Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k_cat/K_m ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k_cat/K_m values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k_cat/K_m ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.     

Performance of a recombinant strain of<Emphasis Type="Italic"> Streptomyces lividans</Emphasis> for bioconversion of penicillin G to deacetoxycephalosporin G

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO₄ and α-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO₄ and 1.3 mM α-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO₄ and 1.9 mM α-ketoglutarate. Electronic Publication     

New strategy of site-directed mutagenesis identifies new sites to improve <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> deacetoxycephalosporin C synthase activity toward penicillin G

Based on multiple sequence alignment of different deacetoxycephalosporin C synthase (DAOCSs) and the crystal structure of Streptomyces clavuligerus DAOCS, 2-oxoglutarate, and penicillin G triple complex, ten residues (Y184, V245, S261, C37, T42, V51, S59, A61, Q126, and T213) not directly involved in substrate recognition were selected as mutational targets. Twenty one mutants were generated and characterized, and five (Q126M, T213V, S261M, S261A, and Y184A) showed improved activity toward penicillin G, with 1.45- to 4.50-fold increment in the k _cat/K _m. Q126, T213, and S261 are identified for the first time, as sites with significant effect on enzyme activity.     

Directed Evolution of Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Enhancement of Penicillin G Expansion

The deacetoxycephalosporin C synthase from Streptomyces clavuligerus was directly modified for enhancement of penicillin G expansion into phenylacetyl-7-aminodeacetoxycephalosporanic acid, an important intermediate in the industrial manufacture of cephalosporin antibiotics. Nine new mutants, mutants M73T, T91A, A106T, C155Y, Y184H, M188V, M188I, H244Q, and L277Q with 1.4- to 5.7-fold increases in the k_cat/K_m ratio, were obtained by screening 6,364 clones after error-prone PCR-based random mutagenesis. Subsequently, DNA shuffling was carried out to screen possible combinations of substitutions, including previous point mutations. One quaternary mutant, the C155Y/Y184H/V275I/C281Y mutant, which had a k_cat/K_m ratio that was 41-fold higher was found after 10,572 clones were assayed. The distinct mutants obtained using different mutagenesis methods demonstrated the complementarity of the techniques. Interestingly, most of the mutated residues that result in enhanced activities are located within or near the unique small barrel subdomain, suggesting that manipulation of this subdomain may be a constructive strategy for improvement of penicillin expansion. Several mutations had very distinct effects on expansion of penicillins N and G, perhaps due to different penicillin-interacting modes within the enzyme. Thus, the present study provided not only promising enzymes for cephalosporin biosynthesis but also a large number of mutants, which provided new insights into the structure-function relationship of the protein that should lead to further rational engineering.     

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K_m for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.     

高浓度底物下青霉素扩环酶的活性评价与底物抑制现象

本文对青霉素扩环酶(Penicillin expandase,也称Deacetoxycephalosporin C synthase,DAOCS)在高浓度青霉素G下的底物抑制现象进行初步评价与表征,筛选适合工业应用条件的高活力突变体。我们通过HPLC对已报道的几个DAOCS高活力突变体在青霉素G浓度5.6至500 mmol/L间的比活力进行定量测定,并与不同催化反应动力学模型的理论推测变化趋势比较,发现DAOCS野生型酶及高活力突变体H4、H5、H6与H7在高浓度青霉素G条件下均表现出明显的底物抑制现象,但是变化趋势不同。野生型酶与突变体H4的比活力先上升后下降,与竞争性抑制模型预测不符。突变体H5、H6与H7的比活力变化呈现更复杂的变化趋势。在所有测试的突变体中,H6的活性显著高于其他突变体酶。青霉素G对野生型DAOCS的底物抑制现象符合非竞争性抑制模型的预测。而部分突变体表现出复杂的底物抑制行为,表明其具有更复杂的作用机制。在高底物浓度下筛选具有较强催化活性的青霉素扩环酶突变体对于推动其在工业生产中的应用具有重要指导作用。     

Characterization and expression in Streptomyces lividans of cefD and cefE genes from Nocardia lactamdurans: the organization of the cephamycin gene cluster differs from that in Streptomyces clavuligerus

