3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag+, Pb2+ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

There are four nitrogen atoms in l-arginine molecule and the nitrogen content is 32.1%. By now, metabolic engineering for l-arginine production strain improvement was focused on carbon flux optimization. In previous work, we obtained an l-arginine-producing Corynebacterium crenatum SDNN403 (ARG) through screening and mutation breeding. In this paper, a strain engineering strategy focusing on nitrogen supply and ammonium assimilation for l-arginine production was performed. Firstly, the effects of nitrogen atom donor (l-glutamate, l-glutamine and l-aspartate) addition on l-arginine production of ARG were studied, and the addition of l-glutamine and l-aspartate was beneficial for l-arginine production. Then, the glutamine synthetase gene glnA and aspartase gene aspA from E. coli were overexpressed in ARG for increasing the l-glutamine and l-aspartate synthesis, and the l-arginine production was effectively increased. In addition, the l-glutamate supply re-emerged as a limiting factor for l-arginine biosynthesis. Finally, the glutamate dehydrogenase gene gdh was co-overexpressed for further enhancement of l-arginine production. The final strain could produce 53.2 g l−1 of l-arginine, which was increased by 41.5% compared to ARG in fed-batch fermentation.

Most available methods for modifying the catalytic properties of enzymes are costly and time-consuming, as they rely on the information of enzyme crystal structure or require handling large amounts of mutants. This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.

•An acidic hetero-exopolysaccharide (BMPS) was produced and purified from Bacillus mucilaginosus SM-01.•BMPS was mainly compromised of Glc, Man, GlcA with Mw of 2.67 × 106 Da.•A neutral fragment (BMPS-H) was obtained by mild acid hydrolysis of BMPS.•The chemical structure of BMPS-H was characterized to be•Figure optionsIn this study, an acidic hetero-exopolysaccharide, designated as BMPS, was isolated from the culture supernatant of bacteria Bacillus mucilaginosus SM-01 by ion-exchange and size-exclusion chromatography. The molecular weight of BMPS was measured to be 2.67 × 106 Da. To analyze the structure of the polysaccharide, the low-molecular weight fragment (BMPS-H) of BMPS was obtained under mild hydrolysis. BMPS-H was determined to be a liner neutral fraction with an average molecular weight of 1621 Da. Monosaccharide analysis indicated that BMPS-H contained glucose and mannose in a molar ratio of 1.5:1. Based on methylation analysis and NMR spectroscopy data, the following structure of BMPS-H was established:Figure optionsIn addition, MALDI-TOF-MS analysis showed the hydroxyl group on C-2 of 1,4-linked-glucosyl residues was partially acetylated.

Hydroxylation of dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA) by Colletotrichum lini ST-1 is an essential step in the synthesis of many steroidal drugs, while low DHEA concentration and 7α,15α-diOH-DHEA production are tough problems to be solved urgently in industry. In this study, the significant improvement of 7α,15α-diOH-DHEA yield in 5-L stirred fermenter with 15 g/L DHEA was achieved. To maintain a sufficient quantity of glucose for the bioconversion, glucose of 15 g/L was fed at 18 h, the 7α,15α-diOH-DHEA yield and dry cell weight were increased by 17.7 and 30.9 %, respectively. Moreover, multi-step DHEA addition strategy was established to diminish DHEA toxicity to C. lini, and the 7α,15α-diOH-DHEA yield raised to 53.0 %. Further, a novel strategy integrating glucose-feeding with multi-step addition of DHEA was carried out and the product yield increased to 66.6 %, which was the highest reported 7α,15α-diOH-DHEA production in 5-L stirred fermenter. Meanwhile, the conversion course was shortened to 44 h. This strategy would provide a possible way in enhancing the 7α,15α-diOH-DHEA yield in pharmaceutical industry.

7α,15α-diOH-DHEA is a key precursor of the novel oral contraceptive Yasmin. Colletotrichum lini could catalyze dehydroepiandrosterone (DHEA) at the 7α and 15α positions. In this work, C. lini resting cells were applied in the bioconversion of DHEA to 7α,15α-diOH-DHEA. In the presence of 2 % (w/v) Tween-80, the conversion efficiency of DHEA increased drastically. The DHEA conversion and the 7α,15α-diOH-DHEA yield increased by 34.6 and 87.0 %, respectively, at the DHEA concentration of 10 g/L. Furthermore, the effects of Tween-80 on substrate solubility and C. lini physiological properties were studied. Results showed that the DHEA solubility with 2 % Tween-80 increased by 7.8 times. Meanwhile, the mycelia were integrated and full in the presence of 2 % Tween-80. The analysis on fatty acid profile of the C. lini cell membrane indicated that Tween-80 increases the content of unsaturated fatty acid. All above results suggested that the enhanced product yield caused by Tween-80 was mainly associated with easier substrate-molecule transportation across the cell membrane of C. lini.

Corynebacterium crenatum SYPA5-5, an l-arginine high-producer obtained through multiple mutation-screening steps, had been deregulated by the repression of ArgR that inhibits l-arginine biosynthesis at genetic level. Further study indicated that feedback inhibition of SYPA5-5 N-acetylglutamate kinase (CcNAGK) by l-arginine, as another rate-limiting step, could be deregulated by introducing point mutations. Here, we introduced two of the positive mutations (H268N or R209A) of CcNAGK into the chromosome of SYPA5-5, however, resulting in accumulation of large amounts of the intermediates (l-citrulline and l-ornithine) and decreased production of l-arginine. Genetic and enzymatic levels analysis involved in l-arginine biosynthetic pathway of recombinants SYPA5-5-NAGKH268N (H-7) and SYPA5-5-NAGKR209A (R-8) showed that the transcription levels of argGH decreased accompanied with the reduction of argininosuccinate synthase and argininosuccinase activities, respectively, which led to the metabolic obstacle from l-citrulline to l-arginine. Co-expression of argGH with exogenous plasmid in H-7 and R-8 removed this bottleneck and increased l-arginine productivity remarkably. Compared with SYPA5-5, fermentation period of H-7/pDXW-10-argGH (H-7-GH) reduced to 16 h; meanwhile, the l-arginine productivity improved about 63.6 %. Fed-batch fermentation of H-7-GH in 10 L bioreactor produced 389.9 mM l-arginine with the productivity of 5.42 mM h−1. These results indicated that controlling the transcription of argGH was a key factor for regulating the metabolic flux toward l-arginine biosynthesis after deregulating the repression of ArgR and feedback inhibition of CcNAGK, and therefore functioned as another regulatory mode for l-arginine production. Thus, deregulating all these three regulatory modes was a powerful strategy to construct l-arginine high-producing C. crenatum.

