Heterologous expression of a deacetylase and its application in L-glufosinate preparation.

With potent herbicidal activity, biocatalysis synthesis of L-glufosinate has drawn attention. In present research, NAP-Das2.3, a deacetylase capable of stereoselectively resolving N-acetyl-L-glufosinate to L-glufosinate mined from Arenimonas malthae, was heterologously expressed and characterized. In Escherichia coli, NAP-Das2.3 activity only reached 0.25 U/L due to the formation of inclusive bodies. Efficient soluble expression of NAP-Das2.3 was achieved in Pichia pastoris. In shake flask and 5 L bioreactor fermentation, NAP-Das2.3 activity by recombinant P. pastoris reached 107.39 U/L and 1287.52 U/L, respectively. The optimum temperature and pH for N-acetyl-glufosinate hydrolysis by NAP-Das2.3 were 45 °C and pH 8.0, respectively. The Km and Vmax of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 µmol mg-1 min-1, respectively. Within 90 min, 92.71% of L-enantiomer in 100 mM racemic N-acetyl-glufosinate was converted by NAP-Das2.3. L-glufosinate with high optical purity (e.e.P above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for L-glufosinate biosynthesis.

Strain Screening and Particle Formation: a Lysinibacillus boronitolerans for Self-Healing Concrete.

Microbial-induced calcite precipitation is a promising technology to solve the problem of cracks in soil concrete. The most intensively investigated microorganisms are urease-producing bacteria. Lysinibacillus that is used as urease-producing bacteria in concrete repair has rarely been reported. In this study, Lysinibacillus boronitolerans with a high urease activity was isolated from soil samples. This strain is salt- and alkali-tolerance, and at pH 13, can grow to ~OD600 2.0 after 24 h. At a salt concentration of 6%, the strain can still grow to ~OD600 1.0 after 24 h. The feasibility of using this strain in self-healing concrete was explored. The data showed that cracks within ~0.6 mm could be repaired naturally with hydration when spores and substrates were added to the concrete in an appropriate proportion. Moreover, the number and morphology of CaCO3 crystals that were produced by bacteria can be influenced by the concrete environment. An efficiency method to elucidate the process of microbial-induced calcium carbonate crystal formation was established with Particle Track G400. This study provides a template for future studies on the theory of mineralization based on microorganisms. IMPORTANCE The formation of calcium carbonate crystals in concrete by urease-producing bacteria is not understood fully. In this study, a Lysinibacillus boronitolerans strain with a high urease activity was isolated and used to analyze the counts and sizes of the crystals and the relationship with time. The data showed that the number of crystal particles increases exponentially in a short period with sufficient substrate, after which the crystals grow, precipitate or break. In concrete, the rate-limiting steps of calcium carbonate crystal accumulation are spore germination and urease production. These results provided data support for the rational design of urease-producing bacteria in concrete repair.

Improvement of R-2-(4-hydroxyphenoxy) propionic acid biosynthesis of Beauveria bassiana by combined mutagenesis.

R-2-(4-hydroxyphenoxy)propionicacid (HPOPA) is a valuable intermediate for the synthesis of enantiomerically pure aryloxyphenoxypropionic acid herbicides. In this work, to improve the HPOPA biosynthesis by Beauveria bassiana ZJB16002 from the substrate R-2-phenoxypropionic acid (POPA), the original HPOPA producer B. bassiana ZJB16002 was subjected to physical mutagenesis with 137 Cs-γ irradiation and chemical mutagen N-methyl-N'-nitro-N-nitrasoguanidine (NTG) induced mutagenesis. The effects of different treatment doses of the mutagens on the lethal rate and positive mutation rate were investigated, and the results showed that the optimal 137 Cs-γirradiation dose and NTG concentration was 850 Gy and 500 µg/mL, respectively. Under these conditions, a mutant strain CCN-7 with the highest HPOPA production capacity was obtained through two rounds of 137 Cs-γ irradiation treatment followed by one round of NTG mutagenesis. At the substrate (POPA) concentration of 50 g/L, HPOPA titer of CCN-7 reached 36.88 g/L, which was 9.73-fold higher than the parental strain. The morphology of the wild-type and mutant strain was compared and the results might provide helpful information in exploration of the correlation of morphology and biochemical features of B. bassiana.