Summary The cefD and cefE genes of Nocardia lactamdurans, which encode isopenicillin N epimerase and deacetoxycephalosporin C synthase respectively, have been located 0.63 kb upstream from the lysine-6-amino-transferase (lat) gene. cefD contains an open reading frame (ORF) of 1197 nucleotides (nt) encoding a protein of 398 amino acids with a M_r of 43 622. The deduced amino acid sequence exhibits 62.2% identity to the cefD gene product of Streptomyces clavuligerus. The sequence SXHKXL in isopenicillin N epimerase resembles the consensus sequence for pyridoxal phosphate binding found in several amino acid decarboxylases from Enterobacteria. cefE contains an ORF of 945 nt encoding a protein of 314 amino acids with a M_r of 34532, which is similar to the deacetoxycephalosporin C synthase of S. clavuligerus. Expression of both genes, cefD and cefE, in S. lividans transformants, results in deacetoxycephalosporin C synthase and isopenicillin N epimerase activities that are 10–12 times higher than those in N. lactamdurans. The cefD and cefE genes of N. lactamdurans are closely linked but the overall organization of the cephamycin gene cluster differs in N. lactamdurans and S. clavuligerus.     

Two alternative modes for optimizing nylon‐6 byproduct hydrolytic activity from a carboxylesterase with a β‐lactamase fold: X‐ray crystallographic analysis of directly evolved 6‐aminohexanoate‐dimer hydrolase

Promiscuous 6-aminohexanoate-linear dimer (Ald)-hydrolytic activity originally obtained in a carboxylesterase with a β-lactamase fold was enhanced about 80-fold by directed evolution using error-prone PCR and DNA shuffling. Kinetic studies of the mutant enzyme (Hyb-S4M94) demonstrated that the enzyme had acquired an increased affinity (K_m = 15 mM) and turnover (k_cat = 3.1 s⁻¹) for Ald, and that a catalytic center suitable for nylon-6 byproduct hydrolysis had been generated. Construction of various mutant enzymes revealed that the enhanced activity in the newly evolved enzyme is due to the substitutions R187S/F264C/D370Y. Crystal structures of Hyb-S4M94 with bound substrate suggested that catalytic function for Ald was improved by hydrogen-bonding/hydrophobic interactions between the Ald—COOH and Tyr370, a hydrogen-bonding network from Ser187 to , and interaction between and Gln27-O_ɛ derived from another subunit in the homo-dimeric structure. In wild-type Ald-hydrolase (NylB), Ald-hydrolytic activity is thought to be optimized by the substitutions G181D/H266N, which improve an electrostatic interaction with (Kawashima et al., FEBS J 2009; 276:2547–2556). We propose here that there exist at least two alternative modes for optimizing the Ald-hydrolytic activity of a carboxylesterase with a β-lactamase fold.     

Evolving enzyme technology for pharmaceutical applications: case studies

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent. Received 07 March 1997/ Accepted in revised form 15 June 1997     

Enhancement of the activity and alkaline pH stability of <Emphasis Type="Italic">Thermobifida fusca</Emphasis> xylanase A by directed evolution

Directed evolution has been used to enhance the catalytic activity and alkaline pH stability of Thermobifida fusca xylanase A, which is one of the most thermostable xylanases. Under triple screened traits of activity, alkaline pH stability and thermostability, through two rounds of random mutagenesis using DNA shuffling, a mutant 2TfxA98 with approximately 12-fold increased k _cat/K _m and 4.5-fold decreased K _m compared with its parent was obtained. Moreover, the alkaline pH stability of 2TfxA98 is increased significantly, with a thermostability slightly lower than that of its parent. Five amino acid substitutions (T21A, G25P, V87P, I91T, and G217L), three of them are near the catalytic active site, were identified by sequencing the genes encoding this evolved enzyme. The activity and stabilizing effects of each amino acid mutation in the evolved enzyme were evaluated by site-directed mutagenesis. This study shows a useful approach to improve the catalytic activity and alkaline pH stability of T. fusca xylanase A toward the hydrolysis of xylan.     

Enhancement of the activity of l-aspartase from Escherichia coli W by directed evolution

l-Aspartase was modified by directed evolution. After four rounds of error-prone PCR and three rounds of DNA shuffling, an evolved enzyme purified from the final round showed a 28-fold increased k(cat)/K(m) and 4.6-fold decreased K(m). The thermostability and stable pH range were also enhanced. The DNA sequence of the evolved aspartase gene showed seven base changes, resulting in three amino acid changes from the native enzyme: N217K, T233R, V367G. The mechanism of the enhancement of activity was analyzed.     

