Isolation and characterization of a novel soil strain, Pseudomonas cepacia BY21, with glutaryl-7-aminocephalosporanic acid acylase activity

A search was undertaken to screen microorganisms in soil which produce an enzyme capable of deacylating glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) to 7-aminocephalosporanic acid (7-ACA). To facilitate screening, a model substrate, glutaryl-p-nitroanilide, and a 7-ACA sensitive strain, Enterobacter taylorae BY312, were used as a color indicator and bioassay, respectively. An isolate, Pseudomonas cepacia BY21, was found to produce glutaryl-7-ACA acylase, of which the activity was optimal at pH 8.0 and 45°C.     

Systems metabolic engineering design: Fatty acid production as an emerging case study

Increasing demand for petroleum has stimulated industry to develop sustainable production of chemicals and biofuels using microbial cell factories. Fatty acids of chain lengths from C₆ to C₁₆ are propitious intermediates for the catalytic synthesis of industrial chemicals and diesel‐like biofuels. The abundance of genetic information available for Escherichia coli and specifically, fatty acid metabolism in E. coli, supports this bacterium as a promising host for engineering a biocatalyst for the microbial production of fatty acids. Recent successes rooted in different features of systems metabolic engineering in the strain design of high‐yielding medium chain fatty acid producing E. coli strains provide an emerging case study of design methods for effective strain design. Classical metabolic engineering and synthetic biology approaches enabled different and distinct design paths towards a high‐yielding strain. Here we highlight a rational strain design process in systems biology, an integrated computational and experimental approach for carboxylic acid production, as an alternative method. Additional challenges inherent in achieving an optimal strain for commercialization of medium chain‐length fatty acids will likely require a collection of strategies from systems metabolic engineering. Not only will the continued advancement in systems metabolic engineering result in these highly productive strains more quickly, this knowledge will extend more rapidly the carboxylic acid platform to the microbial production of carboxylic acids with alternate chain‐lengths and functionalities. Biotechnol. Biotechnol. Bioeng. 2014;111: 849–857. © 2014 Wiley Periodicals, Inc.     

Screening and characterization of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas sp.

Summary Three screening methods were used to isolate GL-7-ACA acylase-producing strains. Three positive isolates were identified with Pseudomonas nitroreducens CCRC 11041 possessing the highest activity, against GL-7-ACA and GL-7-ADCA. No activity was detected when Ceph C or succinyl-7-ACA was used as substrate; glutaric acid was found to be inhibitory. CCRC 11041 could produce maximal GL-7-ACA acylase activity when cultivated on meat extract medium II. The enzyme had a pH optimum of 5.0 and a temperature optimum of 42°C.     

产GL-7-ACA酰化酶重组菌的构建及高表达研究

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。     

Nocardia sp. Carboxylic Acid Reductase: Cloning, Expression, and Characterization of a New Aldehyde Oxidoreductase Family

We have cloned, sequenced, and expressed the gene for a unique ATP- and NADPH-dependent carboxylic acid reductase (CAR) from a Nocardia species that reduces carboxylic acids to their corresponding aldehydes. Recombinant CAR containing an N-terminal histidine affinity tag had K_m values for benzoate, ATP, and NADPH that were similar to those for natural CAR, and recombinant CAR reduced benzoic, vanillic, and ferulic acids to their corresponding aldehydes. car is the first example of a new gene family encoding oxidoreductases with remote acyl adenylation and reductase sites.     

Cloning and co-expression of D-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase genes in Escherichia coli

To convert cephalosporin C to 7-aminocephalosporin (7-ACA), a D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis and a glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase) gene from Pseudomonas were cloned and expressed in recombinant Escherichia coli. For DAAO recombinant strain BL21(DE3)/pET-DAAO, a high DAAO activity of 250 U ml⁻¹ was obtained by a fed-batch culture. A GL-7-ACA acylase gene, in which the signal peptide sequence was deleted, was also successfully expressed in a recombinant E. coli BL21(DE3)/pET-ACY with a high expression level of 3000 U l⁻¹. A novel recombinant strain, BL21(DE3)/pET-DA, harboring both genes of DAAO and GL-7-ACA acylase, was further constructed, and a rather high DAAO activity of 140 U ml⁻¹ and GL-7-ACA acylase activity of 950 U l⁻¹ were simultaneously obtained. This recombinant strain, in which two genes are co-expressed, made it possible to catalyze cephalosporin C into 7-ACA directly.     