1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. Identifying the bottlenecks for BT biosynthesis is expected to contribute to improving the efficiency of this process. In this work, we first constructed a prototype strain for BT production by assembling a four-step synthetic pathway and disrupting the competing pathways for xylose in Escherichia coli BW25113. Using this prototype strain, we conducted systematic fine-tuning of the pathway enzyme expression level to identify the potential bottlenecks and optimize the BT biosynthesis. Production conditions were also optimized by exploring the effects of temperature, pH and cell density on BT titer. BT production was increased by 4.3-fold from the prototype strain, achieved a final titer of 1.58 g/L with a yield of 7.9 % after 72-h biotransformation.

Microbial keratinase is a well-recognized enzyme that can specifically degrade insoluble keratins. A keratinase-producing bacterium was isolated from a duck ranch soil and identified as Acinetobacter sp. R-1 based on the biochemical characteristics and 16S rDNA gene sequencing. It showed high keratinase activity and low collagenase activity. The keratinase was purified to electrophoretic homogeneity with 6.69 % recovery, 2.68-fold purification and an estimated molecular weight of 25 kDa. Additionally, the keratinase showed optimal activity at 50 °C and pH11. Keratinase activity of Acinetobacter sp. significantly increased in the presence of Li+, Na+, and Ca2+, while it was completely inhibited by EDTA, indicating it was a metallo-keratinase. Moreover, the crude keratinase from Acinetobacter sp. R-1 could thoroughly depilate goat skin and simultaneously modify the wool surface, which indicated its applicable potential in leather and textile industries.

A comparative genomic analysis was performed to study the genetic variations between the l-serine-producing strain Corynebacterium glutamicum SYPS-062 and the mutant strain SYPS-062-33a, which was derived from SYPS-062 by random mutagenesis with enhanced l-serine production. Some variant genes between the two strains were reversely mutated or deleted in the genome of SYPS-062-33a to verify the influences of the gene mutations introduced by random mutagenesis. It was found that a His-594 → Tyr mutation in aceE was responsible for the more accumulation of by-products, such as l-alanine and l-valine, in SYPS-062-33a. Furthermore, the influence of this point mutation on the l-serine production was investigated, and the results suggested that this point mutation led to a better growth profile and a higher l-serine production in the high-yield strain 33a∆SSAAI, which was derived from SYPS-062-33a by metabolic engineering with the highest l-serine production to date.

Surfactant-stable keratinases with good properties are promising candidates for extensive applications in detergent industries. A novel fungal keratinase-producing strain, Gibberella intermedia CA3-1, is described in this study. The keratinase production medium was optimized and composed of 10 g L−1 of wool powder, 5 g L−1 of tryptone, 10 g L−1 of maltodextrin and 0.5 g L−1 of NaCl. Keratinase activity was increased up to 109 U mL−1 from 15 U mL−1 by culture optimization. The optimal reaction pH and temperature of the enzyme were 9.0 and 60°C, respectively. The keratinase activity could be improved by sodium dodecyl sulphate (SDS), and it remained stable in the presence of several surfactants and commercial detergents. G. intermedia keratinase was proved to completely remove blood stains from cotton cloth when combined with detergents. These findings indicate that this fungal keratinase is a promising catalyst for the application in detergent industry. To our knowledge, this is the first report on keratinase production by Gibberella genus.

Comparative studies of immobilized and free cells of Gibberellaintermedia CA3-1 in bioconversion of 3-cyanopyridine to nicotinic acid were performed. Entrapping method was chosen based on the advantages in enzymatic activity recovery, mechanical strength and preparation procedure. Four entrapment matrices were investigated and sodium alginate was screened to be the most suitable material. Maximal nitrilase activity of alginate immobilized cells was obtained under conditions of 2 % alginate, 0.6 % CaCl2, 0.4 g cell/g alginate, 1.8 mm bead size. The immobilized cells showed excellent substrate tolerance even when the 3-cyanopyridine concentration was 700 mM. The half-lives of immobilized cells at 30, 40 and 50 °C were 315, 117.5 and 10.9 h, respectively, correspondingly 1.4, 1.6 and 1.7-fold compared with that of the free cells. Efficient reusability of immobilized cells up to 28 batches was achieved and 205.7 g/(g dcw) nicotinic acid was obtained with 80.55 % enzyme activity preserved.

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity.

The direct fermentative production of l-serine from sugar has attracted increasing attention. Corynebacterium glutamicum SYPS-062 can directly convert sugar to l-serine. In this study, the effects of exogenous and endogenous regulation of cofactor folate on C. glutamicum SYPS-062 growth and l-serine accumulation were investigated. For exogenous regulation, the inhibitor (sulfamethoxazole) or precursor (p-aminobenzoate) of folate biosynthesis was added to the medium, respectively. For endogenous regulation, the gene (pabAB) that encodes the key enzyme of folate biosynthesis was knocked out or overexpressed to obtain the recombinant C. glutamicum SYPS-062 ΔpabAB and SYPS-062(pJC-tac-pabAB), respectively. The results indicated that decreased levels of cofactor folate supported l-serine accumulation, whereas increased levels of cofactor folate aided in cell growth of C. glutamicum SYPS-062. Thus, this study not only elucidated the role of folate in C. glutamicum SYPS-062 growth and l-serine accumulation, but also provided a novel and convenient approach to regulate folate biosynthesis in C. glutamicum.

