Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Computer-Aided Reconstruction and Application of Bacillus halodurans S7 Xylanase with Heat and Alkali Resistance.

ß-1,4-Endoxylanase is the most critical hydrolase for xylan degradation during lignocellulosic biomass utilization. However, its poor stability and activity in hot and alkaline environments hinder its widespread application. In this study, BhS7Xyl from Bacillus halodurans S7 was improved using a computer-aided design through isothermal compressibility (ßT) perturbation engineering and by combining three thermostability prediction algorithms (ICPE-TPA). The best variant with remarkable improvement in specific activity, heat resistance (70 °C), and alkaline resistance (both pH 9.0 and 70 °C), R69F/E137M/E145L, exhibited a 4.9-fold increase by wild-type in specific activity (1368.6 U/mg), a 39.4-fold increase in temperature half-life (458.1 min), and a 57.6-fold increase in pH half-life (383.1 min). Furthermore, R69F/E137M/E145L was applied to the hydrolysis of agricultural waste (corncob and hardwood pulp) to efficiently obtain a higher yield of high-value xylooligosaccharides. Overall, the ICPE-TPA strategy has the potential to improve the functional performance of enzymes under extreme conditions for the high-value utilization of lignocellulosic biomass.

In-situ substitution and community dynamics modeling for enhanced safety in Chinese rice wine brewing.

This research paper focuses on the application of the "Design-Build-Test-Learn" framework to design and evaluate a synthetic microbial community aimed at studying the impact of Lactic Acid Bacteria (LAB) interactions and fitness on the formation of biogenic amines (BAs) in Chinese rice wine (CRW). The study reveals a close correlation between the assembly model of LAB and the accumulation of BAs in CRW, and multiple interactions were observed between amine-producing and non-amine-producing LAB, including commensalism, amensalism, and competition. The commensalism among amine-producing LAB was found to promote BAs accumulation through metabolic cross-feeding of amino acids. Moreover, the higher-order interaction community was designed to regulate the BAs formation effectively. For instance, the interference of Lactiplantibacillus plantarum (ACBC271) resulted in the elimination of amine-producing LAB viability, resulting in a 22% decrease (not exceeding 43.54 mg/L) in the total amount of BAs. Simulation of community dynamics models further suggests that LAB with quantitative social interactions can effectively control LAB accumulation in CRW by forecasting fluctuation in BAs generation through fitness competition and metabolic interference. Overall, this study provides valuable insights into the complex interaction networks within microbial communities in traditional fermentation ecosystems. It also proposes a novel approach for quality control of nitrogen food safety factors in fermented foods.

Cadmium Induces Kidney Iron Deficiency and Chronic Kidney Injury by Interfering with the Iron Metabolism in Rats.

Cadmium (Cd) is a common environmental pollutant and occupational toxicant that seriously affects various mammalian organs, especially the kidney. Iron ion is an essential trace element in the body, and the disorder of iron metabolism is involved in the development of multiple pathological processes. An iron overload can induce a new type of cell death, defined as ferroptosis. However, whether iron metabolism is abnormal in Cd-induced nephrotoxicity and the role of ferroptosis in Cd-induced nephrotoxicity need to be further elucidated. Sprague Dawley male rats were randomly assigned into three groups: a control group, a 50 mg/L CdCl2-treated group, and a 75 mg/L CdCl2-treated group by drinking water for 1 month and 6 months, respectively. The results showed that Cd could induce renal histopathological abnormalities and dysfunction, disrupt the mitochondria's ultrastructure, and increase the ROS and MDA content. Next, Cd exposure caused GSH/GPX4 axis blockade, increased FTH1 and COX2 expression, decreased ACSL4 expression, and significantly decreased the iron content in proximal tubular cells or kidney tissues. Further study showed that the expression of iron absorption-related genes SLC11A2, CUBN, LRP2, SLC39A14, and SLC39A8 decreased in proximal tubular cells or kidneys after Cd exposure, while TFRC and iron export-related gene SLC40A1 did not change significantly. Moreover, Cd exposure increased SLC11A2 gene expression and decreased SLC40A1 gene expression in the duodenum. Finally, NAC or Fer-1 partially alleviated Cd-induced proximal tubular cell damage, while DFO and Erastin further aggravated Cd-induced cell damage. In conclusion, our results indicated that Cd could cause iron deficiency and chronic kidney injury by interfering with the iron metabolism rather than typical ferroptosis. Our findings suggest that an abnormal iron metabolism may contribute to Cd-induced nephrotoxicity, providing a novel approach to preventing kidney disease in clinical practice.

Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi.

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

Effective degradation of anthraquinones in Folium Sennae with Monascus fermentation for toxicity reduce and efficacy enhancement.

Folium Sennae are widely used around the world, mainly in purging and removal of endogenous active substances, such as anthraquinone and its derivatives. However, the potential toxicity of anthraquinones to the liver, kidney, and intestinal limits the application of Folium Sennae. In this study, we aimed at safe regulation of Folium Sennae to degrade anthraquinones, boosting medicinal properties and reducing toxicity and potency with Monascus fermentation. Monascus strains H1102 for Folium Sennae fermentation were selected as the initial strain which was capable of producing high yields of functional pigment and low yields of hazardous citrinin. The anthraquinone degradation rate reached 41.2%, with 212.2 U mL-1 of the pigment and approximately 0.038 mg L-1 of the citrinin under optimal fermentation conditions followed by response surface streamlining, which met the requirements of reducing toxicity, increasing efficiency of Monascus fermented Folium Sennae. Furthermore, the Monascus/Folium Sennae culture had no observable toxic effect on HK-2 and L-02 cells in vitro and further inhibited cell apoptosis and necrosis. Overall, our results showed that Monascus fermentation could provide an alternative strategy for toxicity reduction of herbal medicines as well as efficacy enhancement.

Efficient 2-O-α-D-glucopyranosyl-sn-glycerol production by single whole-cell biotransformation through combined engineering and expression regulation with novel sucrose phosphorylase from Leuconostoc mesenteroides ATCC 8293.

2-O-α-D-glucopyranosyl-sn-glycerol (2-αGG) is a high value product with wide applications. Here, an efficient, safe and sustainable bioprocesses for 2-αGG production was designed. A novel sucrose phosphorylase (SPase) was firstly identified from Leuconostoc mesenteroides ATCC 8293. Subsequently, SPase mutations were processed with computer-aided engineering, of which the activity of SPaseK138C was 160% higher than that of the wild-type. Structural analysis revealed that K138C was a key functional residue moderating substrate binding pocket and thus influences catalytic activity. Furthermore, Corynebacterium glutamicum was employed to construct microbial cell factories along with ribosome binding site (RBS) fine-tuning and a two-stage substrate feeding control strategy. The maximum production of 2-αGG by these combined strategies reached 351.8 g·L-1 with 98% conversion rate from 1.4 M sucrose and 3.5 M glycerol in a 5-L bioreactor. This was one of the best performance reported in single-cell biosynthesis of 2-αGG, which paved effective ways for industrial-scale preparation of 2-αGG.

Behavior of enzymes under high pressure in food processing: mechanisms, applications, and developments.

High pressure processing (HPP) offers the benefits of safety, uniformity, energy-efficient, and low waste, which is widely applied for microbial inactivation and shelf-life extension for foods. Over the past forty years, HPP has been extensively researched in the food industry, enabling the inactivation or activation of different enzymes in future food by altering their molecular structure and active site conformation. Such activation or inactivation of enzymes effectively hinders the spoilage of food and the production of beneficial substances, which is crucial for improving food quality. This paper reviews the mechanism in which high pressure affects the stability and activity of enzymes, concludes the roles of key enzymes in the future food processed using high pressure technologies. Moreover, we discuss the application of modified enzymes based on high pressure, providing insights into the future direction of enzyme evolution under complex food processing conditions (e.g. high temperature, high pressure, high shear, and multiple elements). Finally, we conclude with prospects of high pressure technology and research directions in the future. Although HPP has shown positive effects in improving the future food quality, there is still a pressing need to develop new and effective combined processing methods, upgrade processing modes, and promote sustainable lifestyles.

Redesign of γ-glutamyl transpeptidase from Bacillus subtilis for high-level production of L-theanine by cavity topology engineering.

