Valuable Biocatalysts Research Articles

Cloning and characterization of a novel recombinant multifunctional glycoside hydrolase β-D-xylosidase/α-L-arabinopyranosidase/β-glucosidase from Bifidobacterium adolescentis and its application on the biotransformation of ginsenoside Rb3

β-D-xylosidase, an important glycoside hydrolase, hydrolyzes xylo-oligosaccharides to yield xylose and biotransforms specific saponins by cleaving β-xylose at the non-reducing end. It is extensively employed in renewable resources, including fuels, food, and pharmaceuticals, as a xylanolytic enzyme. In this study, a 1197 bp β-xylosidase gene (BaXyl5B) derived from Bifidobacterium adolescentis was cloned and expressed in E. coli BL21. Following purification, the recombinant β-xylosidase exhibited a distinct band on SDS-PAGE; its molecular weight was determined to be 46.4 kDa. The optimal activity of BaXyl5B was observed at a pH of 6.0 and a temperature of 30 °C. Purified BaXyl5B demonstrated multifunctional activities involving p-nitrophenyl-β-D-xylopyranoside (pNPβXyl), p-nitrophenyl-α-L-arabinopyranoside (pNPαArap), and p-nitrophenyl-β-D-glucopyranoside (pNPβGlc). A significant finding of this study was that BaXyl5B exhibited high selectivity towards ginsenoside Rb3, which could hydrolyze xylose at the C-20 position of Rb3 to yield Rd and F2. Overall, this research explored a novel β-xylosidase, BaXyl5B, that could serve as a valuable biocatalyst for converting ginsenosides and synthesizing active glycosides and aglycones, making it a promising enzyme for further exploration and utilization.

Novel, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase from Hymenobacter siberiensis PAMC 29290, a psychrotolerant bacterium from Arctic marine sediment.

Endo-β-1,3-glucanase is a glycoside hydrolase (GH) that plays an essential role in the mineralization of β-glucan polysaccharides. In this study, the novel gene encoding an extracellular, non-modular GH16 endo-β-1,3-glucanase (GluH) from Hymenobacter siberiensis PAMC 29290 isolated from Arctic marine sediment was discovered through an in silico analysis of its whole genome sequence and subsequently overexpressed in Escherichia coli BL21. The 870-bp GluH gene encoded a protein featuring a single catalytic GH16 domain that shared over 61% sequence identity with uncharacterized endo-β-1,3-glucanases from diverse Hymenobacter species, as recorded in the National Center for Biotechnology Information database. The purified recombinant endo-β-1,3-glucanase (rGluH: 31.0 kDa) demonstrated peak activity on laminarin at pH 5.5 and 40°C, maintaining over 40% of its maximum endo-β-1,3-glucanase activity even at 25°C. rGluH preferentially hydrolyzed D-laminarioligosaccharides and β-1,3-linked polysaccharides, but did not degrade D-laminaribiose or structurally unrelated substrates, confirming its specificity as a true endo-β-1,3-glucanase without ancillary GH activities. The biodegradability of various substrate polymers by the enzyme was evaluated in the following sequence: laminarin > barley β-glucan > carboxymethyl-curdlan > curdlan > pachyman. Notably, the specific activity (253.1 U mg-1) and catalytic efficiency (k cat /K m : 105.72 mg-1 s-1 mL) of rGluH for laminarin closely matched its specific activity (250.2 U mg-1) and k cat /K m value (104.88 mg-1 s-1 mL) toward barley β-glucan. However, the k cat /K m value (9.86 mg-1 s-1 mL) of rGluH for insoluble curdlan was only about 9.3% of the value for laminarin, which correlates well with the observation that rGluH displayed weak binding affinity (< 40%) to the insoluble polymer. The biocatalytic hydrolysis of D-laminarioligosaccharides with a degree of polymerization between 3 and 6 and laminarin generally resulted in the formation of D-laminaribiose as the predominant product and D-glucose as the secondary product, with a ratio of approximately 4:1. These findings suggest that highly active rGluH is an acidic, cold-adapted D-laminaribiose- and D-glucose-releasing GH16 endo-β-1,3-glucanase, which can be exploited as a valuable biocatalyst for facilitating low temperature preservation of foods.