Dihydroxylation of dehydroepiandrosterone (DHEA) is an essential step in the synthesis of many important pharmaceutical intermediates. However, the solution to the problem of low biohydroxylation conversion in the biotransformation of DHEA has yet to be found. The effects of natural oils on the course of dihydroxylation of DHEA to 3β,7α,15α-trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) were studied. With rapeseed oil (2 %, v/v) addition, the bioconversion efficiency was improved, and the 7α,15α-diOH-DHEA yield was increased by 40.8 % compared with that of the control at DHEA concentration of 8.0 g/L. Meantime, the ratio of 7α,15α-diOH-DHEA to 7α-OH-DHEA was also increased by 4.5 times in the rapeseed oil-containing system. To explain the mechanism underlying the increase of 7α,15α-diOH-DHEA yield, the effects of rapeseed oil on the pH of the bioconversion system, the cell growth and integrity of Gibberella intermedia CA3-1, as well as the membrane composition were systematically studied. The addition of rapeseed oil enhanced the substrate dispersion and maintained the pH of the system during bioconversion. Cells grew better with favorable integrity. The fatty acid profile of G. intermedia cells revealed that rapeseed oil changed the cell membrane composition and improved cell membrane permeability for lipophilic substrates.

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.

Antrodia camphorata is a well-known Chinese medicinal mushroom that protects against diverse health-related conditions. Submerged fermentation of A. camphorata is an alternative choice for the effective production of bioactive metabolites, but the effects of nutrition and environment on mycelial morphology are largely unknown. In this study, we show that A. camphorata American Type Culture Collection 200183 can form arthrospores in the end of liquid fermentation. Different morphologies of A. camphorata in submerged culture were analyzed using scanning electron microscopy. The optimal carbon and nitrogen sources for sporulation were soluble starch and yeast extract. We found that a carbon-to-nitrogen ratio (C/N) of 40:1, MgSO4 (0.5 g/l), KH2PO4 (3.0 g/l), an initial pH 5.0, and an inoculum size of 1.5 × 105 spores/ml led to maximum production of arthroconidia. Our results will be useful in the regulation and optimization of A. camphorata cultures for efficient production of arthroconidia in submerged culture, which can be used as inocula in subsequent fermentation processes.

Nitrilase is one of the most important members in the nitrilase superfamily and it is widely used for bioproduction of commodity chemicals and pharmaceutical intermediates as well as bioremediation of nitrile-contaminated wastes. However, its application was hindered by several limitations. Searching for new nitrilases and improving their application performances are the driving force for researchers. Genetic data resources in various databases are quite rich in post-genomic era. Besides, more than 99 % of microbes in the environment are unculturable. Metagenomic technology and genome mining are thus becoming burgeoning areas and provide unprecedented opportunities for searching more useful novel nitrilases due to the abundance of already existing but unexplored gene resources, namely uncharacterized genome information in the database and unculturable microbes in the natural environment. These techniques seem to be innovative and highly efficient. This study reviews the current status and future directions of metagenomics and genome mining in nitrilase exploration. Moreover, it discussed their utilization in coping with the challenges for nitrilase application. In the next several years, with the rapid development of nitrile biocatalysis, these two techniques would be bound to attract increasing attentions and even become a dominant trend for finding more novel nitrilases. Also, this review would provide guidance for exploitation of other commercially important enzymes.

This study investigated the effects of hydroxylase cyptochrome P450 inducers on the efficiency of the biotransformation of dehydroepiandrosterone (DHEA) to 3β, 7α, 15α-trihydroxy-5-androsten-17-one (7α,15α-diOHDHEA) by Colletotrichum lini ST-1. Special attention was given to the substrate DHEA being the best inducer and the fact that it could improve the yield of 7α, 15α-diOHDHEA. Based on the effects of the DHEA pre-induction phases and additional concentrations on 7α, 15α-diOHDHEA production, a substrate pre-induction process was established as follows: 2 g/L DHEA was added for the first time after 12 h of inoculation, followed by the second addition of 6 g/L DHEA after 12 h later. The results showed that this substrate pre-induction process improved the content of cytochrome P450 and that the 7α, 15α-diOH-DHEA yield reached 90.1%, which was 26.9% higher than that obtained in the original process.

We developed (GLP-1A2G)2-HSA (GGH) analogs that are resistant to degradation and also show high serum glucose-reducing activity in vivo. Five analogs with N-terminal extensions were designed based on the protein GGH. Next, we constructed recombinant plasmids capable of expressing the five analogs in methylotrophic yeast Pichia pastoris GS115. Expression reached 150 mg/L in a small-scale incubation. Fusion proteins were successfully purified from the supernatant using ultrafiltration concentration, affinity absorption chromatography, hydrophobic chromatography, ion exchange chromatography and gel filtration. A single band was observed on SDS-PAGE and the purity was 97%. Activity test results suggested that both A-GGH and G-GGH showed better activity in vitro and that their cAMP levels were significantly increased by 10-fold compared to GGH without N-terminal extension. Additionally, A-GGH efficiently enhanced the glucoselowering effect, which was maintained after the administration for 24 h. A-GGH is a potential drug for treating type 2 diabetes.

