γ-lactamase is used for the resolution of γ-lactam which is utilized in the synthesizing of abacavir and peramivir. In some cases, enzymatic method is the most utilized method because of its high efficiency and productivity. The cellulose binding domain (CBD) of cellulose is often used as the bio-specific affinity matrix for enzyme immobilization. Cellulose is cheap and it has excellent chemical and physical properties. Meanwhile, binding between cellulose and CBD is tight and the desorption rarely happened.

The “bidirectional gene pair” indicates a particular head-to-head gene organization in which transcription start sites of two genes are located on opposite strands of genomic DNA within a region of one kb. Despite bidirectional gene pairs are well characterized, little is known about their expression profiles and regulation features in tumorigenesis. We used RNA-seq data from The Cancer Genome Atlas (TCGA) dataset for a systematic analysis of the expression profiles of bidirectional gene pairs in 13 cancer datasets. Gene pairs on the opposite strand with transcription end site distance within one kb or on the same strand with the distance of two genes between 1–10 kb and gene pairs comprising two randomly chosen genes were used as control gene pairs (CG1, CG2, and random). We identified and characterized up-/down-regulated genes by comparing the expression level between tumors and adjacent normal tissues in 13 TCGA datasets. There were no consistently significant difference in the percentage of up-/down-regulated genes between bidirectional and control/random genes in most of TCGA datasets. However, the percentage of bidirectional gene pairs comprising two up- or two down-regulated genes was significantly higher than gene pairs from CG1/2 in 12/11 analyzed TCGA datasets and the random gene pairs in all 13 TCGA datasets. Then we identified the methylation correlated bidirectional genes to explore the regulatory mechanism of bidirectional genes. Like the differentially expressed gene pairs, the bidirectional genes in a pair were significantly prone to be both hypo- or hyper-methylation correlated genes in 12/13 TCGA datasets when comparing to the CG2/random gene pairs despite no significant difference between the percentages of hypo-/hyper-methylation correlated genes in bidirectional and CG2/random genes in most of TCGA datasets. Finally, we explored the correlation between bidirectional genes and patient’s survival, identifying prognostic bidirectional genes and prognostic bidirectional gene pairs in each TCGA dataset. Remarkably, we found a group of prognostic bidirectional gene pairs in which the combination of two protein coding genes with different expression level correlated with different survival prognosis in survival analysis for OS. The percentage of these gene pairs in bidirectional gene pair were significantly higher than the gene pairs in controls in COAD datasets and lower in none of 13 TCGA datasets.

Background: Corynebacterium ammoniagenesis an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A byC. ammoniagenesATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain.Results: In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (P_rpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase fromPseudomonas putida(PpcoaA) was overexpressed inC. ammoniagenesunder the control of P_rpl21, CoA yield increased approximately 4.4 times.Conclusions: This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit forC. ammoniagenesand should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity inC. ammoniagenesATCC 6871 for industrial applications.

R‐1,3‐butanediol (R‐1,3‐BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R‐1,3‐BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio‐selectivity is not high enough for further organic synthesis efforts. Here, a short‐chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4‐hydroxy‐2‐butanone (4H2B), residues were selected for virtual mutagenesis, their substrate‐binding energies were compared, and four sites were selected for saturation mutagenesis. High‐throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer‐aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B‐binding pocket. The number of hydrogen bonds between the stabilized 4H2B‐binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild‐type enzyme, while the substrate‐binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio‐selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate‐binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.