Genome-Wide Identification of the Cytochrome P450 Superfamily Genes and Targeted Editing of BnCYP704B1 Confers Male Sterility in Rapeseed.

The cytochrome P450 (CYP450) monooxygenase superfamily, which is involved in the biosynthesis pathways of many primary and secondary metabolites, plays prominent roles in plant growth and development. However, systemic information about CYP450s in Brassica napus (BnCYP450) was previously undiscovered and their biological significance are far from understood. Members of clan 86 CYP450s, such as CYP704Bs, are essential for the formation of pollen exine in plant male reproduction, and the targeted mutagenesis of CYP704B genes has been used to create new male sterile lines in many crops. In the present study, a total of 687 BnCYP450 genes were identified in Brassica napus cultivar "Zhongshuang 11" (ZS11), which has nearly 2.8-fold as many CYP450 members as in Arabidopsis thaliana. It is rationally estimated since Brassica napus is a tetraploid oil plant with a larger genome compared with Arabidopsis thaliana. The BnCYP450 genes were divided into 47 subfamilies and clustered into nine clans. Phylogenetic relationship analysis reveals that CYP86 clan consists of four subfamilies and 109 BnCYP450s. Members of CYP86 clan genes display specific expression profiles in different tissues and in response to ABA and abiotic stresses. Two BnCYP450s within the CYP704 subfamily from CYP86 clan, BnCYP704B1a and BnCYP704B1b, display high similarity to MS26 (Male Sterility 26, also known as CYP704B1). These two BnCYP704B1 genes were specifically expressed in young buds. We then simultaneously knocked-out these two BnCYP704B1 genes through a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome engineering system. The edited plants displayed a pollenless, sterile phenotype in mature anthers, suggesting that we successfully reproduced genic male sterility (GMS, also known as nuclear male sterility) lines in Brassica napus. This study provides a systemic view of BnCYP450s and offers a strategy to facilitate the commercial utility of the CRISPR/Cas9 system for the rapid generation of GMS in rapeseed via knocking-out GMS controlling genes.

Progress in the microbial production of S-adenosyl-L-methionine.

S-Adenosyl-L-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.

Improving methionine and ATP availability by MET6 and SAM2 co-expression combined with sodium citrate feeding enhanced SAM accumulation in Saccharomyces cerevisiae.

S-adenosyl-L-methionine (SAM), biosynthesized from methionine and ATP, exhibited diverse pharmaceutical applications. To enhance SAM accumulation in S. cerevisiae CGMCC 2842 (wild type), improvement of methionine and ATP availability through MET6 and SAM2 co-expression combined with sodium citrate feeding was investigated here. Feeding 6 g/L methionine at 12 h into medium was found to increase SAM accumulation by 38 % in wild type strain. Based on this result, MET6, encoding methionine synthase, was overexpressed, which caused a 59 % increase of SAM. To redirect intracellular methionine into SAM, MET6 and SAM2 (encoding methionine adenosyltransferase) were co-expressed to obtain the recombinant strain YGSPM in which the SAM accumulation was 2.34-fold of wild type strain. The data obtained showed that co-expression of MET6 and SAM2 improved intracellular methionine availability and redirected the methionine to SAM biosynthesis. To elevate intracellular ATP levels, 6 g/L sodium citrate, used as an auxiliary energy substrate, was fed into the batch fermentation medium, and an additional 19 % increase of SAM was observed after sodium citrate addition. Meanwhile, it was found that addition of sodium citrate improved the isocitrate dehydrogenase activity which was associated with the intracellular ATP levels. The results demonstrated that addition of sodium citrate improved intracellular ATP levels which promoted conversion of methionine into SAM. This study presented a feasible approach with considerable potential for developing highly SAM-productive strains based on improving methionine and ATP availability.

[Relationship between FAS/FASL gene polymorphisms and susceptibility of coal worker's pneumoconiosis].

OBJECTIVE: To investigate the effects of FAS and FASL gene polymorphisms on genetic susceptibility of coal worker's pneumoconiosis and their relationship to the pulmonary fibrosis. METHODS: 340 with coal worker's pneumoconiosis (CWP) and 312 coal mine workers (controls) exposed to the coal dusts were selected. FAS-1377G > A, FAS-670A > G and FASL-844T > C gene polymorphisms were analyzed by PCR-RFLP techniques. RESULTS: The distribution frequencies of genotypes of FAS-1377, FAS-670, FASL-844 genotypes in CWP had no significant differences compared to the control. Compared to CWP patients with exposure year > or = 25, the risk of pneumoconiosis with FAS-1377 GA/AA genotype was significantly higher than those with FAS-1377GG in the patients working age < 25 years (P = 0.098, 95% CI: 0.932 approximately 2.298); the risk of CWP in those with FAS-670AG genotype was higher than those with FAS-670GG genotype (P = 0.098, 95% CI: 0.928 approximately 2.404) the risks of CWP in those with FASL-844TT genotype and FASL-844TC genotype were respectively higher than those with FASL-844CC genotype (P = 0.039, 95% CI: 1.088 approximately 27.358, P = 0.089, 95% CI: 0.852 approximately 2.101). The frequencies of genotypes of FASL-844T > C were significantly different between CWP patients with exposure year > or = 25 and < 25. The risk of CWP with FASL-844TT genotype was significantly higher than that of FASL-844TT + TC (P = 0.054, 95% CI: 0.971 approximately 23.833). The risk of CWP patients with FASL-844TT/CT + FAS-1377GA genotype was 1.810-fold than the patients with FASL-844CC + FAS-1377GG genotype. The risk of CWP patients with FASL-844TT/CT + FAS-670AG genotype was 2.117-fold than the patients with FASL-844CC + FAS-670AA genotype. The risk of CWP patients with FASL-844TT/TC + FAS-1377GA/AA + FAS-670AG/GG genotype was 2.043-fold than the patients with FASL-844CC + FAS-1377GG+FAS-670AA genotype. CONCLUSION: FAS-1377G > A, FAS-670A > G and FASL-844T > C gene polymorphisms may not be associated with the susceptibility of CWP in Han nationality, but these three gene polymorphisms and their joint actions may influence on the progression of CWP.