Corynebacterium crenatum SYA is an aerobic, Gram-positive, non-sporulating coryneform bacterium, and the mutant C. crenatum strain SYPA 5–5 can produce 30 g/l L-arginine under optimal culture conditions. In this study, the evolution of the cluster argCJBDFRGH (argC~H) involved in arginine biosynthesis in C. crenatum SYA, and SYPA 5–5 was investigated. Compared to the argR of its wild type C. crenatum SYA, a nucleotide substitution (C→T) within the argR gene of the mutant C. crenatum strain SYPA 5–5 was found. The inactivation of ARGR resulted in increased enzyme activities involved in L-arginine biosynthesis and increased L-arginine production in C. crenatum. In contrast, constructing an overexpressing argR C. crenatum/pTR, a complete and functional ARGR decreased the expression of enzymes, depressed transcriptional level of the argC~H cluster, and reduced the production of L-arginine in C. crenatum. It was thus evident that the inactivation of an ARGR suppressor could relieve a bottleneck in downstream steps of the L-arginine biosynthetic pathway, providing a good strategy for improving L-arginine production.

Corynebacterium crenatum SYPA is an aerobic and industrial l-arginine producer. A lysE deletion mutant was constructed searching a natural function of this in C. crenatum SYPA. Tracking determination of intracellular and extracellular l-arginine in the minimum culture CGXII showed that LysE did play an important role in transporting l-arginine from intracellular to extracellular, without which the accumulation of intracellular l-arginine of C. crenatum SYPA could reach an extremely high concentration. Nevertheless, the lower intracellular concentration of l-arginine could decrease the feedback inhibition in the biosynthesis of l-arginine. To improve the yield from excretion, we attempted to achieve overexpress in the l-arginine transporter in C. crenatum SYPA. Thus, the heterologous and homologous expressions of EcargO and CclysE, respectively, were carried out by means of the shuttle plasmid pJCtac in C. crenatum SYPA. It was found that l-arginine production was improved by 13.6 % because of the overexpression of lysE in the strain (SYPA/pJCE) contrast to the wild-type strain and reached 35.9 g/L, whereas l-arginine production of the strain SYPA/pJCO was scarcely changed. Consequently, we succeeded in obtaining a mutant form of LysE which has an effect to decrease the intracellular concentration of l-arginine in an l-arginine producer of C. crenatum SYPA and found that the production of l-arginine by C. crenatum carrying lysE was improved. These results show the importance of the factor (lysE) involved in the excretion of l-arginine on its over-production in C. crenatum.

Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-l-glutamate (NAG) to N-acety-l-glutamy-l-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the l-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the l-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I0.5R 50–60 fold, and the G287D and R209A mutants could increase the I0.5R 30–40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition.

In this work a headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS) and GC–olfactometry (GC–O) was developed to evaluate the profile of the volatile compounds that contribute to the aroma of Antrodia camphorata in submerged culture. For this purpose, the HS-SPME sampling method for the volatile compounds of A. camphorata in submerged culture was optimised by a D-optimal design. A HS extraction of the culture broth of A. camphorata followed by incubation on a carboxen/polydimethylsiloxane (CAR/PDMS) fibre during 31.8 min at 54.6 °C gave the most effective and accurate extraction of the volatile compounds. By the optimised method, a total of 49 volatile compounds were identified in culture broth of A. camphorata, while a total of 55 volatile compounds were identified in the mycelia. A series of C8 aliphatic compounds (mushroom-like odour), several lactones (fruity odour) and l-linalool (citrus-like odour) were the most potent key odourant in both the mycelia and culture broth. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor volatile compounds associated to A. camphorata.Research highlights► Volatiles of fermented Antrodia camphorata were analysed by HS-SPME–GC–MS. ► Aroma of fermented A. camphorata was evaluated by HS-SPME–GC–O. ► HS-SPME condition for the volatiles was extensively optimised by a D-optimal design.

In this study, alteration in morphology of submergedly cultured Antrodia camphorata ATCC 200183 including arthroconidia, mycelia, external and internal structures of pellets was investigated. Two optimization models namely response surface methodology (RSM) and artificial neural network (ANN) were built to optimize the inoculum size and medium components for intracellular triterpenoid production from A. camphorata. Root mean squares error, R2, and standard error of prediction given by ANN model were 0.31%, 0.99%, and 0.63%, respectively, while RSM model gave 1.02%, 0.98%, and 2.08%, which indicated that fitness and prediction accuracy of ANN model was higher when compared to RSM model. Furthermore, using genetic algorithm (GA), the input space of ANN model was optimized, and maximum triterpenoid production of 62.84 mg l−1 was obtained at the GA-optimized concentrations of arthroconidia (1.78 × 105 ml−1) and medium components (glucose, 25.25 g l−1; peptone, 4.48 g l−1; and soybean flour, 2.74 g l−1). The triterpenoid production experimentally obtained using the ANN–GA designed medium was 64.79 ± 2.32 mg l−1 which was in agreement with the predicted value. The same optimization process may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoid production from A. camphorata and to improve the production of bioactive metabolites from potent medicinal fungi by changing the fermentation parameters.

Microbial transformation of glycinonitrile into glycine by nitrile hydrolase is of considerable interest to green chemistry. A novel fungus with high nitrile hydrolase was newly isolated from soil samples and identified as Fusarium oxysporum H3 through 18S ribosomal DNA, 28S ribosomal DNA, and the internal transcribed spacer sequence analysis, together with morphology characteristics. After primary optimization of culture conditions including pH, temperature, carbon/nitrogen sources, inducers, and metal ions, the enzyme activity was greatly increased from 326 to 4,313 U/L. The preferred carbon/nitrogen sources, inducer, and metal ions were glucose and yeast extract, caprolactam, and Cu2+, Mn2+, and Fe2+, respectively. The maximum enzyme formation was obtained when F. oxysporum H3 was cultivated at 30 °C for 54 h with the initial pH of 7.2. There is scanty report about the optimization of nitrile hydrolase production from nitrile-converting fungus.

