Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.

中文翻译：

---

调控区域的突变导致苏云金芽孢杆菌中链霉素抗性和角蛋白酶合成增加。

角蛋白酶是一类具有重要工业意义的蛋白酶。为了充分利用芽孢杆菌作为一种固有的优质蛋白酶微生物生产者，我们进行了核糖体工程，通过诱导链霉素抗性来提高野生型苏云金芽孢杆菌的角蛋白酶合成能力。突变体 Bt(Str-O) 被确定为稳定的角蛋白酶过度生产者。两种菌株的比较特征表明，尽管在 MIC 中对链霉素的耐药性增加了八倍，但突变体对其他常用抗生素的耐药性并未受到影响。此外，当在液体 LB 培养基中培养时，突变体表现出增强的角蛋白酶合成（1.5 倍）。在整个羽毛降解实验中，突变体可以将双重角蛋白酶分泌到培养基中，每 107 CFU 达到 640 U/mL。相比之下，在扫描电子显微镜分析和孢子形成实验中没有发现显着差异。为了了解导致这些表型变化的遗传因素，我们克隆并分析了 rpsL 基因。没有观察到突变。我们随后确定了这两种菌株的基因组序列。比较 rpsL 基因表明，链霉素耐药性的出现不一定取决于公认的“热点”中的突变。全基因组分析表明，突变体的表型变化是调控区域和非编码 RNA 基因发生遗传变异的共同结果。