Identification of a robust bacterial pyranose oxidase that displays an unusual pH dependence

Pyranose oxidases are valuable biocatalysts, yet only a handful of bacterial pyranose oxidases are known. These bacterial enzymes exhibit noteworthy distinctions from their extensively characterized fungal counterparts, encompassing variations in substrate specificity and structural attributes. Herein a bacterial pyranose oxidase from Oscillatoria princeps (OPOx) was biochemically characterized in detail. In contrast to the fungal pyranose oxidases, OPOx could be well expressed in Escherichia coli as soluble, fully flavinylated and active oxidase. It was found to be highly thermostable (melting temperature >90 ⁰C) and showed activity on glucose, exhibiting an exceptionally low KM value (48 μM). Elucidation of its crystal structure revealed similarities with fungal pyranose oxidases, such as being a tetramer with a large central void leading to a narrow substrate access tunnel. In the active site, the FAD cofactor is covalently bound to a histidine. OPOx displays a relatively narrow pH optimum for activity with a sharp decline at relatively basic pH values which is accompanied with a drastic change in its flavin absorbance spectrum. The pH-dependent switch in flavin absorbance features and oxidase activity was shown to be fully reversible. It is hypothesized that a glutamic acid helps to stabilize the protonated form of the histidine that is tethered to the FAD. OPOx presents itself as a valuable biocatalyst as it is highly robust, well-expressed in E. coli, shows low KM values for monosaccharides and has a peculiar pH dependent “on-off switch”.

A novel, cost-effective approach for the production of hydrogenase enzymes and molecular hydrogen from recycled whey-based by-products

Cupriavidus necator produces O2-tolerant [NiFe]-hydrogenases (Hyds), and is a valuable biocatalyst for biological fuel cells, while Escherichia coli produces H2 during mixed-acid fermentation. A mixture of curd and cheese whey (CW) was used to explore two-phase growth with C. necator H16 and E. coli. Enhanced biomass, as well as Hyd activity and electrical potential (∼0.65 V) were shown for growth of C. necator in the CW medium with added glycerol. The residual growth medium made available after the cultivation of C. necator and removal of cells was then used for cultivation of E. coli. Maximum fermentative growth of E. coli was attained after 72 h and with a H2 yield of ∼6 mmol/L/g dry whey after 48 h. This study demonstrates the economically viable production of biomass, hydrogenase enzymes and H₂ using cheap, industrially produced whey within the 3R concept.

Engineered Imine Reductase Catalyzed Enantiodivergent Synthesis of Alkylated Amphetamines.

Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.

Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones

Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.

An Active and Versatile Electron Transport System for Cytochrome P450 Monooxygenases from the Alkane Degrading Organism Acinetobacter sp. OC4.

Cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the oxyfunctionalization of non-activated carbon-hydrogen bonds. Most CYPs rely on electron transport proteins as redox partners. In this study, the ferredoxin reductase (FdR) and ferredoxin (FD) for a cytochrome P450 monooxygenase from Acinetobacter sp. OC4 are investigated. Upon heterologous production of both proteins independently in Escherichia coli, spectral analysis showed their reduction capability towards reporter electron acceptors, e. g., cytochrome c. The individual proteins' specific activity towards cytochrome c reduction was 25 U mg-1. Furthermore, the possibility to enhance electron transfer by artificial fusion of the units was elucidated. FdR and FD were linked by helical linkers [EAAAK]n, flexible glycine linkers [GGGGS]n or rigid proline linkers [EPPPP]n of n=1-4 sequence repetitions. The system with a glycine linker (n=4) reached an appreciable specific activity of 19 U mg-1 towards cytochrome c. Moreover, their ability to drive different members of the CYP153A subfamily is demonstrated. By creating artificial self-sufficient P450s with FdR, FD, and a panel of four CYP153A representatives, effective hydroxylation of n-hexane in a whole-cell system was achieved. The results indicate this protein combination to constitute a functional and versatile surrogate electron transport system for this subfamily.