•Low intracellular NADPH/NADP was demonstrated to be a limiting factor during DHEA biotransformation.•Hydroxylation yield from DHEA was increased significantly by a dual cosubstrate-coupled system.•High substrate concentration (15 g/L) and high yield were obtained by a fed-batch transformation model.Hydroxylation of DHEA to 7α,15α-diOH-DHEA was catalyzed by NADPH-dependent cytochrome P450 monooxygenase from Colletotrichum lini. By adding coenzyme precursor nicotinic acid, the NADPH/NADP ratio was significantly increased, and the 7α,15α-diOH-DHEA molar conversion was enhanced from 37.37% to 50.85%. To improve the availability of intracellular NADPH, a dual cosubstrate-coupled system consisting of nicotinic acid and glucose was investigated in C. lini. Using 20 mM nicotinic acid and 15 g/L glucose as cosubstrate for NADPH regeneration, the 7α,15α-diOH-DHEA molar conversion was dramatically increased by 74.58%. The conversion course was simultaneously shortened by 30 h. Moreover, a fed-batch transformation model was established to diminish DHEA toxicity to C. lini and further increase DHEA concentration. The maximum concentration of DHEA was elevated to 15 g/L using a three-batch transformation in a coenzyme regeneration system, and 7α,15α-diOH-DHEA production of 11.21 g/L could be achieved after 60 h of biotransformation. These results demonstrated that this strategy was promising for steroids hydroxylation.Download high-res image (125KB)Download full-size image

•Patterns of bacterial succession showed no significant batch-to-batch difference.•Vinegar microbiota gradually evolved into a structure similar to the starter.•Dynamics of Acetobacter and Lactobacillus were highly similar among different batches of acetic acid fermentation.•Dynamics of main 16 flavor metabolites in different batches of acetic acid fermentation were stable.Solid-state fermentation of traditional Chinese vinegar is a mixed-culture refreshment process that proceeds for many centuries without spoilage. Here, we investigated bacterial community succession and flavor formation in three batches of Zhenjiang aromatic vinegar using pyrosequencing and metabolomics approaches. Temporal patterns of bacterial succession in the Pei (solid-state vinegar culture) showed no significant difference (P > 0.05) among three batches of fermentation. In all the batches investigated, the average number of community operational taxonomic units (OTUs) decreased dramatically from 119 ± 11 on day 1 to 48 ± 16 on day 3, and then maintained in the range of 61 ± 9 from day 5 to the end of fermentation. We confirmed that, within a batch of fermentation process, the patterns of bacterial diversity between the starter (took from the last batch of vinegar culture on day 7) and the Pei on day 7 were similar (90%). The relative abundance dynamics of two dominant members, Lactobacillus and Acetobacter, showed high correlation (coefficient as 0.90 and 0.98 respectively) among different batches. Furthermore, statistical analysis revealed dynamics of 16 main flavor metabolites were stable among different batches. The findings validate the batch-to-batch uniformity of bacterial community succession and flavor formation accounts for the quality of Zhenjiang aromatic vinegar. Based on our understanding, this is the first study helps to explain the rationality of age-old artistry from a scientific perspective.

Zhenjiang aromatic vinegar is one of the most famous Chinese traditional vinegars. In this study, change of the microbial community during its fermentation process was investigated. DGGE results showed that microbial community was comparatively stable, and the diversity has a disciplinary series of changes during the fermentation process. It was suggested that domestication of microbes and unique cycle-inoculation style used in the fermentation of Zhenjiang aromatic vinegar were responsible for comparatively stable of the microbial community. Furthermore, two clone libraries were constructed. The results showed that bacteria presented in the fermentation belonged to genus Lactobacillus, Acetobacter, Gluconacetobacter, Staphylococcus, Enterobacter, Pseudomonas, Flavobacterium and Sinorhizobium, while the fungi were genus Saccharomyces. DGGE combined with clone library analysis was an effective and credible technique for analyzing the microbial community during the fermentation process of Zhenjiang aromatic vinegar. Real-time PCR results suggested that the biomass showed a “system microbes self-domestication” process in the first 5 days, then reached a higher level at the 7th day before gradually decreasing until the fermentation ended at the 20th day. This is the first report to study the changes of microbial community during fermentation process of Chinese traditional solid-state fermentation of vinegar.Highlights► Microbial community variation during vinegar fermentation was studied by PCR-DGGE. ► Microbes in DGGE profiles were identified by bacterial and fungal clone libraries. ► Change of microbe amount during vinegar fermentation was analyzed by real-time PCR.

•Broad substrate spectrum of Colletotrichum lini ST-1.•High conversion rate (70–85%), high stereo-selectivity and high substrate concentration (4 g/L) of C. lini ST-1.•3 novel 11α,15α-dihydroxy-steroid compounds.•Systematic analysis of hydroxylation mechanism.Biotransformation of a series of steroid compounds (estradiol, estrone, androst-4-en-3,17-dione, testosterone, canrenone, 16α,17α-epoxyprogesterone and progesterone) with Colletotrichum lini ST-1 as biocatalyst was investigated. With the exception of estradiol, estrone and progesterone, the microorganism could selectively hydroxylate steroid substrates (4 g/L) with 70–85% conversion rate and 60–76% total products yield. The different hydroxylation sites between androst-4-en-3,17-dione (3) and testosterone (4) suggested that the hydroxyl group or carbonyl group on the substrate at C17 had profound influence on the location of introduced hydroxyl groups. Transformations of 3-keto-steroid (3, 4, 5, 6 and 7) included monohydroxylation or dihydroxylation at 11α and 15α positions, while hydroxylations of 3-hydroxy-steroid (DHEA) were hydroxylation at 7α and 15α positions. Moreover, time course experiments demonstrated dihydroxylation of androst-4-en-3,17-dione (3), canrenone (5) and 16α,17α-epoxyprogesterone (6) were all initiated by hydroxylation on ring-D (C15) followed by attack on ring-C (C11). In this study, several new hydroxylation products were discovered, including 11α,15α-dihydroxyandrost-4-en-3,17-dione (9), 11α,15α-dihydroxy-canrenone (12) and 11α,15α-dihydroxy-16α,17α-epoxyprogesterone (14). The breadth of substrate spectrum and the excellent conversion rates achieved with this fungus indicated that C. lini ST-1 was a potential microorganism for production of valuable pharmaceutical ingredients and precursors.Download full-size image