Enhancement of glutaryl-7-aminocephalosporanic acid acylase activity of γ-glutamyltranspeptidase of Bacillus subtilis

Semisynthetic cephalosporins, the best-selling antibiotics worldwide, are derived from 7-aminocephalosporanic acid (7-ACA). Currently, in the pharmaceutical industrie, 7-ACA is mainly produced from cephalosporin C by sequential application of D -amino acid oxidase and cephalosporin acylase. Here we study the potential of industrially amenable enzyme γ-glutamyltranspeptidase from Bacillus subtilis for 7-ACA production, since the wild-type γ-glutamyltranspeptidase of B. subtilis has inherent glutaryl-7-aminocephalosporanic acid acylase activity with a k_cat value of 0.0485 s^-1. Its activity has been enhanced by site directed and random mutagenesis. The k_cat/K_m value was increased to 3.41 s^-1 mM^-1 for a E423Y/E442Q/D445N mutant enzyme and the kcat value was increased to 0.508 s^-1 for a D445G mutant enzyme. Consequently, the catalytic efficiency and the turnover rate were improved up to about 1000-fold and 10-fold, compared with the wildtype γ-glutamyltranspeptidase of B. subtilis.     

代谢工程改造大肠杆菌合成己二酸

己二酸是一种具有重要应用价值的二元羧酸,是合成尼龙-66的关键前体。目前,生物法生产己二酸存在生产周期长、生产效率低的问题。本研究选择一株野生型高产琥珀酸菌株大肠杆菌(Escherichia coli) FMME N-2为底盘细胞,首先通过引入逆己二酸降解途径的关键酶,成功构建了可合成0.34 g/L己二酸的E. coli JL00菌株;接着,对合成路径限速酶进行表达优化,使E. coli JL01菌株在摇瓶发酵条件下产量达到0.87 g/L;随后,通过敲除sucD基因、过表达acs基因和突变lpd基因的组合策略平衡己二酸合成前体的供应,优化菌株E. coli JL12己二酸产量进一步提升至1.51 g/L;最后,在5 L发酵罐上对己二酸发酵工艺进行优化。工程菌株经72 h分批补料发酵,己二酸的产量达到22.3 g/L,转化率为0.25 g/g,生产强度为0.31 g/(L·h),具备了一定的应用潜力。本研究可为包括己二酸在内的多种二元羧酸细胞工厂的构建提供理论依据和技术基础。     

Bispecific chimeric antigen receptors targeting the CD4 binding site and high-mannose Glycans of gp120 optimized for anti–human immunodeficiency virus potency and breadth with minimal immunogenicity

Background aims

Chimeric antigen receptors (CARs) offer great potential toward a functional cure of human immunodeficiency virus (HIV) infection. To achieve the necessary long-term virus suppression, we believe that CARs must be designed for optimal potency and anti-HIV specificity, and also for minimal probability of virus escape and CAR immunogenicity. CARs containing antibody-based motifs are problematic in the latter regard due to epitope mutation and anti-idiotypic immune responses against the variable regions.

Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

We performed a comparative characterization of three new cephalosporin acylases which were prepared from E. coli recombinant strains and found originally from Pseudomonas sp. A14, Bacillus laterosporus J1 and Pseudomonas diminuta N176. Both A14 and N176 acylases consisted of two non-identical subunits (α, β) whose molecular weights were 28,000 (α), 61,000 (β) and 26,000 (α), 58,000 (β), respectively, whereas J1 acylase consisted of a single peptide with molecular weight of 70,000. The maximum specific activities of A14, J1 and N176 acylases for glutaryl 7-ACA were 7.1, 5.3 and 100 units/mg, respectively, and that of N176 acylase for cephalosporin C was 3.1 units/mg. The K_m values of glutaryl 7-ACA for A14, J1 and N176 acylases were 2.1, 3.2 and 2.6 mM, respectively, and that of cephalosporin C for N176 acylase was 4.8 mM. A14, J1 and N176 acylases exhibited differential activities for cephalosporins having an aliphatic dicarboxylic acid in the acyl side chain and only N176 acylase showed an activity for cephalosporin C. N176 acylase as well as A14 acylase also showed a weak activity for a cephalosporin derivative having a heterocyclic carboxylic acid in the side chain. A14, J1 and N176 acylases catalyzed the reverse reaction to synthesize glutaryl 7-ACA from 7-ACA and glutaric acid, although the rate of the synthesis was 10 to 10⁵ fold slower than that of hydrolysis. The activities of the cephalosporin acylases were considerably inhibited by the reaction products, 7-ACA and glutaric acid. The types of the inhibition by 7-ACA and glutaric acid were both competitive. A14, J1 and N176 acylases were thermostable, their residual activities exceeding more than 90% after treatment at 50°C for 1 h at their optimal pHs.     

Batch production of deacetyl 7-aminocephalosporanic acid by immobilized cephalosporin-C deacetylase

Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The K_m^app value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.     

Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device

A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: K_m = 2.8 × 10^?3M, K_ia acetate = 5 × 10^?2M, and K_id deacetyl-7-ACA = 3.6 × 10^?2M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. where S_t = mg/ml 7-ACA, A_t = mg/ml acetate, D_t = mg/ml deacetyl-7-ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (>97%) hydrolysis of a 24 mg/ml 7-ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7-ACA solutions containing 4 to 24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactivation occurred.     

Biotransformation of cephalosporin C to 7-aminocephalosporanic acid with coimmobilized biocatalyst in a batch bioreactor

The permeabilized cells of Trigonopsis variabilis CCY 15-1-3 having D-amino acid oxidase (DAAO) activity were used to convert cephalosporin C (CPS-C) into 7-(-ketoadipyl amido) cephalosporanic acid (CO-GL-7-ACA) in a batch bioreactor with good aeration and stirring during the process. The deacylation of 7--(4-carboxybutanamido)-cephalosporanic acid (GL-7-ACA) to 7-cephalosporanic acid (7-ACA) by permeabilized cells of Pseudomonas species 3635 having 4--(4-carboxybutamido)-cephalosporanic acid acylase (GL-7-ACA acylase) activity was performed in a batch bioreactor. A spectrophotometric method for the determination of CO-GL-7-ACA and 7-ACA was proposed. Experimental data were fitted by non-linear regression with parameters optimization. The sorption method (without reaction) was applied for the determination of cephalosporin effective diffusion coefficients in Ca-pectate gel beads. These beads were prepared by dropping a potassium pectate gel suspension of inactive permeabilized cells of Trigonopsis variabilis and Pseudomonas species, crosslinked with glutaraldehyde, into a stirred 0.2 M calcium chloride solution. Concentrations of appropriate cephem components were measured by the refractive method. Values of effective diffusion coefficients were calculated by the Fibbonacci optimization method.List of Symbols c _L mol/dm³ concentration on the surface of a bead - c _L0 mol/dm³ initial cephalosporin concentration - c _L mol/dm³ equilibrium cephalosporin concentration in the solution - c _s1 mol/dm³ concentration of CPS-C - c _s2 mol/dm³ concentration of GL-7-ACA - D _ei m²/s effective diffusion coefficient of the components - K _i mol/dm³ inhibition parameter in Eq. (2) - K _{m i} mol/dm³ Michaelis constant in Eq. (1) - K _{m 2} mol/dm³ Michaelis constant in Eq. (2) - n number of beads - q _n nonzero positive roots in Eq. (7) - r ₁ mol/(dm³·s) rate of the conversion of CPS-S to CO-GL-7-ACA - r ₂ mol/(dm³·s) rate of the conversion of GL-7-ACA to 7-ACA - R m radius of the bead - S( ) symbol for total residual sum of squares in Eq. (1) - t s time - V _{m 1} mol/(dm³·s) max. reaction rate in Eq. (1) - V _{m 2} mol/(dm³·s) max. reaction rate in Eq. (2) - V _L dm³ volume of the solution excluding the space occupied by beads - V _s dm³ volume of beads - y _i mol/(dm³ · s) symbol for experimental data in Eq. (1) - _i mol/(dm³· s) symbol for calculated data in Eq. (1) - _P porosity, defined by Eq. (5) - dimensionless parameter, defined by Eq. (6) The authors wish to thank Dr. P. Gemeiner of Slovak Academy of Sciences for rendering of pectate gel. This work is supported by Ministry of Education (Grant No. 1/990 935/93)     