Production of (R)-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana ZJB16007 in solid state fermentation using rice bran.

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key chiral intermediate for phenoxypropionic acid herbicide synthesis. In this study, to improve the production of R-HPPA with B. bassiana ZJB16007, the cultivation conditions in solid-state fermentation (SSF) were investigated. The effects of various substrates on R-HPPA production were evaluated and the process parameters were also optimized. The results showed that rice bran was the optimal substrate for R-HPPA production. The optimal medium components and cultivation conditions were: rice bran: silkworm chrysalis powder = 5.25: 2.25 (g: g), nutrient salts solution 12 mL which contained 50 g/L R-PPA, pH 5.0, and cultivated at 28 °C for 11 days. Under the optimized conditions, the transformation of R-HPPA was significantly improved and the yield of R-HPPA reached 77.78%, which was 15.14% higher than that of the control (67.55%). Therefore, SSF may serve as an alternative for R-HPPA production by B. bassiana ZJB16007.

A high-throughput screening method for improved R-2-(4-hydroxyphenoxy)propionic acid biosynthesis.

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key intermediate of the enantiomerically pure phenoxypropionic acid herbicides. R-HPPA could be biosynthesized through selective introduction of a hydroxyl group (-OH) into the substrate R-2-phenoxypropionic acid (R-PPA) at C-4 position, facilitated by microorganisms with hydroxylases. In this study, an efficient high-throughput screening method for improved R-HPPA biosynthesis through microbial hydroxylation was developed. As a hydroxylated aromatic product, R-HPPA could be oxidized by oxidant potassium dichromate to form brown-colored quinone-type compound. The concentration of R-HPPA can be quantified according to the absorbance of the colored compound at a suitable wavelength of 570 nm; and the R-HPPA biosynthetic capability of microorganism strains could also be rapidly evaluated. After optimization of the assay conditions, the high-throughput screening method was successfully used in identification of Beauveria bassiana mutants with enhanced R-HPPA biosynthesis capacity. A positive mutant C-7 with high tolerance to 20 g/L R-PPA was rapidly selected from 1920 mutants. The biomass and R-HPPA titer were 12.5- and 38.19-fold higher compared with the original strain at 20 g/L R-PPA. This high-throughput screening method developed in this work could also be a potential tool for screening strains producing other important phenolic compounds.

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41 g/L vs 1.39 g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2 g/L SAM in a 5-L fermenter after 96 h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15-33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g-1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L-1L-met and 0.60 g L-1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L-1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L-1 to 10.1 g L-1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

Asymmetric synthesis of (R)-1,3-butanediol from 4-hydroxy-2-butanone by a newly isolated strain Candida krusei ZJB-09162.

Biocatalytic asymmetric preparation of (R)-1,3-butanediol has been attracting much attention in pharmaceuticals industry. A new ideal strain, ZJB-09162, which exhibited high reduction activity and excellent (R)-stereospecificity towards 4-hydroxy-2-butanone, has been successfully isolated from soil samples. Based on morphology, physiological tests (API 20 C AUX), and 5.8S-ITS sequence, the isolate was identified as Candida krusei. Kinetic characterization demonstrated that carbonyl reductase from C. krusei ZJB-09162 preferred NADH to NADPH as cofactor, indicating it might be a new carbonyl reductase. (R)-1,3-Butanediol was produced in 19.8 g/L, 96.6% conversion, and 99.0% ee at optimal pH 8.5, 35 °C with a 2:1 molar ratio of glucose to 4H2B. In order to achieve higher product titer, the substrate loading was optimized in fixed catalysts and fixed substrate/catalysts ratio mode. The bioreduction of 4-hydroxy-2-butanone at a concentration of 45.0 g/L gave (R)-1,3-butanediol in 38.7 g/L and 83.9% conversion. Therefore, C. krusei ZJB-09162 was, for the first time, proven to be a promising biocatalyst for enzymatic preparation of (R)-1,3-butanediol.

