Molar Conversion Rate Research Articles

An engineered dual-functional L-DOPA decarboxylase enables a minimized hydroxytyrosol cascade

Hydroxytyrosol has been proven beneficial to human health. However, the process involving the conversion of L-DOPA to 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) in hydroxytyrosol biosynthesis typically required the simultaneous use of decarboxylase and oxidative deaminase. In addition, phenylacetaldehyde reductase from Solanum lycopersicum (SlPAR) in hydroxytyrosol biosynthesis exhibits poor thermal stability. In this study, we unexpectedly discovered that L-DOPA decarboxylase from Pseudomonas putida (PpDODC) exhibits weak dual-functional activity for both decarboxylation and oxidative deamination of L-DOPA. Through a dual-function reshaping strategy, the best dual-functional mutant PpDODC/Y79F/Y324F achieved an enzyme activity of 0.95 U/mg and exhibited a 256.8-fold increase in activity compared to the wild-type. Through rational design of SlPAR, the optimal mutant SlPAR/G52D/V188I/N234W/Q286P (SlPAR-M4) maintained stability after 12 h' treatment at 40 °C. Based on these mutants, we established a simplified cascade to synthesize hydroxytyrosol from L-DOPA, achieving a hydroxytyrosol yield of 31.4 mM from 32 mM L-DOPA in a 5-hour reaction. This process achieved the highest molar conversion rate (98.2 %) currently reported for the synthesis of hydroxytyrosol from L-DOPA. This study provides a novel solution for hydroxytyrosol synthesis from a new perspective, and the mutants hold promise for widespread application in biotransformation studies of hydroxytyrosol and other structurally-similar compounds.

Metabolic engineering of Escherichia coli for upcycling of polyethylene terephthalate waste to vanillin.

Polyethylene terephthalate (PET) waste presents a significant environmental challenge due to its durability and resistance to degradation. Innovative approaches for upcycling PET waste into high-value chemicals can mitigate these issues while contributing to a circular economy. In this study, we developed a multi-enzyme cascade system in E. coli to convert PET-derived monomer terephthalic acid (TPA) into vanillin (VAN). The metabolic engineering approach was then employed to increase VAN production, including 1) inhibition of VAN degradation by knocking out endogenous aldehyde reductases and alcohol dehydrogenases and 2) enhancement of TPA uptake by modifying membrane proteins to increase cell permeability. The engineered E. coli demonstrated a VAN production of 658.55 mg/L from 1992 mg/L of TPA with a molar conversion rate of 71.1 %, representing the highest production of VAN using TPA as the substrate. Additionally, the engineered E. coli effectively converted post-consumer PET waste into VAN under mild conditions, with the highest production of 259.2 mg/L in 20× diluted PET hydrolysates, highlighting its potential for application in PET waste upcycling. This approach not only provides an environmentally friendly alternative to traditional chemical synthesis but also offers substantial economic benefits by transforming low-value waste into high-value chemicals.

Production of neohesperidin from hesperetin by an engineered strain of Escherichia coli

Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana, rhamnose synthase VvRHM-NRS from Vitis vinifera, and rhamnose transferase Cm1,2RhaT from Citrus maxima. After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced 4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

Cloning and Direct Evolution of a Novel 7-O-Glycosyltransferase from Cucurbita moschata and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-O-glucoside.

Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Dihydro-β-ionone production by a one-pot enzymatic cascade of a short-chain dehydrogenase NaSDR and enoate reductase AaDBR1

Dihydro-β-ionone, a high-value compound with distinctive fragrance, is widely utilized in the flavor and fragrance industries. However, its low abundance in plant sources poses a significant challenge to its application through traditional extraction methods. Development of an enzyme cascade reaction with artificial design offers a promising alternative. Herein, a short-chain dehydrogenase NaSDR, was identified from Novosphingobium aromaticivorans DSM 12444, which exhibited a high activity in converting β-ionol to β-ionone. A novel biosynthesis route to produce dihydro-β-ionone from β-ionol was developed, by utilizing alcohol dehydrogenase NaSDR and enoate reductase AaDBR1. Under the optimized conditions (0.29 mg/mL NaSDR, 0.39 mg/mL AaDBR1, 1 mM NADP+ and 2.5 mM β-ionol at 40 °C for 2 h), a maximum yield (173.11 mg/L) of dihydro-β-ionone was achieved with a molar conversion rate of 35.6 %, which was 2.7-fold higher than that before optimization. Additionally, this cascade reaction achieved self-sufficient NADPH regeneration through the actions of NaSDR and AaDBR1. This study offered a fresh perspective for achieving a green and sustainable synthesis of dihydro-β-ionone and could inspire on another natural products biosynthesis.