L-Theanine is a multifunctional nonprotein amino acid found naturally in tea leaves. It has been developed as a commercial product for a wide range of applications in the food, pharmaceutical, and healthcare industries. However, L-theanine production catalyzed by γ-glutamyl transpeptidase (GGT) is limited by the low catalytic efficiency and specificity of this class of enzymes. Here, we developed a strategy for cavity topology engineering (CTE) based on the cavity geometry of GGT from B. subtilis 168 (CGMCC 1.1390) to obtain an enzyme with high catalytic activity and applied it to the synthesis of L-theanine. Three potential mutation sites, M97, Y418, and V555, were identified using the internal cavity as a probe, and residues G, A, V, F, Y, and Q, which may affect the shape of the cavity, were obtained directly by computer statistical analysis without energy calculations. Finally, 35 mutants were obtained. The optimal mutant Y418F/M97Q showed a 4.8-fold improvement in catalytic activity and a 25.6-fold increase in catalytic efficiency. The recombinant enzyme Y418F/M97Q exhibited a high space-time productivity of 15.4 g L-1 h-1 by whole-cell synthesis in a 5 L bioreactor, which was one of the highest concentrations reported so far at 92.4 g L-1. Overall, this strategy is expected to enhance the enzymatic activity associated with the synthesis of L-theanine and its derivatives.Key points • Cavity topology engineering was used to modify the GGT for L-theanine biocatalysis. • The catalytic efficiency of GGT was increased by 25.6-fold. • Highest productivity of L-theanine reached 15.4 g L -1 h-1 (92.4 g L-1) in a 5 L bioreactor.

Isothermal Compressibility Perturbation as a Protein Design Principle for T1 Lipase Stability-Activity Trade-Off Counteracting.

Given the widely existing stability-activity trade-off in enzyme evolution, it is still a goal to obtain enzymes embracing both high activity and stability. Herein, we employed an isothermal compressibility (ßT) perturbation engineering (ICPE) strategy to comprehensively understand the stability-activity seesaw-like mechanism. The stability and activity of mutants derived from ICPE uncovered a high Pearson correlation (r = 0.93) in a prototypical enzyme T1 lipase. The best variant A186L/L188M/A190Y exhibited a high Tm value up to 78.70 °C, catalytic activity of 474.04 U/mg, and a 73.33% increase in dimethyl sulfoxide resistance compared to the wild type, one of the highest comprehensive performances reported to date. The elastic activation mechanism mediated by conformational change with a ΔßT range of -6.81 × 10-6 to -1.90 × 10-6 bar-1 may account for the balancing of stability and activity to achieve better performing enzymes. The ICPE strategy deepens our understanding of stability-activity trade-off and boosts its applications in enzyme engineering.

Inside Out Computational Redesign of Cavities for Improving Thermostability and Catalytic Activity of Rhizomucor Miehei Lipase.

Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (Tm) of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The Tm of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg-1), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.

Improving ethanol tolerance of ethyl carbamate hydrolase by diphasic high pressure molecular dynamic simulations.

Ethyl carbamate (EC) is mainly found in fermented foods and fermented alcoholic beverages, which could cause carcinogenic potential to humans. Reducing EC is one of the key research priorities to address security of fermented foods. Enzymatic degradation of EC with EC hydrolase in food is the most reliable and efficient method. However, poor tolerance to ethanol severely hinders application of EC hydrolase. In this study, the mutants of EC hydrolase were screened by diphasic high pressure molecular dynamic simulations (dHP-MD). The best variant with remarkable improvement in specific activity and was H68A/K70R/S325N, whose specific activity was approximately 3.42-fold higher than WT, and relative enzyme activity under 20% (v/v) was 5.02-fold higher than WT. Moreover, the triple mutant increased its stability by acquiring more hydration shell and forming extra hydrogen bonds. Furthermore, the ability of degrading EC of the immobilized triple mutant was both detected in mock wine and under certain reaction conditions. The stability of immobilized triple mutant and WT were both improved, and immobilized triple mutant degraded nearly twice as much EC as that of immobilized WT. Overall, dHP-MD was proved to effectively improve enzyme activity and ethanol tolerance for extent application at industrial scale.

Protein-Glutaminase Engineering Based on Isothermal Compressibility Perturbation for Enhanced Modification of Soy Protein Isolate.

Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions.

[Mechanisms and applications of enzyme-catalyzed protein cross-linking].

Protein cross-linking plays important roles in food, chemical, medicine and other fields. Enzyme-catalyzed protein cross-linking is an efficient and economically viable alternative to physical and chemical cross-linking. However, detailed analysis of enzyme-catalyzed protein cross-linking at molecular level is still lacking. This review summarized the mechanisms of enzyme-catalyzed protein cross-linking, its effects on protein structure, and its applications in food, chemical and pharmaceutical fields.

Semi-quantitative activity assays for high-throughput screening of higher activity gamma glutamyl transferase and enzyme immobilization to efficiently synthesize L-theanine.

