Reclassification of Streptomyces griseostramineus as a synonym of Streptomyces griseomycini based on a polyphasic taxonomic approach.

In the present work, the taxonomic relationship between Streptomyces griseomycini and Streptomyces griseostramineus was reevaluated by a comprehensive comparison of phenotypic, chemotaxonomic and genomic characteristics, as well as phylogeny. Phylogenetic analysis based on 16S rRNA gene sequences and whole-genome sequences indicated that Streptomyces griseostramineus JCM 4385T was clustered together with Streptomyces griseomycini JCM 4382T, suggesting they were closely related to each other. However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between their genomes were 99.7 and 97.5 %, respectively, much larger than the recommended threshold values of 96.7 % ANI and 70 % dDDH for Streptomyces species delineation. In addition, the morphological, cultural, physio-biochemical and chemotaxonomic features of these two species further demonstrated that they belonged to the same genome species. Based on the above data and the principle of priority in nomenclature, it is proposed that S. griseostramineus (Preobrazhenskaya et al. 1957) Pridham et al. 1958 (Approved Lists 1980) is a later heterotypic synonym of S. griseomycini (Preobrazhenskaya et al. 1957) Pridham et al. 1958 (Approved Lists 1980).

Mining of a novel reductase and its application for asymmetric reduction of p-methoxyacetophenone.

(R)-1-(4-methoxyphenyl) ethanol [(R)-1b] is an essential precursor for the synthesis of aryl propanoic acids' anti-inflammatatory drugs. Biocatalysts for (R)-1b preparation are limited and reductase has problems of low substrate concentration and low conversion rate. As a result, there is a constant need for discovering novel biocatalysts with excellent catalytic performances. In this study, a novel reductase LpSDR from Lacisediminihabitans profunda for the biocatalytic reduction of p-methoxyacetophenone (1a) to (R)-1b was obtained based on gene-mining technology, and some key reaction parameters were also investigated to improve the conversion rate of 1a using whole cells of recombinant Escherichia coli expressing reductase LpSDR as biocatalysts. It was found that the optimal concentration of isopropanol, ZnSO4·7H2O solution, 1a, and recombinant E. coli resting cells, the optimal reaction temperature, buffer pH, and reaction time were 1.95 mol l-1, 0.75 mmol l-1, 75 mmol l-1, 250 g (wet weight) l-1, 28°C, 7.0, and 21 h, respectively. Under the above conditions, a conversion rate of 99.5% and an enantiomeric excess of 99.6% were obtained, which were superior to the corresponding values previously reported. This study provides a novel reductase LpSDR, which is helpful in reducing 1a to (R)-1b.

Identification of a newly isolated Sphingomonas sp. LZ1 and its application to biosynthesize chiral alcohols.

A strain LZ1, which showed efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone to enantiopure (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol, which is the key intermediate for the synthesis of a receptor antagonist and antidepressant, was isolated from a soil sample. Based on its morphological, 16S rDNA sequence, and phylogenetic analysis, the strain LZ1 was identified to be Sphingomonas sp. LZ1. To our knowledge, this is the first reported case of the species Sphingomonas exhibiting stricter S-enantioselectivity and its use for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone. Some key reaction parameters involved in the bioreduction catalyzed by whole cells of Sphingomonas sp. LZ1 were subsequently optimized, and the optimized conditions for the synthesis of (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol were determined to be as follows: phosphate buffer pH 7.5, 70 mM of 3,5-bis(trifluoromethyl) acetophenone, 30 g/L of glucose as a co-substrate, 300 g (wet weight)/L of resting cell as the biocatalyst, and a reaction for 24 h at 30°C and 180 rpm. Under the above conditions, a best yield of 94% and an excellent enantiomeric excess of 99.6% were obtained, respectively. Sphingomonas sp. LZ1 could also asymmetrically reduce a variety of prochiral ketones to their corresponding optical alcohols with excellent enantioselectivity. These results indicated that Sphingomonas sp. LZ1 had a remarkable capacity to reduce 3,5-bis(trifluoromethyl)acetophenone to its corresponding (S)-[3,5-bis(trifluoromethyl)phenyl]ethanol, and might be a new potential biocatalyst for the production of valuable chiral alcohols in industry.

Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency.

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is a valuable chiral intermediate for the synthesis of antiemetic drug Aprepitant and Fosaprepitant. A Leifsonia xyli HS0904-derived carbonyl reductase (LXCAR), an effective biocatalyst for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-BTPE, was overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis indicated that the amino acid sequence of recombinant LXCAR showed 89 % similarity to short-chain dehydrogenase/reductase. E. coli recombinant carbonyl reductase crude extract showed a specific activity of 1.54 U/mg, which was 62 times higher than that of L. xyli HS0904 crude extract. By using error-prone polymerase chain reaction and site-directed mutagenesis, the engineered LXCAR demonstrated superior catalytic activity toward BTAP, and the obtained mutant LXCAR-S154Y exhibited nearly 13-fold, 5.4-fold, and 2.3-fold increase in k cat/K m value, k cat value, and specific activity toward BTAP, respectively, compared to the recombinant LXCAR. Additionally, the reduction of BTAP by whole cells of mutant LXCAR-S154Y afforded a best yield of 99.6 % for (R)-BTPE within 2 h at 200 mM BTAP, which was shortened by 28 and 2 h compared to those catalyzed by L. xyli HS0904 cells and recombinant E. coli cells expressing LXCAR, respectively. Moreover, a yield of 82.5 % for (R)-BTPE was achieved within 12 h at an increased BTAP concentration of up to 1,000 mM (256 g/l), representing a 1.9-fold increase over the recombinant LXCAR. Homology modeling and docking analysis revealed the molecular basis for the high catalytic activity of mutant LXCAR-S154Y toward BTAP. The results present here provide a promising alternative for economical and efficient production of chiral alcohols by engineered LXCAR.

Tsukamurella sp. E105 as a new biocatalyst for highly enantioselective hydrolysis of ethyl 2-(2-oxopyrrolidin-1-yl) butyrate.

A new bacterial strain, E105, has been introduced as a biocatalyst for the enantioselective hydrolysis of ethyl (R,S)-2-(2-oxopyrrolidin-1-yl) butyrate, (R,S)-1, to (S)-2-(2-oxopyrrolidin-1-yl) butyric acid, (S)-2. This strain was isolated from 60 soil samples using (R,S)-1 as the sole carbon source. The isolate was identified as Tsukamurella tyrosinosolvens E105, based on its morphological characteristics, physiological tests, and 16S rDNA sequence analysis. The process of cell growth and hydrolase production for this strain was then investigated. The hydrolase activity reached its maximum after cultivation at 200 rpm and 30 °C for 36 h. Furthermore, the performance of the enantioselective hydrolysis of (R,S)-1 was studied. The optimal reaction temperature, initial pH, substrate concentration, and concentration of suspended cells were 30 °C, 6.8, 10 and 30 g/l (DCW), respectively. Under these conditions, a high conversion (>45 %) of the product (S)-2 with an excellent enantiomeric excess (ee) (>99 %), and a satisfied enantiomeric ratio (E) (>600) as well were obtained. This study showed that the bacterial isolate T. tyrosinosolvens E105 displayed a high enantioselectivity towards the hydrolysis of racemic ethyl 2-(2-oxopyrrolidin-1-yl) butyrate.