Construction and Application of a Multienzyme System for Synthesis of L-malate.

This study aimed to develop a multienzymatic system for synthesis of L-malate. First, recombinant Escherichia coli strains were constructed expressing maleic acid cis-trans isomerase (MaiA) or fumarase C (FumC) from different sources. Serratia marcescens MaiA (SMaiA) and E. coli FumC (ECFumC) showed good catalytic performance. Next, six co-expression systems for SMaiA and ECFumC were constructed. E. coli BL21 (DE3)-pRSFDuet-1-ecfumC-smaiA (named strain pFM2) had the highest L-malate catalytic activity. In 7-L fed-batch fermentation, the SMaiA and ECFumC activities of strain pFM2 wet cells were 43.4 and 154.5 U/g, respectively, 2.4- and 10.7-fold the values that were obtained in shaken flasks. Finally, a whole-cell catalytic process was established for the production of L-malate by strain pFM2 with maleate as the substrate. When the dose of pFM2 wet cells was 0.5g/100mL and 1mol/L maleate was the substrate, the catalytic process was completed within 4h. Notably, the intermediate fumarate was almost absent during the conversion process. The concentration of L-malate reached 143.8g/L with a yield of 0.60g/(L·min). The molar conversion rate of the substrate was 98.4%. These findings lay a foundation for the industrial application of multienzymatic synthesis of L-malate.

Enzymatic production of 1,4-cyclohexanedimethylamine

1,4-cyclohexanedimethylamine (1,4-BAC) is an important monomer for bio-based materials, it finds wide applications in various fields including organic synthesis, medicine, chemical industry, and materials. At present, its synthesis primarily relies on chemical method, which suffer from issues such as expensive metal catalyst, harsh reaction conditions, and safety risks. Therefore, it is necessary to explore greener alternatives for its synthesis. In this study, a two-bacterium three-enzyme cascade conversion pathway was successfully developed to convert 1,4-cyclohexanedicarboxaldehyde to 1,4-cyclohexanedimethylamine. This pathway used Escherichia coli derived aminotransferase (EcTA), Saccharomyces cerevisiae derived glutamate dehydrogenase (ScGlu-DH), and Candida boidinii derived formate dehydrogenase (CbFDH). Through structure-guided protein engineering, a beneficial mutant, EcTAF91Y, was obtained, exhibiting a 2.2-fold increase in specific activity and a 1.9-fold increase in kcat/Km compared to that of the wild type. By constructing recombinant strains and optimizing reaction conditions, it was found that under the optimal conditions, a substrate concentration of 40 g/L could produce (27.4±0.9) g/L of the product, corresponding to a molar conversion rate of 67.5%±2.1%.

Enhancing the Thermostability and Catalytic Activity of the Lipase from Rhizopus oryzae via Introducing N-Glycosylation.

Lipase from Rhizopus oryzae (ROL) exhibits remarkable sn-1,3 stereoselectivity and catalytic activity, but its poor thermostability limits its applications in the production of 1,3-dioleoyl-2-palmitoyl glycerol (OPO, a high-quality substitute for human milk fat). In this work, a semirational method was proposed to engineer the thermostability and catalytic activity of 4M (ROL mutant in our previous study). First, a computer-aided design is performed using 4M as a template, and N-glycosylation mutants are then recombinantly expressed and screened in Pichia pastoris, the optimal mutant N227 exhibited a half-life of 298.8 h at 45 °C, which is 7.23-folds longer than that of 4M. Its catalytic activity also reached 1043.80 ± 61.98 U/mg, representing a 29.2% increase compared to 4M (808.02 ± 47.02 U/mg). Molecular dynamics simulations of N227 suggested that the introduction of glycan enhanced the protein rigidity, while the strong hydrogen bonds formed between the glycan and the protein stabilized the lipase structure, thereby improving its thermostability. The acidolysis reaction between oleic acid (OA) and glycerol tripalmitate (PPP) was successfully carried out using immobilized N227, achieving a molar conversion rate of 90.2% for PPP. This engineering strategy guides the modification of lipases, while the glycomutants obtained in this study have potential applications in the biosynthesis of OPO.