The bio-production of theanine is currently of significant interest due to its wide applications in food and healthcare products. Gamma glutamyl transferase (GGT) has been widely applied in L-theanine synthesis, but L-theanine yields remain prohibitively low for commercial production. In this study, a robust high-throughput screening process for isolating GGT mutants was developed through a combination of error-prone PCR techniques and a colorimetric reaction. The co-expression of PrsA lipoprotein enhances the secretion of GGT, thus GGT could be obtained quickly and easily without crushing cells. Random mutations on ggt genes were introduced by using error-prone PCR kits to build a large mutant library. A colorless compound generated by the reaction between NH4+ (released from L-theanine synthesis) and OPA was measured quantitatively by UV/visible spectroscopy when mixed with TCA and DMSO. Approximately 30 positive clones with improved color formation on the 96-well plates were identified, and mutants T413P and T463S with more than by 30 % higher transpeptidation activity versus the original GGT were isolated. To improve the operational stability and economical use, mutant GGT was immobilized on a prepared oxidized cellulose nanofiber membrane. The remaining activity of immobilized GGT was 88 % versus 72 % of free enzyme over 15 h. A fed-batch conversion was performed with the immobilized GGT, and over 70 g/L L-theanine could be accumulated within 18 h after feeding twice. Versus other studies, this is one of the best L-theanine synthesis systems using immobilized GGT.

Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli.

Several approaches for efficient production of cadaverine, a bio-based diamine with broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-type WT (181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations.

[Effect of key notes of TCA cycle on L-glutamate production].

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Puerarin restores the autophagic flux to alleviate cadmium­induced endoplasmic reticulum stress in NRK­52E cells.

Cadmium (Cd) is a heavy metal that can accumulate and cause damage to a variety of tissues and organs. The kidney is the primary target organ for Cd accumulation and toxic damage. Autophagy, which is a critical intracellular process, serves an important role in maintaining the homeostasis of the intracellular environment. Endoplasmic reticulum stress (ERS) is another key process that functions to promote cell survival or results in cell injury and death. Both autophagy and ERS are associated with oxidative stress; however, the mechanism by which ERS is regulated by autophagy in Cd­induced nephrotoxicity remains unclear. The present study employed a rat NRK­52E cell model, where alterations in cell morphology, density and viability, the accumulation of reactive oxygen species, an increase in malondialdehyde generation and a decrease in antioxidant enzyme activity and apoptosis were induced by Cd treatment. Cd induced the activation of nuclear factor erythroid 2­related factor 2 (NRF2), an obstruction of autophagic flux and ERS, which were attenuated by puerarin administration. Furthermore, puerarin failed to alleviate ERS following knockdown of autophagy­related protein 7 in NRK­52E cells. Overexpression of Ras­related protein Rab­7, which promotes the fusion of autophagosomes and lysosomes, efficiently reduced ERS. Taken together, these results indicate that puerarin administration restored the autophagic flux to alleviate ERS, via blocking the activation of NRF2.

Engineered disulfide bonds improve thermostability and activity of L-isoleucine hydroxylase for efficient 4-HIL production in Bacillus subtilis 168.

4-Hydroxyisoleucine, a promising drug, has mainly been applied in the clinical treatment of type 2 diabetes in the pharmaceutical industry. l-Isoleucine hydroxylase specifically converts l-Ile to 4-hydroxyisoleucine. However, due to its poor thermostability, the industrial production of 4-hydroxyisoleucine has been largely restricted. In the present study, the disulfide bond in l-isoleucine hydroxylase protein was rationally designed to improve its thermostability to facilitate industrial application. The half-life of variant T181C was 4.03 h at 50°C, 10.27-fold the half-life of wild type (0.39 h). The specific enzyme activity of mutant T181C was 2.42 ± 0.08 U/mg, which was 3.56-fold the specific enzyme activity of wild type 0.68 ± 0.06 U/mg. In addition, molecular dynamics simulation was performed to determine the reason for the improvement of thermostability. Based on five repeated batches of whole-cell biotransformation, Bacillus subtilis 168/pMA5-ido T181C recombinant strain produced a cumulative yield of 856.91 mM (126.11 g/L) 4-hydroxyisoleucine, which is the highest level of productivity reported based on a microbial process. The results could facilitate industrial scale production of 4-hydroxyisoleucine. Rational design of disulfide bond improved l-isoleucine hydroxylase thermostability and may be suitable for protein engineering of other hydroxylases.

Directed Evolution of Ornithine Cyclodeaminase Using an EvolvR-Based Growth-Coupling Strategy for Efficient Biosynthesis of l-Proline.

l-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of l-proline is from l-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, l-proline can be also synthesized in l-ornithine and/or l-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving l-proline production from l-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCDK205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate l-proline biosynthesis in l-arginine overproducing Corynebacterium crenatum. The strain PS6 with best performance reached 15.3 g/L l-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual l-ornithine and/or l-arginine.