羧酸还原酶的研究进展

羧酸还原酶(carboxylic acid reductases,CARs)可以催化羧酸还原为相应的醛,反应条件温和、拥有广阔的底物范围且副反应较少。本文旨在综述近年来羧酸还原酶系统发育、结构与催化机理、蛋白质工程和固定化工程等方面的研究成果,揭示其作为重要工具酶在生物转化及合成生物学中的应用前景。     

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO₂-eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.     

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C₆–C₁₀ carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C₆–C₁₀ dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C₆–C₁₀ ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.     

Inhibition of ribonuclease A by nucleoside–dibasic acid conjugates

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside–dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK_a of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments indicate a competitive mode of inhibition with inhibition constant (K_i) value of 132 ± 3 μM for Oxa-aT. Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen bonding distance of His-12, Lys-41 and His-119.     

A new ferulic acid ester, a new ellagic acid derivative, and other constituents from pachycentria formosana: effects on neutrophil pro-inflammatory responses

A new ferulic acid ester derivative, tetracosane‐1,24‐diyl di[(Z)‐ferulate] ( 1 ), and a new ellagic acid derivative, 3,4 : 3′,4′‐bis(O,O‐methylene)ellagic acid ( 2 ), have been isolated from leaves and twigs of Pachycentria formosana, together with eight known compounds. Their structures were determined by in‐depth spectroscopic and mass‐spectrometric analyses. Among the isolated compounds, oleanolic acid ( 6 ), ursolic acid acetate ( 7 ), and 3‐epibetulinic acid ( 9 ) exhibited potent inhibition (IC₅₀ values ≤21.8 μM ) of O₂^⋅− generation by human neutrophils in response to N‐formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB). In addition, oleanolic acid ( 6 ), 3‐O‐[(E)‐feruloyl]ursolic acid ( 8 ), 3‐epibetulinic acid ( 9 ), and lawsonic acid ( 10 ) also inhibited fMLP/CB‐induced elastase release with IC₅₀ values ≤18.6 μM .     

NSAIDs do not require the presence of a carboxylic acid to exert their anti-inflammatory effect – why do we keep using it?

The carboxylic acid group (–COOH) present in classical NSAIDs is partly responsible for the gastric toxicity associated with the administration of these drugs. This concept has been extensively proven using NSAID prodrugs. However, the screening of NSAIDs with no carboxylic acid at all has been neglected. The goal of this work was to determine if new NSAID derivatives devoid of acidic moieties would retain the anti-inflammatory activity of the parent compound, without causing gastric toxicity. To test this concept, we replaced the carboxylic acid group in ibuprofen, flurbiprofen, and naproxen with three ammonium moieties. We tested the resulting water-soluble NSAID derivatives for anti-inflammatory and ulcerogenic activity in vitro and in vivo. In this regard, we observed that all non-acidic NSAIDs exerted a potent anti-inflammatory activity, suggesting that the acid group in commercial 2-phenylpropionic acid NSAIDs not be an essential requirement for anti-inflammatory activity. These data provide complementary evidence supporting the discontinuation of ulcerogenic acidic NSAIDs.     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.