases (BVMOs) are able to catalyse the asymmetric oxidation of sulfides. This property has made them attractive catalysts for the synthesis of chiral sulfoxide drugs. Here, we have designed and synthesised an exhaustive combinatorial mutant library of the previously identified lansoprazole sulfide monooxygenase CbBVMOV1. From this synthetic combinatorial mutant library, the best mutant, CbBVMOV3, was selected with a specific activity of approximately 1 U mg-1 for lansoprazole sulfoxides. We then optimised the reaction conditions of a two-phase system, achieving the enzymatic asymmetric synthesis of (R)-lansoprazole in a space-time yield of 213 g L-1 d-1 and an enantiomeric excess of >99% (R) with no detectable by-products. In addition, CbBVMOV3 showed higher activity towards other prazole sulfides. These results indicate the potential application of CbBVMO in the chiral sulfoxide drug industry.
Subject(s)
Mixed Function Oxygenases , Sulfoxides , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Sulfides , LansoprazoleABSTRACT
Cytochrome P450 monooxygenases (CYP450s) play an important role in the biosynthesis of natural products by activating inert C-H bonds and inserting hydroxyl groups. However, the activities of most plant-derived CYP450s are extremely low, limiting the heterologous biosynthesis of natural products. Traditional enzyme engineering methods, either rational or screening-based, are not suitable for CYP450s because of the lack of crystal structures and high-throughput screening methods for this class of enzymes. CYP725A4 is the first hydroxylase involved in the biosynthesis pathway of Taxol. Its low activity, promiscuity, and multispecificity make it a bottleneck in Taxol biosynthesis. Here, we identified key amino acids that affect the in vivo activity of CYP725A4 by constructing the ancestral enzymes of CYP725A4. We obtained positive mutants that showed an improved yield of hydroxylated products based on the key amino acids identified, providing guidance for the modification of other CYP450s involved in the biosynthesis of natural products.
Subject(s)
Amino Acids , Biological Products , Amino Acids/genetics , Cytochrome P-450 Enzyme System/metabolism , Paclitaxel/chemistry , Paclitaxel/metabolismABSTRACT
(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6â Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3â mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.
Subject(s)
Oxygenases , Podophyllotoxin , Oxygenases/metabolism , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Substrate SpecificityABSTRACT
Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/âµQ375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.
Subject(s)
Formate Dehydrogenases , NAD , NADP/metabolism , NAD/metabolism , Formate Dehydrogenases/genetics , Formate Dehydrogenases/metabolism , Protein Engineering/methods , Oxidation-ReductionABSTRACT
Cytochrome P450 monooxygenases CYP120As are the unique non-membrane P450s, which are extensively involved in retinoid biodegradation. As the O-functionalized 1,3,3-trimethylcyclohex-1-ene moiety exists in many bioactive compounds which could only be catalyzed by Class II P450s, exploration of the catalytic repertoire of CYP120As is therefore highly attractive. However, up to date, only one bacteriogenic candidate (CYP120A1) was demonstrated for the hydroxylation of C16 and C17 of retinoic acid, by utilizing the integral membrane protein cytochrome P450 reductase redox partner for the electron transfer. Herein, we provided an efficient prokaryotic functional expression system of CYP120As in E. coli by expression of the CYP120A1 coupled with several reductase partners. Fusion redox partners to the C-terminal of the heme-domain are also working on other CYP120A members. Among them, the fusion protein of CYP120A29 and FAD/FMN reductase from Bacillus megaterium P450BM3 (CYP101A2) showed the highest expression level. Based on the available translational fusion systems, the regioselectivity and the substrate scope of the CYP120As have also been explored. This work represents a good starting point for further expanding the catalytic potential of CYP120 family. KEY POINTS: ⢠Characterization of CYP120As in E. coli is firstly achieved by constructing fusion proteins. ⢠The feasibility of three P450 reductase domains to CYP120As was evaluated. ⢠Hydroxylated products of retinoic acid by six CYP120As were sorted and analyzed.
