Enhanced synthesis of chloramphenicol intermediate L-threo-p-nitrophenylserine using engineered L-threonine transaldolase and by-product elimination.

L-threo-p-nitrophenylserine (component 2) is an important intermediate during synthesis of chloramphenicol. However, its biosynthesis is limited by enzyme activity and stereoselectivity. In this study, we achieved a breakthrough in the high-efficiency production of 2 by employing engineered Chitiniphilus shinanonensis L-threonine transaldolase (ChLTTA) in conjunction with a by-product elimination system within a one-pot reaction. Notably, a novel visual stepwise high-throughput screening method was developed for the directed evolution of ChLTTA, leveraging its characteristic color. The engineered mutant F70D/F59A (Mu6 variant) emerged as a star performer, exhibiting a remarkable 2.6-fold increase in catalytic efficiency over the wild-type ChLTTA, coupled with an outstanding 91.5 % diastereoisomer excess (de). Molecular dynamics (MD) simulations unraveled the mechanism responsible for the enhanced catalytic performance observed in the Mu6 variant. Meanwhile, the Mu6 variant was coupled with Saccharomyces cerevisiae ethanol dehydrogenase (ScADH) and Candida boidinii formate dehydrogenase (CbFDH) to create a high-efficiency cascade system (E.coli/pRSF-Mu6-ScADH-CbFDH). Under optimized conditions, this cascade system demonstrated unparalleled performance, yielding 201.5 mM of 2 with an impressive conversion of 95.9 % and a de value of 94.5 %. This achievement represents the highest reported yield to date. This study offers a novel insight into the sustainable and efficient production of chloramphenicol intermediate.

Rational Design of l-Threonine Transaldolase-Mediated System for Enhanced Florfenicol Intermediate Production.

l-threo-p-methylsulfonylphenylserine (compound 1b) is the main intermediate of florfenicol, and its efficient synthesis has been the subject of current research. Herein, Burkholderia diffusa l-threonine transaldolase (BuLTTA) was rationally designed based on the sequence-structure-function relationship. A mutant M4 (Asn35Ser/Thr352Asn) could produce 35.5 mM 1b with 88.8% conversion and 93.8% diastereoselectivity, 314 and 129% of the values observed for wild-type BuLTTA. Molecular dynamics simulations indicated that the shortened distance between key active site residues and the transition state (PLP-1b) and the improved hydrogen bond force enhanced the catalytic performance of the M4 variant. Then, the mutant M4 was combined with K. kurtzmanii alcohol dehydrogenase (KkADH) to eliminate the BuLTTA-inhibiting byproduct acetaldehyde, and a cosubstrate was added to regenerate the ADH cofactor NADH. Under optimized conditions, the yield of 1b reached 115.2 mM with a conversion of 96% and a diastereoselectivity of 95.5%. This work provides a new strategy for the efficient and sustainable production of 1b.

Candida yunnanensis sp. nov. and Candida parablackwelliae sp. nov., two yeast species in the Candida albicans/Lodderomyces clade.

During studies on the yeast communities associated with rotting wood in the Xishuangbanna Tropical Rainforest in PR China, four novel yeast strains were found. Phylogenetic analysis based on the concatenated sequences of the D1/D2 domains of the large subunit rRNA gene and the ITS regions showed that these strains represented two novel species in the Candida albicans/Lodderomyces clade. The novel species, represented by strains NYNU 17948 and NYNU 17981, formed a clade with Candida maltosa and Candida baotianmanensis, with 1-1.8% sequence divergence in the D1/D2 domains and 8.9-10% sequence divergence in the ITS regions. The other novel species, represented by NYNU 17105 and NYNU 17763, is most closely related to Candida blackwelliae with 0.7 % sequence divergence in the D1/D2 domains and 6.9 % sequence divergence in the ITS regions. The two novel species could be distinguished from their closest described species in terms of physiological traits. The two novel species are described as Candida yunnanensis sp. nov. (holotype NYNU 17948) and Candida parablackwelliae sp. nov. (holotype NYNU 17763).

Saturnispora galanensis sp. nov., a yeast species isolated from rotting wood.

Three strains of a novel yeast species were isolated from rotting wood in the Xishuangbanna Tropical Rainforest, Yunnan Province, PR China. Sequence analysis of the D1/D2 domains of the large subunit rRNA gene and the internal transcribed spacer (ITS) regions showed that the novel species represents a member of the genus Saturnispora. It differed from its closest known species, Saturnispora sekii CBS 10931T, by 1.3 % nucleotide substitutions in the D1/D2 domains and by 2.2 % nucleotide substitutions in the ITS regions, respectively. In contrast to Saturnispora sekii, the novel yeast species was unable to assimilate glycerol, dl-lactate, succinate and citrate, and grow at 37 °C. The name Saturnispora galanensis sp. nov. is proposed to accommodate these strains, with NYNU 1797 as the holotype.

