Enhanced cobalamin biosynthesis in Ensifer adhaerens by regulation of key genes with gradient promoters

Cobalamin is an essential human vitamin widely used in the pharmaceutical,food,and feed additive industries and currently produced by bacteria or archaea.Ensifer adhaerens HY-1 is an industrial strain that also produces cobalamin.However production outputs are poor and the specific synthesis pathways require characterization.In this study,the whole genome sequence of E.adhaerens HY-1 was generated and annotated,and genes associated with cobalamin biosynthesis were identified.Then,three genes,CobSV,CobQ,and CobW were identified as the most efficient ones for enhancing cobalamin synthesis.By transcriptome sequencing of E.adhaerens HY-1 cells at different growth stages,65 endogenous promoters with different gradient strengths were identified.After combined expression of different strength promoters and key genes,a high cobalamin-producing recombinant strain,‘hmm’(genotype:PmetH-CobSV-PibpA-CobQ-Pmdh-CobW),was generated.Cobalamin production was 143.8 mg/L in shaking flasks,which was 41.0%higher than the original strain.Cobalamin production was further enhanced to 171.2 mg/L using fed-batch fermentation.Importantly,our data and novel approach provide important references for the analysis of cobalamin synthesis and other metabolites in complex metabolic pathways.

枸溶性钾肥与解钾菌液配施对烤烟生长及钾素营养状况的影响

为明确枸溶性钾肥对烤烟生长和钾素吸收的作用效果,采用盆栽试验研究了枸溶性钾肥与解钾菌液(Ensifer和Bacillus cereus strain)配施对烤烟生长及钾素吸收积累的影响。结果表明,枸溶性钾肥中配施解钾菌可促进烤烟生长发育,有利于烟株的早生快发;与单施硫酸钾肥相比,枸溶性钾肥与菌液配施的烟株根系鲜质量最高增加15.01%,烤烟烟叶叶绿素含量增加16.27%,烤烟根系活力提高17.07%;枸溶性钾肥与解钾菌液配施能够促进烟株对钾素的吸收,使钾素在烟株体内的分配更加合理;同时枸溶性钾肥与解钾菌液配施还可改善烤烟农艺性状,以枸溶性钾肥与Ensifer菌液配合施用的效果最佳。

一株甲拌磷降解菌的诱变选育与降解特性研究

从甲拌磷污染的土壤中驯化分离得到1株能够以甲拌磷为唯一碳源生长的革兰氏阴性细菌JZ1-黏着剑菌(Ensifer adhaerenssp.)。最初,该菌株在24 h内对200mg/L甲拌磷的降解率为42.2%。当驯化质量浓度为800 mg/L时,JZ1对200 mg/L甲拌磷的降解率达到56.3%。以JZ1为出发菌经化学诱变和紫外诱变后获得菌株JZ1-Ⅱ。JZ1-Ⅱ对甲拌磷的降解作用明显增加:当盐酸羟胺质量分数为2%时,降解率提高至67.8%;进一步经紫外照射45 s,降解率提高至83.2%。气相色谱法测定甲拌磷的降解动态,在JZ1-Ⅱ的作用下,甲拌磷在12～24 h内下降迅速,24 h后降解率基本稳定在83%。采用氯化亚锡法测定培养基中总磷和无机磷的含量,分析甲拌磷的降解途径,甲拌磷的降解过程应为:甲拌磷(O,O-二乙基-S-乙硫基甲基二硫代磷酸酯)首先降解为二乙基磷酸,继而转变为磷酸。

一株草甘膦高效降解菌的筛选和表征研究

为研究利用细菌微生物降解草甘膦农药污染,从沈阳某地区农田土壤中分离得到一株草甘膦高效降解菌Ensifer sp.BRY。基于16S rDNA检测,BRY被鉴定为剑菌属(Ensifer sp.)。BRY能在以草甘膦(最高浓度400 mg·L^(-1))为唯一碳源的无机盐培养基中生长,在50 h对300 mg·L^(-1)草甘膦的降解率可达到69.60%。在30℃、pH 6.0、10%初始接种量时,菌株BRY在50 h内的草甘膦(100 mg·L^(-1))降解率达到91.93%,当相同条件下调节初始接种量为20%时,菌株BRY的草甘膦降解率升高。当培养体系加入其他碳源(葡萄糖、蔗糖)时,草甘膦降解率降低。菌株BRY对不同浓度草甘膦的降解过程符合Haldane方程,其最大比生长速率μmax为1.68 h^(-1),半饱和常数Ks为167.80 mg·L^(-1),抑制常数Ksi为50.55 mg·L^(-1),Ksi/Ks为0.30。研究表明,菌株BRY对草甘膦具有较高的耐受能力和降解能力,通过优化培养条件可以提高降解效率,在用于草甘膦污染环境的生物修复过程中,菌株BRY具有独特潜力。