Gene Cascade Shift and Pathway Enrichment in Rat Kidney Induced by Acarbose Through Comparative Analysis.

Acarbose is an effective anti-diabetic drug to treat type 2 diabetes mellitus (T2DM), a chronic degenerative metabolic disease caused by insulin resistance. The beneficial effects of acarbose on blood sugar control in T2DM patients have been confirmed by many studies. However, the effect of acarbose on patient kidney has yet to be fully elucidated. In this study, we report in detail the gene expression cascade shift, pathway and module enrichment, and interrelation network in acarbose-treated Rattus norvegicus kidneys based on the in-depth analysis of the GSE59913 microarray dataset. The significantly differentially expressed genes (DEGs) in the kidneys of acarbose-treated rats were initially screened out by comparative analysis. The enriched pathways for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were further identified. The protein-protein interaction (PPI) analysis for DEGs was achieved through the STRING database mining. Pathway interrelation and hub genes for enriched pathways were further examined to uncover key biological effects of acarbose. Results revealed 44 significantly up-regulated genes and 86 significantly down-regulated genes (130 significant differential genes in total) in acarbose-treated rat kidneys. Lipid metabolism pathways were considerably improved by acarbose, and the physical conditions in chronic kidney disease (CKD) patients were improved possibly through the increase of the level of high-density lipoprotein (HDL) by lecithin-cholesterol acyl-transferase (LCAT). These findings suggested that acarbose may serve as an ideal drug for CKD patients, since it not only protects the kidney, but also may relieve the complications caused by CKD.

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier.

Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R - 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S - 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl(2), 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R - 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35 degrees C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S - 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S - 1.

Screening of Fungi Isolates for C-4 Hydroxylation of R-2-Phenoxypropionic Acid Based on a Novel 96-Well Microplate Assay Method.

R-2-(4-Hydroxyphenoxy)propionic acid (R-HPPA) is a pivotal intermediate for the synthesis of aryloxyphenoxypropionate (APP) herbicide. To rapidly screen microbial isolates with the capacity of hydroxylating R-2-phenoxypropionic acid to R-HPPA from various environmental samples, a convenient and safe 96-well microplate assay method with sodium nitrite (NaNO2) as chromogenic reagent was proposed and optimized. The optimum assay conditions were as follows: the detection wavelength was 420 nm, the concentration of NaNO2 solution was 6.0 g/L, color reaction temperature was 60 °C, the pH of the NaNO2 solution was 2.4, and the reaction time was 40 min. With the aid of this method, screening for microorganisms with C-4-specific hydroxylation activity of R-PPA was conducted. As a result, 23 strains among 3744 single colonies isolated from various samples exhibited the hydroxylation activity. Among these strains, the highest bioconversion rate was achieved by Penicillium oxalicum A5 and Aspergillus versicolor A12, respectively. After 72-h cultivation in shake flask, their conversion rates of R-HPPA from 10 g/L R-PPA reached 21.18% and 40.24%, respectively. The established method was effective in rapid screening of microbes capable of biosynthesizing R-HPPA through hydroxylation of R-PPA, and the obtained two fungi species could be potentially used for R-HPPA production.

Enhanced (R)-2-(4-Hydroxyphenoxy)Propionic Acid Production by Beauveria bassiana: Optimization of Culture Medium and H2O2 Supplement Under Static Cultivation.