The enzyme 3-ketosteroid-Δ1-dehydrogenase (KSDD), involved in steroid metabolism, catalyzes the transformation of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD) specifically. Its coding gene was obtained from Mycobacterium neoaurum JC-12 and expressed on the plasmid pMA5 in Bacillus subtilis 168. The successfully expressed KSDD was analyzed by native-PAGE. The activities of the recombinant enzyme in B. subtilis were 1.75 U/mg, which was about 5-fold that of the wild type in M. neoaurum. When using the whole-cells as catalysts, the products were analyzed by tin-layer chromatography and high-performance liquid chromatography. The recombinant B. subtilis catalyzed the biotransformation of AD to ADD in a percent conversion of 65.7% and showed about 18 folds higher than M. neoaurum JC-12. The time required for transformation of AD to ADD was about 10 h by the recombinant B. subtilis, much shorter than that of the wild-type strain and other reported strains. Thus, the efficiency of ADD production could be improved immensely. For industrial applications, the recombinant B. subtilis containing KSDD provides a new pathway of producing steroid medicines.Highlights► The gene ksdd from Mycobacterium neoaurum JC-12 was successfully expressed in Bacillus subtilis 168. ► The enzyme activities in recombinant were about 1.75 ± 0.09 U/mg, about 5 folds higher than that of M. neoaurum JC-12. ► The recombinant can catalyze AD to ADD by the whole-cell transformation. ► The maximum transformation rate of AD reached 65.7% in 10 h by the recombinant while 25% in 132 h by M. neoaurum JC-12.

•An inclusion complex between CDs and dehydroepiandrosterone (DHEA) could be formed at a 1:1 molar ratio.•Hydroxylation yield from DHEA by Colletotrichum lini ST-1 was increased significantly by cyclodextrin complexation technique.•The mechanisms of CD complexation technique for enhancing the hydroxylation yield of DHEA were analyzed systematic.The cyclodextrins (CDs) complexation technique was performed for the enhancement of hydroxylation yield from dehydroepiandrosterone (DHEA) by Colletotrichum lini ST-1. Using DHEA/methyl-β-cyclodextrin (M-β-CD) or DHEA/hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complexes as substrate (10 g/L), the hydroxylation yields were increased by 14.98% and 20.54% respectively, and the biotransformation course was shortened by 12 h. X-ray diffractometry, differential scanning calorimetry, and phase solubility analyses showed an inclusion complex was formed between CDs and DHEA at a molar ratio of 1:1, which remarkably increased the solubility of DHEA, and then improved substrate biotransformation efficiency and hydroxylation yield. Meanwhile, results of thermodynamic parameters (ΔG, ΔH, ΔS and Ks) analysis revealed the complexation process was spontaneous and DHEA/CDs inclusion complex was stable. Scanning electron microscopy and transmission electron microscopy showed that the enhancement of DHEA hydroxylation yield also depended on the improvement of cell permeability through interaction between cytomembrane and CDs. These results suggested that the CDs complexation technique was a promising method to enhance steroids hydroxylation yield by increasing steroids solubility and decreasing membrane resistance of substrate and product during biotransformation process.Download full-size image

•Aqueous extracts and methanol extracts from birch stimulated steroid production of I. obliquus.•Aqueous extract from birch bark could simultaneously stimulated biomass and steroid content.•Methanol extract from birch bark only elevated the steroid content.•Six steroids in I. obliquus were simultaneously stimulated.Steroids was considered as one of the bioactive components in Inonotus obliquus, while this kind of secondary metabolites are less accumulated in cultured mycelia. In this study, effect of extracts from bark and core of host-related species, birch (Betula platyphylla Suk.), on steroid production of I. obliquus in submerged culture were evaluated. The results showed that all dosages (0.01 and 0.1 g/L) of aqueous extracts and methanol extracts from birch bark and birch core possessed significantly stimulatory effect on steroid production of I. obliquus (P < 0.05). Among the eight extracts, the aqueous extract (0.01 g/L) from birch bark gave the highest steroid production (225.5 ± 8.7 mg/L), which is 97.3% higher than that of the control group. The aqueous extract (0.01 and 0.1 g/L) from birch bark could simultaneously stimulated mycelial growth and steroid content, while the methanol extract from birch bark only elevated the steroid content. High performance liquid chromatography analysis showed that productions of betulin, ergosterol, cholesterol, lanosterol, stigmasterol, and sitosterol in I. obliquus simultaneously increased in the presence of aqueous extract and methanol extract from birch bark. The results presented herein indicate that extracts from birch bark could act as an inducer for steroid biosynthesis of I. obliquus.

•C. glutamicum SYPS-062 had mutation in the 308 residue (Met→Ile) compared with the model strain.•Significantly lower PYK activity was found in C. glutamicum SYPS-062.•This mutation site in the 308 residue closely associated with its activity.•The lower activity of PYK can improve L-serine production.•The pyk-deleted mutant did not affect the cell growth, and L-serine increased 12%.Pyruvate kinase (PYK) is an important enzyme in the intermediary metabolism and has attracted much attention as a target for metabolic engineering of Corynebacterium glutamicum. Genome sequencing revealed that the 308 residue of PYK was mutated from methionine in model strain C. glutamicum ATCC14067 to isoleucine in L-serine-producing strain C. glutamicum SYPS-062. Consequently, a significantly lower PYK activity (77%) was noted in C. glutamicum SYPS-062, when compared with that in C. glutamicum ATCC14067. To confirm the role of this point mutation, pyk in both C. glutamicum SYPS-062 and C. glutamicum SYPS-062-33aΔSSAA was reversely mutated to restore the PYK enzyme activity, which led to a 33.1% and 28.8% decrease in L-serine titer, respectively. This is the first report to show that the (Met-308→Ile) mutation site of pyk is closely associated with its activity and apparently affected L-serine production. Furthermore, pyk was deleted in strain C. glutamicum SYPS-062-33aΔSSAA, and the resulting strain did not show alteration in growth rate and presented a 12% increase in L-serine production.Download high-res image (138KB)Download full-size image

