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.

中文翻译：

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利用氨诱导启动子在地衣芽孢杆菌中过表达酶

氢氧化铵通常用作酶制造过程中的碱性试剂和具有成本效益的氮源。然而，迄今为止，很少有氨诱导酶表达系统被描述。在这项研究中，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，从而鉴定出 1443 个不同表达的基因，其中 859 个基因上调，584 个下调。随后，分析了氨诱导启动子的核苷酸序列，并显示了它们功能介导的 amyL 表达，编码 α-淀粉酶。TRNA_RS39005 (copA), TRNA_RS41250 (sacA), TRNA_RS23130 (pdpX), TRNA_RS42535 (ald), TRNA_RS31535 (plp), 和 TRNA_RS23240 (dfp) 从 859 个上调基因中选出，在氨和葡萄糖存在下，每个基因都显示出比对照更高的转录水平（FPKM 值）。启动子，来自 copA 的 PcopA、来自 sacA 的 PsacA、来自 pdpX 的 PpdpX、来自 ald 的 Pald 和来自 plp 的 Pplp，除了来自 dfp 的 Pdfp，能够介导 amyL 的表达并且被氨显着诱导。最高的酶表达水平是由 Pplp 介导的，在 5 L 发酵罐中被氨诱导后，α-淀粉酶活性增加了 23%。总之，地衣芽孢杆菌具有不依赖葡萄糖的氨诱导型启动子，可用于介导酶的表达，从而提高发酵过程中的酶产量，这些发酵通常用氨来调节 pH 值和供氮。