Optimizing Continuous‐Flow Biocatalysis with 3D‐Printing and Inline IR Monitoring

AbstractEnzymatic biocatalysis typically generates less waste, uses less water, and minimizes energy consumption compared to traditional chemical methods. Efficient, cell‐free biosynthesis relies on the reuse of its valuable biocatalysts. Immobilization of enzymes on solid supports, such as enzyme carrier resins (ECRs), offers a reliable and widely deployed approach to maximize enzyme turnover in cell‐free biosynthesis. We focus on two major bottlenecks associated with optimizing cell‐free biocatalysis. First, we apply our lab's 3D‐printed labware to screen ECRs in 96‐well mini‐reactors to optimize enzyme immobilization conditions. Second, we introduce inline infrared spectroscopy to monitor bioreactor output and maximize enzyme productivity. Urease provides a model system for examining immobilization conditions and continuous assessment of biocatalyst performance. As required for the high substrate concentrations to improve process efficiency and minimize waste, urease was studied in unusually high concentrations of its substrate – molar concentrations of urea. The optimized reactor processed 3.24 L of 4.00 M urea at an average volumetric productivity of 13 g ⋅ L−1 ⋅ h−1 over 18 h and achieved an estimated productivity number of &gt;17.4 kg urea processed per g of immobilized urease Type‐IX. This workflow can be generalized to most biocatalytic processes and could accelerate adoption of cell‐free biosynthesis for greater chemical sustainability.

Broadening the Substrate Scope of a Polyphosphate Kinase for Canonical and Non‐Canonical Nucleotides

AbstractPolyphosphate kinases (PPKs) are valuable biocatalysts for ATP cofactor regeneration as well as for the phosphorylation of non‐canonical nucleotides. The versatility of PPKs in the latter application is defined by their substrate scope. In this study, we investigate the substrate spectrum of the PPK2‐III enzyme from Meiothermus ruber (MrPPK) for the conversion of canonical and non‐canonical (deoxy)‐ribonucleotides. The enzyme shows high substrate promiscuity for purine and to minor degree pyrimidine substrates, with an overall preference for the native substrate AMP. With structure guided rational amino acid exchanges in the active site, we produced an MrPPK variant with improved activity for a broad variety of purine nucleotides. While the preference for AMP is lost, conversion of nucleotides without a 6‐amino function at the purine moiety is increased. This MrPPK variant is a versatile biocatalyst for the synthesis of non‐canonical nucleotides and could also be useful as a GTP cofactor regeneration system.

Assessment of the efficiency and stability of enzymatic membrane reaction utilizing lipase covalently immobilized on a functionalized hybrid membrane

Enzyme immobilization in membrane bioreactors has been considered as a practical approach to enhance the stability, reusability, and efficiency of enzymes. In this particular study, a new type of hybrid membrane reactor was created through the phase inversion method, utilizing hybrid of graphene oxide nanosheets (GON) and polyether sulfone (PES) in order to covalently immobilize the Candida rugosa lipase (CRL). The surface of hybrid membrane was initially modified by (3-Aminopropyl) triethoxysilane (APTES), before the use of glutaraldehyde (GLU), as a linker, through the imine bonds. The resulted enzymatic hybrid membrane reactors (EHMRs) were then thoroughly analyzed by using field-emission scanning electron microscopy (FE-SEM), contact angle goniometry, surface free energy analysis, X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, attenuated total reflection (ATR), and energy-dispersive X-ray (EDX) spectroscopy. The study also looked into the impact of factors such as initial CRL concentration, storage conditions, and immobilization time on the EHMR's performance and activity, which were subsequently optimized. The results demonstrated that the CRLs covalently immobilized on the EHMRs displayed enhanced pH and thermal stability compared to those physically immobilized or free. These covalently immobilized CRLs could maintain over 60% of their activity even after 6 reaction cycles spanning 50 days. EHMRs are valuable biocatalysts in developing various industrial, environmental, and analytical processes.

Chemoenzymatic Synthesis of Selegiline: An Imine Reductase-Catalyzed Approach.

(R)-Homobenzylic amines are key structural motifs present in (R)-selegiline, a drug indicated for the treatment of early-stage Parkinson's disease. Herein, we report a new short chemoenzymatic approach (in 2 steps) towards the synthesis of (R)-selegiline via stereoselective biocatalytic reductive amination as the key step. The imine reductase IR36-M5 mutant showed high conversion (97%) and stereoselectivity (97%) toward the phenylacetone and propargyl amine substrates, offering valuable biocatalysts for synthesizing alkylated homobenzylic amines.

Divergent Biosynthesis of Bridged Polycyclic Sesquiterpenoids by a Minimal Fungal Biosynthetic Gene Cluster.