(R)-2-(4-hydroxyphenoxy)propionic acid (HPOPA) is a key intermediate for the preparation of aryloxyphenoxypropionic acid herbicides (R-isomer). In order to improve the HPOPA production from the substrate (R)-2-phenoxypropionic acid (POPA) with Beauveria bassiana CCN-A7, static cultivation and H2O2 addition were attempted and found to be conducive to the task at hand. This is the first report on HPOPA production under static cultivation and reactive oxygen species (ROS) induction. On this premise, the cultivation conditions and fermentation medium compositions were optimized. As a result, the optimal carbon source, organic nitrogen source, and inorganic nitrogen source were determined to be glucose, peptone, and ammonium sulfate, respectively. The optimal inoculum size and fermentation temperature were 13.3% and 28°C, respectively. The significant factors including glucose, peptone, and H2O2, identified based on Plackett-Burman design, were further optimized through Central Composite Design (CCD). The optimal concentrations/amounts were as follows: glucose 38.81 g/l, peptone 7.28 g/l, and H2O2 1.08 ml/100 ml. Under the optimized conditions, HPOPA titer was improved from 9.60 g/l to 19.53 g/l, representing an increase of 2.03- fold. The results obtained in this work will provide novel strategies for improving the biosynthesis of hydroxy aromatics.

Enhancement of gibberellic acid production from Fusarium fujikuroi by mutation breeding and glycerol addition.

Gibberellic acid (GA3) is a natural plant growth hormone that has been widely used in agriculture and horticulture. To obtain higher GA3 producing strains, the method of screening the strains for resistance to simvastatin was used after treatment with nitrosoguanidine (NTG) and gamma rays. The rationale for the strategy was that mutants showing simvastatin resistance were likely to be high GA3 producers, as their activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is relatively more effective. GA3 yield of mutant S109 increased by 14.2% than that of the original strain. The GA3 production ability in mutant S109 remained relatively stable after ten generations. With the addition of 0.4 g glycerol on the 5th day during the fermentation process in Erlenmeyer flask, maximum GA3 production of 2.7 g/L was achieved by this mutant, exhibiting 28.6% increase compared with original strain. Furthermore, we also achieved 2.8 g/L GA3 and had a 33.3% increase with addition 20 g glycerol on the 5th day during the fermentation process in a 5-L bioreactor. Our results indicated efficient GA3 production could be achieved on the condition that the supply of glycerol at the suitable conditions. This study would lay a foundation for industrial production of GA3.

A rapid throughput assay for screening (R)-2-(4-hydroxyphenoxy)propionic acid producing microbes.

(R)-2-(4-hydroxyphenoxy)propionic acid ((R)-HPOPA) is an important intermediate for the synthesis of optically pure aryloxyphenoxypropionic acid herbicides. Regioselective hydroxylation of (R)-2-phenoxypropionic acid ((R)-POPA) by microbes is one of the most useful methods for the industrial production of (R)-HPOPA. In this study, we designed and optimized a rapid throughput assay for screening (R)-HPOPA producing bacterial/fungal strains which can regioselectively hydroxylate (R)-POPA. (R)-HPOPA could react with 4-aminoantipyrine (4-AAP) in the presence of potassium hexacyanoferrate (K3[Fe(CN)6]) to form indoxyl antipyrine, an orange-red chromophore, that can easily spectrophotometrically be determined at 550 nm. During the verification of the assay we observed an average recovery rate of between 97.3% and 104.5%. Apart from the rapid throughput, no obvious differences in detection (R)-HPOPA in the culture broth samples were found between our rapid throughput multiplate assay and a high-performance liquid chromatography method. Our optimized assay method is simple, rapid and accurate with high repeatability. It has the potential for high throughput screening (about 3000-5000 samples/day) of the (R)-HPOPA producing strains.

Improvement of amidase production by a newly isolated Delftia tsuruhatensis ZJB-05174 through optimization of culture medium.

The R-amidase production by a newly isolated strain of Delftia tsuruhatensis ZJB-05174 was optimized in this paper. Effects of factors such as carbon sources, nitrogen sources, and inducers on amidase production were investigated. The medium composition was optimized using central composite designs and response surface analysis. The optimal medium components for enhanced amidase production were found to be as follows: glucose, 8.23 g/l; yeast extract, 11.59 g/l; 2,2-(R,S)-dimethylcyclopropane carboxamide, 1.76 g/l; NaCl, 1 g/l; KH2PO4, 1 g/l; and K2HPO4, 1 g/l. A maximum enzyme production of 528.21 U/l was obtained under the optimized conditions, which was 4.7 times higher than that obtained under initial conditions.

