Shen Peili, Niu Dandan, Permaul Kugen, Tian Kangming, Singh Suren, Wang Zhengxiang
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
Department of Biological Chemical Engineering, College of Chemical Engineering and Materials Science, Tianjin University of Science and Technology, Tianjin 300457, China.
J Ind Microbiol Biotechnol. 2021 Jul 1;48(5-6). doi: 10.1093/jimb/kuab037.
Ammonium hydroxide is conventionally used as an alkaline reagent and cost-effective nitrogen source in enzyme manufacturing processes. However, few ammonia-inducible enzyme expression systems have been described thus far. In this study, genomic-wide transcriptional changes in Bacillus licheniformis CBBD302 cultivated in media supplemented with ammonia were analyzed, resulting in identification of 1443 differently expressed genes, of which 859 genes were upregulated and 584 downregulated. Subsequently, the nucleotide sequences of ammonia-inducible promoters were analyzed and their functionally-mediated expression of amyL, encoding an α-amylase, was shown. TRNA_RS39005 (copA), TRNA_RS41250 (sacA), TRNA_RS23130 (pdpX), TRNA_RS42535 (ald), TRNA_RS31535 (plp), and TRNA_RS23240 (dfp) were selected out of the 859 upregulated genes and each showed higher transcription levels (FPKM values) in the presence of ammonia and glucose than that of the control. The promoters, PcopA from copA, PsacA from sacA, PpdpX from pdpX, Pald from ald, and Pplp from plp, except Pdfp from dfp, were able to mediate amyL expression and were significantly induced by ammonia. The highest enzyme expression level was mediated by Pplp and represented 23% more α-amylase activity after induction by ammonia in a 5-L fermenter. In conclusion, B. licheniformis possesses glucose-independent ammonia-inducible promoters, which can be used to mediate enzyme expression and therefore enhance the enzyme yield in fermentations conventionally fed with ammonia for pH adjustment and nitrogen supply.
氢氧化铵在酶制造过程中通常用作碱性试剂和具有成本效益的氮源。然而，迄今为止，很少有氨诱导型酶表达系统被描述。在本研究中，分析了在添加氨的培养基中培养的地衣芽孢杆菌CBBD302的全基因组转录变化，鉴定出1443个差异表达基因，其中859个基因上调，584个基因下调。随后，分析了氨诱导型启动子的核苷酸序列，并展示了它们对编码α-淀粉酶的amyL的功能介导表达。从859个上调基因中选出了TRNA_RS39005（copA）、TRNA_RS41250（sacA）、TRNA_RS23130（pdpX）、TRNA_RS42535（ald）、TRNA_RS31535（plp）和TRNA_RS23240（dfp），在氨和葡萄糖存在的情况下，它们各自的转录水平（FPKM值）均高于对照。除dfp的Pdfp外，copA的PcopA、sacA的PsacA、pdpX的PpdpX、ald的Pald和plp的Pplp启动子能够介导amyL表达，并被氨显著诱导。最高的酶表达水平由Pplp介导，在5升发酵罐中经氨诱导后，α-淀粉酶活性提高了23%。总之，地衣芽孢杆菌拥有不依赖葡萄糖的氨诱导型启动子，可用于介导酶表达，从而提高在传统上添加氨用于调节pH值和提供氮源的发酵过程中的酶产量。