Enhancing L-asparagine Production Through In Vivo ATP Regeneration System Utilizing Glucose Metabolism of Escherichia coli.

L-asparaginase synthetase, an ATP-dependent enzyme, necessitates ATP for its catalytic activity. However, the integration of L-asparaginase synthetase into industrial processes is curtailed by the prohibitive cost of ATP. To address this limitation, this study explores the construction of an efficient ATP regeneration system using the glucose metabolism of Escherichia coli, synergistically coupled with L-asparaginase synthetase catalysis. The optimal conditions for L-asparagine yield were determined in shake flasks. A total of 2.7 g/L was the highest yield achieved under specific parameters, including 0.1 mol/L of substrate, 0.2 mol/L glucose, 0.01 mol/L MgCl2 at pH 7.5, a temperature of 37 °C, and agitation at 300 r/min over 12 h. The process was then scaled to a 3-L fermenter, optimizing the addition rates of the substrate and magnesium chloride, and employing a constant glucose feed of 10 g/L/h. The scale-up process led to a significant enhancement in the production of L-asparagine. The yield of L-asparagine was increased to 38.49 g/L after 20 h of conversion, and the molar conversion rate reached 29.16%. This strategy has proven to be effective in improving the efficiency of L-asparagine production. When compared to in vitro ATP regeneration methods, this in vivo approach showcased superior efficiency and reduced costs. These findings furnish pivotal insights that may propel the enzymatic synthesis of L-asparagine toward viable industrial application.

Deep eutectic solvent-oriented integration of eco-friendly and efficient indican extraction and enzyme-based indigo preparation

Indigo, one of the oldest dyes, finds widespread usage across multiple industries, encompassing food, medicine, printing, dyeing, as well as cosmetics. Indican is recognized as a precursor substance crucial for the synthesis of indigo, however, the current indican extraction and indigo preparation are largely limited due to the low efficiency of indican extraction, by-product production, as well as serious environmental pollution. To address the serial of issues, we developed deep eutectic solvent (DESs)-oriented integration of the efficient extraction of indican and the green preparation of indigo from Strobilanthes cusia (Nees) Kuntze. DESs were rationally designed and screened to make them able to destroy cell walls to facilitate the precipitation of the desired substance for specific indican extraction. And then optimized to obtain the final extraction rate of 22.8 mg/g, which present the highest so far. More importantly, without additional treatment for DES-containing indican extractive, the β-glucosidase Asbg1 was able to convert indican to indigo at a molar conversion rate of 74.6%. By fully harnessing plant resources, the study introduced an innovative process that bypassed extreme acid-base conditions, ensuring rapidness and minimal pollution. This approach offered a promising avenue for the preparation of indigo, paving the way for sustainable production methods.

Sustainable cellobionic acid biosynthesis from starch via artificial in vitro synthetic enzymatic biosystem

Cellobionic acid (CBA), a kind of aldobionic acid, offers potential applications in the fields of pharmaceutical, cosmetic, food, and chemical industry. To tackle the high cost of the substrate cellobiose in CBA production using quinoprotein glucose dehydrogenase, this study developed a coenzyme-free and phosphate-balanced in vitro synthetic enzymatic biosystem (ivSEBS) to enable the sustainable CBA synthesis from cost-effective starch in one-pot via the CBA-synthesis module and gluconic acid-supply module. The metabolic fluxes of this artificial biosystem were strengthened using design−build−test−analysis strategy, which involved exquisite pathway design, meticulous enzyme selection, module validation and integration, and optimization of the key enzyme dosage. Under the optimized conditions, a remarkable concentration of 6.2 g/L CBA was achieved from initial 10 g/L maltodextrin with a starch-to-CBA molar conversion rate of 60 %. Taking into account that the biosystem simultaneously accumulated 3.6 g/L of gluconic acid, the maltodextrin utilization rate was calculated to be 93.3 %. Furthermore, a straightforward scaling-up process was performed to evaluate the industrial potential of this enzymatic biosystem, resulting in a yield of 21.2 g/L CBA from 50 g/L maltodextrin. This study presents an artificial ivSEBS for sustainable production of CBA from inexpensive starch, demonstrating an alternative paradigm for biomanufacturing of other aldobionic acids.