The bridged polycyclic sesquiterpenoids derived from sativene, isosativene, and longifolene have unique structures, and many chemical synthesis approaches with at least 10 steps have been reported. However, their biosynthetic pathway remains undescribed. A minimal biosynthetic gene cluster (BGC), named bip, encoding a sesquiterpene cyclase (BipA) and a cytochrome P450 (BipB) is characterized to produce such complex sesquiterpenoids with multiple carbon skeletons based on enzymatic assays, heterologous expression, and precursor experiments. BipA is demonstrated as a versatile cyclase with (-)-sativene as the dominant product and (-)-isosativene and (-)-longifolene as minor ones. BipB is capable of hydroxylating different enantiomeric sesquiterpenes, such as (-)-longifolene and (+)-longifolene, at C-15 and C-14 in turn. The C-15- or both C-15- and C-14-hydroxylated products are then further oxidized by unclustered oxidases, resulting in a structurally diverse array of sesquiterpenoids. Bioinformatic analysis reveals the BipB homologues as a discrete clade of fungal sesquiterpene P450s. These findings elucidate the concise and divergent biosynthesis of such intricate bridged polycyclic sesquiterpenoids, offer valuable biocatalysts for biotransformation, and highlight the distinct biosynthetic strategy employed by nature compared to chemical synthesis.

Engineered Zea mays phenylalanine ammonia-lyase for improve the catalytic efficiency of biosynthesis trans-cinnamic acid and p-coumaric acid

Phenylalanine ammonia-lyase (PAL) plays a pivotal role in the biosynthesis of phenylalanine. PAL from Zea mays (ZmPAL2) exhibits a bi-function of direct deamination of L-phenylalanine (L-Phe) or L-tyrosine(-L-Tyr) to form trans-cinnamic acid or p-coumaric acid. trans-Cinnamic acid and p-coumaric acid are mainly used in flavors and fragrances, food additives, pharmaceutical and other fields. Here, the Activity of ZmPAL2 toward L-Phe or L-Tyr was improved by using semi-rational and rational designs. The catalytic efficiency (kcat/Km) of mutant PT10 (V258I/I459V/Q484N) against L-Phe was 30.8 μM−1 s−1, a 4.5-fold increase compared to the parent, and the catalytic efficiency of mutant PA1 (F135H/I459L) to L-tyrosine exhibited 8.6 μM−1 s−1, which was 1.6-fold of the parent. The yield of trans-cinnamic acid in PT10 reached 30.75 g/L with a conversion rate of 98%. Meanwhile, PA1 converted L-Tyr to yield 3.12 g/L of p-coumaric acid with a conversion rate of 95%. Suggesting these two engineered ZmPAL2 to be valuable biocatalysts for the synthesis of trans-cinnamic acid and p-coumaric acid. In addition, MD simulations revealed that the underlying mechanisms of the increased catalytic efficiency of both mutant PT10 and PA1 are attributed to the substrate remaining stable within the pocket and closer to the catalytically active site. This also provides a new perspective on engineered PAL.

Mutability landscape guided engineering of a promiscuous microbial glycosyltransferase for regioselective synthesis of salidroside and icariside D2

Microbial glycosyltransferases efficiently synthesize glucosides and have garnered increasing interest. However, limited regioselectivity has impeded their broad application, particularly in the pharmaceutical industry. In this study, the UDP-glycosyltransferase YjiC from Bacillus licheniformis (BlYjiC) was engineered to achieve the bidirectional regioselective glycosylation of tyrosol and its derivatives. Initially, site-directed saturation mutagenesis was performed on two newly identified substrate-binding cavities in the acceptor pocket of BlYjiC to provide a comprehensive blueprint of the interplay between mutations and function (mutability landscape). Iterative saturation mutagenesis was performed, guided by the mutability landscape. Two highly regioselective mutants M6 (M112L/I325Y/L70R/Q136E/I67E/M77R) and M2’ (M112D/I62L) were generated, exhibiting >99 % regioselectivity toward the alcoholic and phenolic hydroxyl of tyrosol, respectively, compared with the wild-type (product mixture: 51:49 %). Both mutants exhibited excellent regioselectivity toward several dihydroxy phenolic substrates, offering valuable biocatalysts for the regioselective synthesis of glucosides. Their application was confirmed in a short synthesis of salidroside (3.6 g/L) and icariside D2 (2.4 g/L), which exhibited near-perfect regioselectivity. This study provides valuable insights into future protein engineering of similar enzymes and opens new avenues for their practical applications.