•Showed a 951 genera-containing picture of microbial diversity in vinegar fermentation.•Flavour metabolic network in vinegar microbiota was reconstructed.•Distribution discrepancy of microbes in different metabolic pathways was revealed.Multispecies microbial community formed through centuries of repeated batch acetic acid fermentation (AAF) is crucial for the flavour quality of traditional vinegar produced from cereals. However, the metabolism to generate and/or formulate the essential flavours by the multispecies microbial community is hardly understood. Here we used metagenomic approach to clarify in situ metabolic network of key microbes responsible for flavour synthesis of a typical cereal vinegar, Zhenjiang aromatic vinegar, produced by solid-state fermentation. First, we identified 3 organic acids, 7 amino acids, and 20 volatiles as dominant vinegar metabolites. Second, we revealed taxonomic and functional composition of the microbiota by metagenomic shotgun sequencing. A total of 86 201 predicted protein-coding genes from 35 phyla (951 genera) were involved in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Metabolism (42.3%), Genetic Information Processing (28.3%), and Environmental Information Processing (10.1%). Furthermore, a metabolic network for substrate breakdown and dominant flavour formation in vinegar microbiota was constructed, and microbial distribution discrepancy in different metabolic pathways was charted. This study helps elucidating different metabolic roles of microbes during flavour formation in vinegar microbiota.

Aim of the studyThe objectives of this study were to investigate the analgesic and anti-inflammatory effects of the dry matter of culture broth (DMCB) of Termitomyces albuminosus in submerged culture and its crude saponin extract (CSE) and crude polysaccharide extract (CPE).Materials and methodsThe analgesic effects of DMCB, CSE and CPE were evaluated with models of acetic acid-induced writhing response and formalin test in mouse. The anti-inflammatory effects of DMCB, CSE and CPE were evaluated by using models of xylene-induced mouse ear swelling and carrageen-induced mouse paw edema.ResultsThe DMCB, CSE and CPE significantly decreased the acetic acid-induced writhing response and the licking time on the late phase in the formalin test. Treatment of DMCB (1000 mg/kg), CSE (200 mg/kg) or CPE (200 mg/kg) inhibited the mouse ear swelling by 61.8%, 79.0% and 81.6%, respectively. In the carrageen-induced mouse paw edema test, the group treated with indomethacin showed the strongest inhibition of edema formation by 77.8% in the third hour after carrageenan administration, while DMCB (1000 mg/kg), CSE (200 mg/kg) and CPE (200 mg/kg) showed 48.4%, 55.6% and 40.5%, respectively.ConclusionsThe results suggested that DMCB of Termitomyces albuminosus possessed the analgesic and anti-inflammatory activities. Saponins and polysaccharides were proposed to be the major active constituents of Termitomyces albuminosus in submerged culture.

Aim of the studyThe antihyperglycemic and antilipidperoxidative effects of the dry matter of culture broth (DMCB) of Inonotus obliquus were investigated.Materials and methodsThe normal, glucose-induced hyperglycemic and alloxan-induced diabetic mice were used to evaluate the antihyperglycemic and antilipidperoxidative effects of the DMCB of Inonotus obliquus.ResultsTreatment with the DMCB (500 and 1000 mg/kg body weight) exhibited a mild hypoglycemic effect in normal mice, and failed to reduce the peak glucose levels after glucose administration. However, euglycemia was achieved in the DMCB of Inonotus obliquus (1000 mg/kg) and glibenclamide-treated mice after 120 min of glucose loading. In alloxan-induced diabetic mice, the DMCB (500 and 1000 mg/kg body weight for 21 days) showed a significant decrease in blood glucose level, the percentages reduction on the 7th day were 11.90 and 15.79%, respectively. However, feeding of this drug for 3 weeks produced reduction was 30.07 and 31.30%. Furthermore, the DMCB treatment significantly decreased serum contents of free fatty acid (FFA), total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C), whereas effectively increased high density lipoprotein-cholesterol (HDL-C), insulin level and hepatic glycogen contents in liver on diabetic mice. Besides, the DMCB treatment significantly increased catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities except for decreasing maleic dialdehyde (MDA) level in diabetic mice. Histological morphology examination showed that the DMCB restored the damage of pancreas tissues in mice with diabetes mellitus.ConclusionsThe results showed that the DMCB of Inonotus obliquus possesses significant antihyperglycemic, antilipidperoxidative and antioxidant effects in alloxan-induced diabetic mice.

•Sucrose was found to be favorable for cell growth and l-serine accumulation.•The resulting mutant accumulated 65.4% more l-serine (11.0 ± 0.25 g/L).•This mutant harbors a better ability to consume sucrose.•serA modification led to dramatically increase in l-serine production.•The key to enhance l-serine production was the removal of the feedback inhibition.The direct fermentative production of l-serine using Corynebacterium glutamicum from renewable biomass attracts increasing attention. This study was designed to enhance l-serine production in a wild-type C. glutamicum SYPS-062 by using random mutagenesis, optimization of media, and metabolic engineering. The resulting mutant, C. glutamicum SYPS 062-33a, accumulated 65.4% more l-serine (11.0 ± 0.25 g/L) and exhibited higher growth and production of other amino acids (such as l-alanine, l-valine) compared with the parent strain. Interestingly, this mutant harbors a better ability to consume sucrose, which is the main composition of economical molasses. To further enhance l-serine production, single sdaA deletion and serA modification was performed, which led to 14.76 ± 0.82 g/L and 19.42 ± 1.20 g/L l-serine, respectively. While double sdaA deletion and serA modification led to 21.27 ± 1.87 g/L l-serine. In addition, when the modified serA gene was overexpressed in C. glutamicum SYPS-062-33aΔSS, the resultant strain accumulated 24.43 ± 0.95 g/L and 21.6 ± 1.2 g/L l-serine in flask and 5-L bioreactor, respectively. Unlike the previous finding, this study demonstrated that the key to further enhance l-serine production may be the removal of the feedback inhibition of PGDH by l-serine in a high l-serine-producing strain C. glutamicum SYPS-062-33a.