Construction of an engineered Escherichia coli for effective synthesis of 2′-fucosyllactose via the salvage pathway

2′-Fucosyllactose (2′-FL) is one of the important functional oligosaccharides in breast milk. So far, few attempts on biosynthesis of 2′-FL by the salvage pathway have been reported. Herein, the salvage pathway enzyme genes were introduced into the E. coli BL21star(DE3) for synthesis of 2′-FL. The 2′-FL titer increased from 1.56 to 2.13 g/L by deleting several endogenous genes on competitive pathways. The α-1,2-fucosyltransferase (WbgL) was selected, and improved the 2′-FL titer to 2.88 g/L. Additionally, the expression level of pathway enzyme genes was tuned through optimizing the plasmid copy number. Furthermore, the spatial distribution of WbgL was enhanced by fusing with the MinD C-tag. After optimizing the fermentation conditions, the 2′-FL titer reached to 7.13 g/L. The final strain produced 59.22 g/L of 2′-FL with 95% molar conversion rate of lactose and 92% molar conversion rate of fucose in a 5 L fermenter. These findings will contribute to construct a highly efficient microbial cell factory to produce 2′-FL or other HMOs.

Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose

D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s·mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35 ℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

Microbial production of cis,cis-muconic acid from aromatic compounds in engineered Pseudomonas

Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons. Pseudomonas spp. have broad metabolic potential for degrading aromatic compounds. The objective of this study was to develop a “biological funneling” strategy based on genetic modification to convert complex aromatic compounds into cis,cis-muconate (ccMA) using Pseudomonas putida B6-2 and P. brassicacearum MPDS as biocatalysts. The engineered strains B6-2 (B6-2ΔcatBΔsalC) and MPDS (MPDSΔsalC(pUCP18k-catA)) thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA, attaining molar conversions of 95.3% (ccMA/biphenyl) and 100% (ccMA/naphthalene). Under mixed substrates, B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration. Inserting exogenous clusters like bedDC1C2AB and xylCMAB allowed B6-2 recombinant strains to convert benzene and toluene to ccMA. In mixed substrates, constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene; the highest molar conversion rate of ccMA from mixed substrates was 85.2% when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA) incubation with biphenyl and naphthalene. This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.

Heterologous Expression and Characterization of a Thermostable α-L-Rhamnosidase from Thermoclostridium stercorarium subsp. thermolacticum DSM 2910 and Its Application in the Biotransformation of Rutin.

An α-L-rhamnosidase gene from Thermoclostridium. stercorarium subsp. thermolacticum DSM 2910 (TstRhaA) was cloned and expressed. The maximum TstRhaA activity of the protein reached 25.2 U/ml, and the molecular mass was approximately 106.6 kDa. The protein was purified 8.0-fold by Ni-TED affinity with an overall recovery of 16.6% and a specific activity of 187.9 U/mg. TstRhaA activity was the highest at 65°C and pH 6.5. In addition, it exhibited excellent thermal stability, better pH stability, good tolerance to low concentrations of organic reagents, and high catalytic activity for p-nitrophenyl-α-L-rhamnopyranoside (pNPR). Substrate specificity studies showed that TstRhaA exhibited a high specific activity for rutin. At 60°C, pH 6.5, and 0.3 U/ml enzyme dosage, 60 g/l rutin was converted to 45.55 g/l isoquercitrin within 150 min. The molar conversion rate of rutin and the yield of isoquercitrin were 99.8% and 12.22 g/l/h, respectively. The results suggested that TstRhaA could be used for mass production of isoquercitrin.

Efficient production of icariin and baohuoside I from Epimedium Folium flavonoids by fungal α-l-rhamnosidase hydrolysing regioselectively the terminal rhamnose of epimedin C

Industrial application of icariin and baohuoside I has been hindered by the short supply to a great extent. In this work, a novel GH78 α-l-rhamnosidase AmRha catalyzed the bioconversion of low-value epimedin C in crude Epimedium Folium flavonoids (EFs) to icariin and baohuoside I was developed. Firstly, the high-level expression of AmRha in Komagataella phaffii GS115 attained an enzyme activity of 571.04 U/mL. The purified recombinant AmRha could hydrolyze α-1,2-rhamnoside bond between two rhamnoses (α-Rha(2 → 1)α-Rha) in epimedin C to produce icariin with a molar conversion rate of 92.3%, in vitro. Furtherly, the biotransformation of epimedin C to icariin by the recombinant Komagataella phaffii GS115 cells was also investigated, which elevated the EFs concentration by fivefold. In addition, biotransformation of epimedins A-C and icariin in the raw EFs to baohuoside I was fulfilled by a collaboration of AmRha and β-glucosidase/β-xylosidase Dth3. The results obtained here provide a new insight into the preparation of high-value products icariin and baohuoside I from cheap raw EFs.