Computation-guided engineering of distal mutations in an artificial enzyme.

Artificial enzymes are valuable biocatalysts able to perform new-to-nature transformations with the precision and (enantio-)selectivity of natural enzymes. Although they are highly engineered biocatalysts, they often cannot reach catalytic rates akin those of their natural counterparts, slowing down their application in real-world industrial processes. Typically, their designs only optimise the chemistry inside the active site, while overlooking the role of protein dynamics on catalysis. In this work, we show how the catalytic performance of an already engineered artificial enzyme can be further improved by distal mutations that affect the conformational equilibrium of the protein. To this end, we subjected a specialised artificial enzyme based on the lactococcal multidrug resistance regulator (LmrR) to an innovative algorithm that quickly inspects the whole protein sequence space for hotpots which affect the protein dynamics. From an initial predicted selection of 73 variants, two variants with mutations distant by more than 11 Å from the catalytic pAF residue showed increased catalytic activity towards the new-to-nature hydrazone formation reaction. Their recombination displayed a 66% higher turnover number and 14 °C higher thermostability. Microsecond time scale molecular dynamics simulations evidenced a shift in the distribution of productive enzyme conformations, which are the result of a cascade of interactions initiated by the introduced mutations.

Thermoalkalophilic polygalacturonase from a novel Glutamicibacter sp.: Bioprospecting, strain improvement, statistical optimization and applications

The current research discusses a multidimensional bioprocess development, that includes bioprospecting, strain improvement, media optimisation, and applications of the extracted enzyme. A potent alkalophilic polygalacturonase (PG) producing bacterial strain was isolated and identified as a novel Glutamicibacter sp. Furthermore, strain improvement by UV and chemical mutagenesis not only improved the enzyme (PGmut) production but also enhanced its temperature optima from 37 °C to 50 °C. The use of solid substrate fermentation, followed bystatistical optimisation through PB and RSM, substantially increasedPGmut production. A 10-fold increase in enzyme production (632 U/gm) was observed when sugarcane bagasse with a pH of 10.5, 66.8 % moisture, and an inoculum size of 10.15 % was used. The model’s accuracy was supported by p-value (p < 0.0001), and an R2 of 0.9940. A pilot-scale experiment, demonstrated ≈ 62,229 U/100 gm PG activity. Additionally, the enzyme’s efficacy in demucilization of coffee beans, and bioscouring of jute fibre indicated that it is a valuable biocatalyst.

Discovery and structural characterization of a thermostable bacterial monoamine oxidase.

Monoamine oxidases (MAOs) are pivotal regulators of neurotransmitters in mammals, while microbial MAOs have been shown to be valuable biocatalysts for enantioselective synthesis of pharmaceutical compounds or precursors thereof. To extend the knowledge of how MAOs function at the molecular level and in order to provide more biocatalytic tools, we set out to identify and study a robust bacterial variant: a MAO from the thermophile Thermoanaerobacterales bacterium (MAOTb ). MAOTb is highly thermostable with melting temperatures above 73 °C and is well expressed in Escherichia coli. Substrate screening revealed that the oxidase is most efficient with n-alkylamines with n-heptylamine being the best substrate. Presteady-state kinetic analysis shows that reduced MAOTb rapidly reacts with molecular oxygen, confirming that it is a bona fide oxidase. The crystal structure of MAOTb was resolved at 1.5 Å and showed an exceptionally high similarity with the two human MAOs, MAO A and MAO B. The active site of MAOTb resembles mostly the architecture of human MAO A, including the cysteinyl protein-FAD linkage. Yet, the bacterial MAO lacks a C-terminal extension found in human MAOs, which explains why it is expressed and purified as a soluble protein, while the mammalian counterparts are anchored to the membrane through an α-helix. MAOTb also displays a slightly different active site access tunnel, which may explain the specificity toward long aliphatic amines. Being an easy-to-express, thermostable enzyme, for which a high-resolution structure was elucidated, this bacterial MAO may develop into a valuable biocatalyst for synthetic chemistry or biosensing.