BackgroundArmillaria mellea (A. mellea) is a traditional Chinese medicinal and edible mushroom, which is proved to possess a lot of biological activities, including anti-oxidation, immunopotentiation, anti-vertigo and anti-aging activities. However, little information is available in regard to its neuroprotection activity in inflammation-mediated neurodegenerative diseases.PurposeWe have found that A. mellea has an anti-inflammatory activity in LPS-induced RAW264.7 cells in our previous study. The objective of this study is to investigate the anti-neuroinflammatory mechanism of a bioassay-guided fractionation (Fr.2) and its active components/compounds.MethodsCompounds were isolated by preparative high performance liquid chromatography (pre-HPLC) and their structures were established by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analyses. The anti-neuroinflammatory effect of Fr.2 and each compounds were investigated in lipopolysaccharide (LPS)-stimulated murine microglia cell line BV-2.ResultsWe demonstrated that Fr.2 significantly decreased the production of inflammation mediator nitric oxide (NO) and inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) in a dose-dependent manner (10, 30, 100 µg/ml). In addition, Fr.2 markedly down-regulated the phosphorylation levels of nuclear factor kappa B p65 (NF-κB p65), inhibitory κB-α (IκB-α) and c-Jun N-terminal kinases (JNKs) pathways. Sevens compounds were isolated from Fr.2, among them, three compounds, 5-hydroxymethylfurfural (CP1), vanillic acid (CP4) and syringate (CP5) were reported for the first time in A. mellea. NO and inflammatory cytokines (TNF-α, IL-6, IL-1β) secretion indicated that daidzein (CP6) and genistein (CP7) showed a more outstanding anti-inflammation potential at non-toxic concentrations (10, 30, 100 µM) than the other five compounds.ConclusionsIn conclusion, Fr.2 may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and suppress inflammation pathway in activated microglia. Daidzein and genistein may serve as the effective anti-inflammation compounds of Fr.2.Download high-res image (123KB)Download full-size image

In this study, the metabolic flux distribution analysis of a new l-arginine (Arg) overproducing strain, Corynebacterium crenatum, was carried out under various oxygen supply conditions in order to explore the optimized oxygen supply profile. The metabolic flux analysis indicated that a relatively higher l-arginine production could be obtained under high oxygen supply (HOS) condition overall. However, during the late fermentation phases, a much more stable l-arginine production could be rather achieved under medium oxygen supply (MOS) condition. As a result, a two-stage oxygen supply strategy, which maintained HOS condition during early fermentation phase, and then step-wisely reduced agitation to keep a stable, smooth and moderate dissolve oxygen levels (DO) changing profile throughout the production phases, was proposed. With the proposed control strategy, the final l-arginine concentration of the batch fermentation was largely increased and reached to a high level of 36.6 g L−1, which was 16% and 51% higher than those obtained under the HOS and MOS conditions. The two-stage oxygen supply strategy could also accelerate glucose consumption rate and thus shorten fermentation time under the same batch initial fermentation condition. The relevant metabolic flux analysis verified the effectiveness of the proposed control strategy.

•First report on cloning of an aromatic nitrilase from Pseudomonas genus.•Pseudomonas putida nitrilase is distinguished from other Pseudomonas nitrilases.•The nitrilase showed outstanding catalytic activity toward cyanopyridine.Nitrilases have long been considered as an attractive alternative to chemical catalyst in carboxylic acids biosynthesis due to their green characteristics and the catalytic potential in nitrile hydrolysis. A novel nitrilase from Pseudomonas putida CGMCC3830 was purified to homogeneity. pI value was estimated to be 5.2 through two-dimensional electrophoresis. The amino acid sequence of NH2 terminus was determined. Nitrilase gene was cloned through CODEHOP PCR, Degenerate PCR and TAIL-PCR. The open reading frame consisted of 1113 bp encoding a protein of 370 amino acids. The predicted amino acid sequence showed the highest identity (61.6%) to nitrilase from Rhodococcus rhodochrous J1. The enzyme was highly specific toward aromatic nitriles such as 3-cyanopyridine, 4-cyanopyridine, and 2-chloro-4-cyanopyridine. It was classified as aromatic nitrilase. The nitrilase activity could reach up to 71.8 U/mg with 3-cyanopyridine as substrate, which was a prominent level among identified cyanopyridine converting enzymes. The kinetic parameters Km and Vmax for 3-cyanopyridine were 27.9 mM and 84.0 U/mg, respectively. These data would warrant it as a novel and potential candidate for creating effective nitrilases in catalytic applications of carboxylic acids synthesis through further protein engineering.Download full-size image

Most available methods for modifying the catalytic properties of enzymes are costly and time-consuming, as they rely on the information of enzyme crystal structure or require handling large amounts of mutants. This study employs sequence analysis and saturation mutagenesis to improve the catalytic activity and reduce the by-product formation of fungal nitrilase in the absence of structural information. Site-saturation mutagenesis of isoleucine 128 and asparagine 161 in the fungal nitrilase from Gibberella intermedia was performed and mutants I128L and N161Q showed higher catalytic activity toward 3-cyanopyridine and weaker amide forming ability than the wild-type. Moreover, the activity of double mutant I128L–N161Q was improved by 100% and the amount of amide formed was reduced to only one third of that of the wild-type. The stability of the mutants was significantly enhanced at 30 and 40 °C. The catalytic efficiency of the mutant enzymes was substantially improved. In this study, we successfully applied a novel approach that required no structural information and minimal workload of mutant screening for engineering of fungal nitrilase.