Subject(s)
Bacterial Proteins , Escherichia coli , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Proteins/metabolism , Oxidation-Reduction , Electron Transport , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Tretinoin/metabolismABSTRACT
The key precursors for nylon synthesis, that is, 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD), are produced from petroleum-based feedstocks. A sustainable biocatalytic alternative method from bio-based adipic acid has been demonstrated recently. However, the low efficiency and specificity of carboxylic acid reductases (CARs) used in the process hampers its further application. Herein, we describe a highly accurate protein structure prediction-based virtual screening method for the discovery of new CARs, which relies on near attack conformation frequency and the Rosetta Energy Score. Through virtual screening and functional detection, five new CARs were selected, each with a broad substrate scope and the highest activities toward various di- and ω-aminated carboxylic acids. Compared with the reported CARs, KiCAR was highly specific with regard to adipic acid without detectable activity to 6-ACA, indicating a potential for 6-ACA biosynthesis. In addition, MabCAR3 had a lower Km with regard to 6-ACA than the previously validated CAR MAB4714, resulting in twice conversion in the enzymatic cascade synthesis of HMD. The present work highlights the use of structure-based virtual screening for the rapid discovery of pertinent new biocatalysts.
Subject(s)
Aminocaproic Acid , Oxidoreductases , Oxidoreductases/metabolism , AdipatesABSTRACT
Terpenoids are pervasive in nature and display an immense structural diversity. As the largest category of plant secondary metabolites, terpenoids have important socioeconomic value in the fields of pharmaceuticals, spices, and food manufacturing. The biosynthesis of terpenoid skeletons has made great progress, but the subsequent modifications of the terpenoid framework are poorly understood, especially for the functionalization of inert carbon skeleton usually catalyzed by hydroxylases. Hydroxylase is a class of enzymes that plays an important role in the modification of terpenoid backbone. This review article outlines the research progress in the identification, molecular modification, and functional expression of this class of enzymes in the past decade, which are profitable for the discovery, engineering, and application of more hydroxylases involved in the plant secondary metabolism.
ABSTRACT
Penicillium expansum, producer of a wide array of secondary metabolites, has the potential to be a source of new terpene synthases. In this work, a platform was constructed with Escherichia coli BL21(DE3) by enhancing its endogenous 2-methyl-d-erythritol-4-phosphate pathway to supply sufficient terpenoid precursors. Using this precursor-supplying platform, we discovered two sesquiterpene synthases from P. expansum: PeTS1, a new (+)-aristolochene synthase, and PeTS4, the first microbial (+)-bicyclogermacrene synthase. To enhance the sesquiterpene production by PeTS1, we employed a MBP fusion tag to improve the heterologous protein expression, resulting in the increase of aristolochene production up to 50 mg/L in a 72 h flask culture, which is the highest production reported to date. We also realized the first biosynthesis of (+)-bicyclogermacrene, achieving 188 mg/L in 72 h. This work highlights the great potential of this microbial platform for the discovery of new terpene synthases and opens new ways for the bioproduction of other valuable terpenoids.
Subject(s)
Alkyl and Aryl Transferases , Sesquiterpenes , Alkyl and Aryl Transferases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Penicillium , Sesquiterpenes/metabolism , Terpenes/metabolismABSTRACT
Baeyer-Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide-type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high-throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild-type enzyme. This new method is feasible for high-throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.
Subject(s)
High-Throughput Screening Assays , Mixed Function Oxygenases , Biocatalysis , Colorimetry , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Substrate Specificity , Sulfides/metabolism , Sulfoxides/metabolismABSTRACT
Paclitaxel (Taxol™), an alkaloid of diterpenoid family, is one of the most widely used anti-cancer drugs due to its effectiveness against a variety of tumors. Rather than directly extraction and chemical synthesis of paclitaxel or its intermediates from yew plants, construction of a microbial cell factory for paclitaxel biosynthesis will be more efficient and sustainable. The challenge for biosynthesis of paclitaxel lies on the insufficient precursor, such as taxadien-5α-ol. In this study, we report a recombinant Escherichia coli strain constructed with a heterologous mevalonate pathway, a taxadiene synthase from yew, and a cytochrome P450-mediated oxygenation system for the de novo production of taxadien-5α-ol, the first product of the multi-step taxadiene oxygenation metabolism. The key enzymes including taxadiene synthases and cytochrome P450 reductases were screened, and the linker for fusing taxadiene-5α-hydroxylase with its reductase partner cytochrome P450 reductase was optimized. By reducing the metabolic burden and optimizing the fermentation conditions, the final production of total oxygenated taxanes was raised up to 27 mg L-1 in a 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L-1, representing approximately a 12-fold and 23-fold improvements, respectively, as compared with the initial titers. The engineered MVA pathway for the overproduction of terpenoid precursors can serve as an efficient platform for the production of other valuable terpenoids.