Production of R-Mandelic Acid Using Nitrilase from Recombinant E. coli Cells Immobilized with Tris(Hydroxymethyl)Phosphine.

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L-1 day-1 and the space-time productivity of 143.2 mmol L-1 h-1 g-1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.

Significant improvement of the nitrilase activity by semi-rational protein engineering and its application in the production of iminodiacetic acid.

Iminodiacetic acid (IDA) is widely used as an intermediate in the manufacturing of chelating agents, glyphosate herbicides and surfactants. To improve activity and tolerance to the substrate for IDA production, Acidovorax facilis nitrilase was selected for further modification by the gene site saturation mutagenesis method. After screened by a two-step screening method, the best mutant (Mut-F168V/T201N/S192F/M191T/F192S) was selected. Compared to the wild-type nitrilase, Mut-F168V/T201N/S192F/M191T/F192S showed 136% improvement in specific activity. Co2+ stimulated nitrilase activity, whereas Cu2+, Zn2+ and Tween 80 showed a strong inhibitory effect. The Vmax and kcat of Mut-F168V/T201N/S192F/M191T/F192S were enhanced 1.23 and 1.23-fold, while the Km was decreased 1.53-fold. The yield of Mut-F168V/T201N/S192F/M191T/F192S with 453.2 mM of IDA reached 71.9% in 5 h when 630 mM iminodiacetonitrile was used as substrate. This study indicated that mutant nitrilase obtained in this study is promising in applications for the upscale production of IDAN.

Isolation and characterization of Delftia tsuruhatensis ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropanecarboxamide.

R-Enantioselective amidases are of considerable industrial interest due to potential applications in the production of optically active compounds. Strain ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropanecarboxamide, was isolated employing a newly established colorimetric screening method. Based on morphology, physiological tests, ATB system (ID 32 GN) and the 16S rRNA sequence, this strain was identified as Delftia tsuruhatensis. The intracellular amidase exhibited excellent thermostability and was not inhibited by urea, a known inhibitor of many amidases. The enantiomeric ratio of intracellular amidase decreased from 27 to 5 when the reaction temperature was increased from 30 to 46 degrees C. A novel temperature-dependent reversal of stereospecificity was observed when the temperature was increased to 56 degrees C. Interestingly, when the reaction was performed at 46 degrees C, cell suspensions pre-incubated at 56 degrees C exhibited the same inversion of stereospecificity.

Bioleaching of chromium from tannery sludge by indigenous Acidithiobacillus thiooxidans.

Chromium in tannery sludge will cause serious environmental problems and is toxic to organisms. The acidophilic sulfur-oxidizing Acidithiobacillus thiooxidans can leach heavy metals form urban and industrial wastes. This study examined the ability of an indigenous sulfur-oxidizing A. thiooxidans to leach chromium from tannery sludge. The results showed that the pH of sludge mixture inoculated with the indigenous A. thiooxidans decreased to around 2.0 after 4 days. After 6 days incubation in shaking flasks at 30 degrees C and 160 rpm, up to 99% of chromium was solubilized from tannery sludge. When treated in a 2-l bubble column bioreactor for 5 days at 30 degrees C and aeration of 0.5 vvm, 99.7% of chromium was leached from tannery sludge. The results demonstrated that chromium in tannery sludge can be efficiently leached by the indigenous A. thiooxidans.

A spectrophotometric screening method for avermectin oxidizing microorganisms.

A spectrophotometric screening method for avermectin oxidizing microbes by determination of 4″-oxo-avermectin was established based on the reaction between 4″-oxo-avermectin and 2,4-dinitrophenylhydrazine. Combined with a gradient HPLC assay, microorganisms capable of regioselectively oxidizing avermectin to 4″-oxo-avermectin were successfully obtained by this method.