Vanrija jinghongensis sp. nov., an asexual basidiomycetous yeast from rotting wood.

Three strains of a novel basidiomycetous yeast were isolated from the Xishuangbanna Tropical Rainforest, Yunnan Province, PR China. Sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions indicated that the novel species represents a member of the genus Vanrija. It differed from the most closely related known species, Vanrija albida CBS 2839T, by 1.5 % sequence divergence (seven substitutions and two gaps out of 597 bp) in the D1/D2 domains and by 7.4 % sequence divergence (17 substitutions and 20 gaps over 495 bp) in the ITS regions, respectively. The three strains of the novel species reproduced asexually, and no mating could be found. In contrast to V. albida, the novel yeast species was able to assimilate d-glucosamine, inulin, erythritol and galactitol and unable to assimilate raffinose. The name Vanrija jinghongensis sp. nov. is proposed to accommodate these strains, with NYNU 17910T (=CICC 33269=CBS 15229) as the type strain.

LncRNA MALAT1 potentiates autophagy­associated cisplatin resistance by regulating the microRNA­30b/autophagy­related gene 5 axis in gastric cancer.

Gastric cancer (GC) is the fourth most common type of cancer worldwide and chemoresistance is a major obstacle to successful GC treatment. In the present study, reverse transcription­quantitative polymerase chain reaction analysis was used to measure the expression of metastasis­associated lung adenocarcinoma transcript 1 (MALAT1) and microRNA (miR)­30b. Western blot analysis was conducted to detect the protein expression of autophagy­related gene 5 (ATG5), p62 and LC3 (LC3­I and LC3­II). Cell viability and half maximal inhibitory concentration were determined by the Cell Counting Kit­8 assay. The green fluorescent protein (GFP)­LC3­positive cell percentage was determined by the GFP­LC3 puncta experiment. Luciferase reporter and RNA immunoprecipitation assays were used to explore the molecular associations among MALAT1, miR­30b and ATG5. MALAT1 was found to be highly expressed in CDDP­resistant AGS(AGS/CDDP) cells and CDDP­resistant HGC­27 (HGC­27/CDDP) cells. Cell viability was markedly increased in MALAT1­overexpressing AGS/CDDP cells, but was notably reduced in MALAT1­depleted HGC­27/CDDP cells. Moreover, MALAT1 potentiated CDDP resistance by facilitating autophagy in AGS/CDDP and HGC­27/CDDP cells. Further investigations demonstrated that MALAT1 inhibited miR­30b expression by direct interaction. Moreover, miR­30b abolished MALAT1­induced CDDP resistance by inhibiting autophagy in AGS/CDDP and HGC­27/CDDP cells. Furthermore, ATG5 was found to be a target of miR­30b. miR­30b weakened resistance to CDDP by inhibiting autophagy in AGS/CDDP and HGC­27/CDDP cells, while this effect was abrogated by increased ATG5 expression. Additionally, MALAT1 sequestered miR­30b from ATG5 to increase ATG5 expression in AGS/CDDP and HGC­27/CDDP cells. Therefore, MALAT1 potentiated autophagy­related CDDP resistance through suppressing the miR­30b/ATG5 axis in AGS/CDDP and HGC­27/CDDP cells, indicating that it may represent a promising target for the reversal of chemoresistance in GC.

Sugiyamaella xiaguanensis f.a., sp. nov., a yeast species isolated from rotting wood.

Three strains representing a novel yeast species, Sugiyamaella xiaguanensis f.a., sp. nov. (type strain NYNU 161041T=CICC 33167T=CBS 14696T), were isolated from rotting wood samples collected in Henan and Yunnan Provinces, PR China. The novel species is able to assimilate cellobiose, salicin and d-xylose, which was typical of the species of the genus Sugiyamaella. Analysis of the D1/D2 domains of the large subunit rRNA gene and internal transcribed spacer regions of these strains showed that this species was related to Sugiyamaella lignohabitans and Sugiyamaella marionensis, its closest relatives. Su. xiaguanensis sp. nov. differed by 1.4 % nucleotide substitutions from Su. lignohabitans, and by 1.9 % nucleotide substitutions from Su. marionensis in the D1/D2 sequences. The ITS sequences of Su. xiaguanensis sp. nov. displayed more than 6.5 % nucleotide substitutions from the latter two species, showing that it is a genetically separate species.

Ruthenium(III) chloride catalyzed acylation of alcohols, phenols, and thiols in room temperature ionic liquids.

Ruthenium(III) chloride-catalyzed acylation of a variety of alcohols, phenols, and thiols was achieved in high yields under mild conditions (room temperature) in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]). The ionic liquid and ruthenium catalyst can be recycled at least 10 times. Our system not only solves the basic problem of ruthenium catalyst reuse, but also avoids the use of volatile acetonitrile as solvent.