ABSTRACT
AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase (BVMO). We previously obtained the AcCHMOM6 mutant, which oxidizes omeprazole sulfide (OPS) to the chiral sulfoxide drug esomeprazole. To further improve the catalytic efficiency of the AcCHMOM6 mutant, a focused mutagenesis strategy was adopted at the intersections of the FAD-binding domain, NADPH-binding domain, and α-helical domain based on structural characteristics of AcCHMO. By using focused mutagenesis and subsequent global evolution two key residues (L55 and P497) at the intersections of the domains were identified. Mutant of L55Y improved catalytic efficiency significantly, whereas the P497S mutant alleviated substrate inhibition remarkably. AcCHMOM7 (L55Y/P497S) was obtained by combining the two mutations, which increased the specific activity from 18.5 (M6) to 108 U/g, and an increase in the Ki of the substrate OPS from 34 to 265 µM. The results indicate that catalytic performance can be elevated by modification of the sensitive sites at the intersection of the domains of AcCHMO. The results also provided some insights for the engineering of other Type I BVMOs or other multidomain proteins.
Subject(s)
Acinetobacter calcoaceticus/enzymology , Bacterial Proteins/chemistry , Mixed Function Oxygenases/chemistry , Acinetobacter calcoaceticus/genetics , Amino Acid Substitution , Bacterial Proteins/genetics , Mixed Function Oxygenases/genetics , Mutation, Missense , Protein DomainsABSTRACT
Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.
Subject(s)
Aldehyde-Lyases/metabolism , High-Throughput Screening Assays/methods , Nitriles/metabolism , Aldehydes/chemistry , Aldehydes/metabolism , Biocatalysis , Directed Molecular Evolution , Escherichia coli/enzymology , Nitriles/chemistry , Stereoisomerism , Substrate SpecificityABSTRACT
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.
ABSTRACT
Prochiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with > 99% enantiomeric excess.
ABSTRACT
Isoleucine dioxygenase (IDO)-catalyzed hydroxylation of isoleucine is a promising method for the synthesis of the diabetic drug (2S,3R,4S)-4-hydroxyisoleucine [(2S,3R,4S)-4-HIL]. However, the low activity of IDO significantly limits its practical application. In this work, a high-throughput screening method was developed and directed evolution was performed on the IDO from Bacillus subtilis, resulting in a double mutant with improvements in specific activity, protein expression level, and fermentation titer of 3.2-, 2.8-, and 9.4-fold, respectively. l-Isoleucine (228 mM) was completely converted to (2S,3R,4S)-4-HIL by the best variant with a space-time yield of up to 80.8 g L-1 d-1, which is the highest record reported so far. With a further increase of the substrate loading to 1 M, a high conversion of 91% could also be achieved. At last, enzymatic synthesis of (2S,3R,4S)-4-HIL was successfully carried out on a 3 L scale, indicating tremendous potential of the IDO variant I162T/T182N for green and efficient production of (2S,3R,4S)-4-HIL.
Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/genetics , Dioxygenases/genetics , Dioxygenases/metabolism , Isoleucine/analogs & derivatives , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Directed Molecular Evolution , Fermentation , Genetic Engineering , Isoleucine/biosynthesis , Isoleucine/metabolismABSTRACT
Aliphatic α,ω-dicarboxylic acids (DCAs) are a class of useful chemicals that are currently produced by energy-intensive, multistage chemical oxidations that are hazardous to the environment. Therefore, the development of environmentally friendly, safe, neutral routes to DCAs is important. We report an in vivo artificially designed biocatalytic cascade process for biotransformation of cycloalkanes to DCAs. To reduce protein expression burden and redox constraints caused by multi-enzyme expression in a single microbe, the biocatalytic pathway is divided into three basic Escherichia coli cell modules. The modules possess either redox-neutral or redox-regeneration systems and are combined to form E. coli consortia for use in biotransformations. The designed consortia of E. coli containing the modules efficiently convert cycloalkanes or cycloalkanols to DCAs without addition of exogenous coenzymes. Thus, this developed biocatalytic process provides a promising alternative to the current industrial process for manufacturing DCAs.