Psychrophiles as a novel and promising source of cold-adapted industrial enzymes

Psychrophiles are an exclusive group of microbes that thrive in extremely cold environments, such as polar regions and deep-sea. These cold-loving microbes have developed a range of adaptations that enable them to function at low temperatures, including the production of cold-adapted enzymes. These enzymes are highly active and stable in cold environments, making them valuable biocatalysts for various industrial processes. The potential applications of psychrophilic enzymes are vast, including in the food, pharmaceutical, and bioremediation industries. Cold-adapted enzymes are particularly useful in low-temperature applications, such as in the production of coldprocessed foods and cold-water detergents. They can also be used in the production of antibiotics and other pharmaceuticals that require low-temperature conditions. Additionally, psychrophilic enzymes can be used in bioremediation processes, where lowtemperature conditions are often encountered. Metagenomic studies have revealed the potential for discovering new psychrophilic enzymes from uncultivated microorganisms in cold environments. The use of recombinant DNA technology has enabled the production of large quantities of psychrophilic enzymes for industrial applications. Psychrophiles represent a novel and promising source of cold-adapted industrial enzymes. The use of these enzymes in various industries has the potential to significantly reduce energy consumption and environmental impact. With ongoing research and technological advancements, more diverse and efficient cold-adapted enzymes will likely be discovered from psychrophilic microorganisms, further expanding the array of applications for these enzymes in the future.

Synthesis of l-DOPA catalyzed by recombinant Escherichia coli expressing tyrosine phenol lyase gene from Desulfitobacterium hafniense

L-DOPA (L-dihydroxyphenylalanine) is a potential drug for the treatment of Parkinson’s disease, and the demand is increasing every year. Tyrosine phenol-lyase (TPL) is a valuable biocatalyst for the biosynthesis of L-tyrosine and its derivatives, which are valuable intermediates in the pharmaceutical industry. In this study, a new TPL gene (Dh-TPL) was cloned from Desulfitobacterium hafniense, ligated into pJJDuet30 vector, and successfully expressed in Escherichia coli. To increase the yield and stability of L-DOPA, as well as decrease the by-product formation, the enzyme production conditions and the catalytic reaction conditions were studied. The optimal TPL production conditions were as follows: the concentration of IPTG was 0.2 mmol/L and the induction temperature was controlled at 18°C. The optimum medium composition for recombinant bacteria includes yeast extract 64 g/L, tryptone 36 g/L, glycerol 4 g/L, KH2PO4 2.31 g/L and K2HPO4 9.4 g/L. The best biosynthesis of L-DOPA was performed in a reaction mixture containing 10.0 g/L catechol, 20 g/L sodium pyruvate and 20 g/L recombinant E. coli resting cells. The optimal reaction temperature and pH were determined to be 18°C and pH 8.5, respectively. Under these conditions, the yield of L-DOPA was 0.21 mol/L (41.7 g/L) after 3 hours reaction.

Diprenylated cyclodipeptide production by changing the prenylation sequence of the nature’s synthetic machinery

Ascomycetous fungi are often found in agricultural products and foods as contaminants. They produce hazardous mycotoxins for human and animals. On the other hand, the fungal metabolites including mycotoxins are important drug candidates and the enzymes involved in the biosynthesis of these compounds are valuable biocatalysts for production of designed compounds. One of the enzyme groups are members of the dimethylallyl tryptophan synthase superfamily, which mainly catalyze prenylations of tryptophan and tryptophan-containing cyclodipeptides (CDPs). Decoration of CDPs in the biosynthesis of multiple prenylated metabolites in nature is usually initiated by regiospecific C2-prenylation at the indole ring, followed by second and third ones as well as by other modifications. However, the strict substrate specificity can prohibit the further prenylation of unnatural C2-prenylated compounds. To overcome this, we firstly obtained C4-, C5-, C6-, and C7-prenylated cyclo-l-Trp-l-Pro. These products were then used as substrates for the promiscuous C2-prenyltransferase EchPT1, which normally uses the unprenylated CDPs as substrates. Four unnatural diprenylated cyclo-l-Trp-l-Pro including the unique unexpected N1,C6-diprenylated derivative with significant yields were obtained in this way. Our study provides an excellent example for increasing structural diversity by reprogramming the reaction orders of natural biosynthetic pathways. Furthermore, this is the first report that EchPT1 can also catalyze N1-prenylation at the indole ring.Key points• Prenyltransferases as biocatalysts for unnatural substrates.• Chemoenzymatic synthesis of designed molecules.• A cyclodipeptide prenyltransferase as prenylating enzyme of already prenylated products.Graphical