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid.

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications. Herein, by directed screening of a metagenomic library from unnatural amino acid-enriched environments, a robust leucine dehydrogenase, TvLeuDH, was identified, which exhibited high substrate tolerance and excellent enzymatic activity towards 2-oxobutyric acid. In addition, TvLeuDH has strong affinity for NADH. Subsequently, a three-enzyme co-expression system containing L-threonine deaminase, TvLeuDH, and glucose dehydrogenase was established. By optimizing reaction conditions, 1.5M L-threonine could be converted to L-2-ABA with a 99% molar conversion rate and a space-time yield of 51.5g·L-1 ·h-1 . In this process, no external coenzyme was added. The robustness of TvLeuDH allowed the reaction to be performed without the addition of extra salt as the buffer, demonstrating the simplest reaction system currently reported. These unique properties for the efficient and environmentally friendly production of chiral amino acids make TvLeuDH a particularly promising candidate for industrial applications, which reveals the great potential of directed metagenomics for industrial biotechnology.

Sodium-Ion-Free Fermentative Production of GABA with Levilactobacillus brevis CD0817

Gamma-aminobutyric acid (GABA) has positive effects on many physiological processes. Lactic acid bacterial production of GABA is a future trend. This study aimed to produce a sodium-ion-free GABA fermentation process for Levilactobacillus brevis CD0817. In this fermentation, both the seed and fermentation media used L-glutamic acid instead of monosodium L-glutamate as the substrate. We optimized the key factors influencing GABA formation, adopting Erlenmeyer flask fermentation. The optimized values of the key factors of glucose, yeast extract, Tween 80, manganese ion, and fermentation temperature were 10 g/L, 35 g/L, 1.5 g/L, 0.2 mM, and 30 °C, respectively. Based on the optimized data, a sodium-ion-free GABA fermentation process was developed using a 10-L fermenter. During the fermentation, L-glutamic acid powder was continuously dissolved to supply substrate and to provide the acidic environment essential for GABA synthesis. The current bioprocess accumulated GABA at up to 331 ± 8.3 g/L after 48 h. The productivity of GABA was 6.9 g/L/h and the molar conversion rate of the substrate was 98.1%. These findings demonstrate that the proposed method is promising in the fermentative preparation of GABA by lactic acid bacteria.

A Multi-Enzyme Cascade for Efficient Production of Pyrrolidone from l-Glutamate.

Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.

Molecular engineering of transketolase from Escherichia coli and tartaric semialdehyde biosynthesis

Transketolase (EC 2.2.1.1, TK) is a thiamine diphosphate-dependent enzyme that catalyzes the transfer of a two-carbon hydroxyacetyl unit with reversible C-C bond cleavage and formation. It is widely used in the production of chemicals, drug precursors, and asymmetric synthesis by cascade enzyme catalysis. In this paper, the activity of transketolase TKTA from Escherichia coli K12 on non-phosphorylated substrates was enhanced through site-directed saturation mutation and combined mutation. On this basis, the synthesis of tartaric semialdehyde was explored. The results showed that the optimal reaction temperature and pH of TKTA_M (R358I/H461S/R520Q) were 32 ℃ and 7.0, respectively. The specific activity on d-glyceraldehyde was (6.57±0.14) U/mg, which was 9.25 times higher than that of the wild type ((0.71±0.02) U/mg). Based on the characterization of TKTA_M, tartaric acid semialdehyde was synthesized with 50 mmol/L 5-keto-d-gluconate and 50 mmol/L non-phosphorylated ethanolaldehyde. The final yield of tartaric acid semialdehyde was 3.71 g with a molar conversion rate of 55.34%. Hence, the results may facilitate the preparation of l-(+)-tartaric acid from biomass, and provide an example for transketolase-catalyzed non-phosphorylated substrates.