Directed Evolution of E. coli Alkaline Phosphatase Towards Higher Catalytic Activity

Directed evolution was used to enhance the catalytic activity of E. coli alkaline phosphatase (EAP). Through two rounds of error-prone PCR and one round of DNA shuffling followed by a rapid, sensitive screening procedure, several improved variants were obtained. Their enzymatic kinetic properties, thermal stabilities and possible mechanism for the improvement were investigated. In 1.0 M Tris buffer, the specific activity of the most active EAP variant S2163 was 1500 units/mg protein, showing it to be 3.6 times more active than the D101S parent enzyme and ～40 times more active than the wild-type EAP. At the same time, the K_m value of the S2163 variant decreased to 1491 μM from the 2384 μM of the D101S. As a result, the k_cat/K_m ratio of this variant showed a 5.8-fold enhancement over that of D101S parent enzyme. Three activating amino acid substitutions, K167R, G180S and S374C, which were located far away from the center of the catalytic pocket, were identified by sequencing the genes encoding evolved enzymes. Possible explanations for the improvement of activity were analyzed.     

Enhancement of mustard oil yield by cellulolytic pretreatment

Summary When cultivated in chemically defined medium, Streptomyces clavuligerus produces cephamycin C. This biosynthesis is greatly inhibited when the bacteria develop rapidly in batch culture. The decrease in cephamycin C biosynthesis is paralleled by a decrease in expandase biosynthesis. This negative effect can be observed whatever the limiting growth substrate (glycerol, ammonium or phosphate), a phenomenon which was confirmed when S. clavuligerus was cultivated in a chemostat.     

Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (K_m, k_cat and k_cat/K_m) of BliGO were 11.22 mM, 0.08 s⁻¹, and 0.01 mM⁻¹ s⁻¹, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM⁻¹ s⁻¹) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.     

A novel penicillin acylase from the environmental gene pool with improved synthetic properties

A new penicillin acylase was isolated by cloning and functional screening of DNA isolated from a sand soil enrichment culture. Sequence analysis of this enzyme, PAS2, revealed homology to a group of prominent penicillin G acylases, including the intensively studied enzyme of Escherichia coli ATCC 11105. Accordingly, PAS2 was found to be an Ntn-hydrolase with an N-terminal serine as the catalytic nucleophile, located on its 61.9 kDa β-subunit. The α-subunit was shown to have a molecular mass of 25.5 kDa.To evaluate the biocatalytic performance of the new enzyme, the complex kinetic parameters α, β₀, and γ were determined for the kinetically controlled synthesis of a number of important semi-synthetic penicillins and cephalosporins. While α is a measure for the relative affinity of the enzyme for the activated acyl donor (AD), β₀ and γ quantify the efficiency of acyl-transfer to the β-lactam nucleophile. Compared to the penicillin acylase of E. coli, PAS2 showed superior potential for the synthesis of 6-aminopenicillanic acid (6-APA)-derived antibiotics, allowing the accumulation of up to 2.3-fold more target product at significantly higher conversion rates. In the synthesis of amoxicillin, for instance, 1.6-fold more antibiotic was formed using the new enzyme, making PAS2 an interesting candidate for biocatalytic application.     

The pcd Gene Encoding Piperideine-6-Carboxylate Dehydrogenase Involved in Biosynthesis of α-Aminoadipic Acid Is Located in the Cephamycin Cluster of Streptomyces clavuligerus

Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74,094).     

Engineering of glycerol dehydrogenase for improved activity towards 1, 3-butanediol

The objective of this study was to use protein engineering techniques to enhance the catalytic activity of glycerol dehydrogenase (GlyDH) on racemic 1, 3-butanediol (1, 3-BDO) for the bioproduction of the important pharmaceutical intermediate 4-hydroxy-2-butanone. Three GlyDH genes (gldA) from Escherichia coli K-12, Salmonella enterica, and Klebsiella pneumoniae MGH78578 were shuffled to generate a random mutagenesis library. The nitroblue tetrazolium/phenazine methosulfate high throughput screening protocol was used to select four chimeric enzymes with up to a 2.6-fold improved activity towards 1, 3-BDO. A rational design method was also employed to further improve the enzyme activity after DNA shuffling. Based on the homology model of GlyDH (Escherichia coli), Asp121 was predicted to influence 1, 3-BDO binding and replaced with Ala by site-directed mutagenesis. Combination of the mutations from both DNA shuffling and rational design produced the best mutant with a V _max value of 126.6 U/mg, a 26-fold activity increase compared with that of the wild type GlyDH from E. coli.     

Saturation mutagenesis of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase R308 site confirms its role in controlling substrate specificity

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k_cat/K_m ratio and 7-fold increase in relative activity.     

Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K(M) in reactions with the novel para-nitrophenyl-beta-d-fucopyranoside substrate; 39-fold decrease in reactivity with the "native"para-nitrophenyl-beta-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/K(M) in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.