Reducing Cellular Autolysis of Bacillus subtilis to Improve Keratinase Production.

Bacillus subtilis has been shown to be an excellent expression host for keratinases due to its powerful secretion system. However, cellular autolysis limits its production capacity. Here, we selected seven genes with significantly upregulated transcript levels from 15 genes associated with cellular autolysis as knockout targets by qRT-PCR and constructed a total of 127 strains to reduce cellular autolysis. Among them, the biomass of B. subtilis BSΔXLPC-ker deficient in xpf, lytC, pcfA, and cwlC increased by 57%. This was confirmed by cell staining, green fluorescent protein imaging, and extracellular nucleic acid leakage assay. Keratinase activity was increased by 1.46-fold in the 5 L fermenter. In addition, the activities of nattokinase and subtilisin E were also increased by 1.50-fold and 1.43-fold, respectively, in the modified chassis cells, which further confirms the generalizability of the strategy. Thus, reducing cellular autolysis to increase the ability of B. subtilis to produce subtilisins is promising.

Fusion of Substrate-Binding Domains Enhances the Catalytic Capacity of Keratinases and Promotes Enzymatic Conversion of Feather Waste.

The unique role of keratinases in keratin hydrolysis has garnered huge interest in the recovery of feather waste. However, owing to the high hydrophobicity of feather keratins, the catalytic capacity of keratinases for hydrolyzing feathers is typically low. In this study, we aimed to improve the keratinase feather hydrolysis efficiency by fusing a substrate-binding domain into the enzyme. We screened several carbohydrate-binding modules (CBMs) and linking peptides. We selected the most promising candidates to construct, clone, and express a fusion keratinase enzyme KerZ1/CBM-L8 with a feather hydrolysis efficiency of 7.8 × 10-8 g/U. Compared with those of KerZ1, KerZ1/CBM-L8 has a feather hydrolysis efficiency that is 2.71 times higher, a kcat value that is 179% higher, which translates to higher catalytic efficiency, and Km and binding constant (K) values that are lower, which indicate a higher KerZ1/CBM-L8-keratin binding affinity. Moreover, the number of binding sites to the substrate (N), determined using isothermal titration calorimetry, was 24.1 times higher than that of KerZ1. Thus, the fusion of the substrate-binding domain improved the binding ability of the keratinase enzyme to the hydrophobic substrate, which improved its feather hydrolysis efficiency. Therefore, using the fusion keratinase would significantly improve the recovery of feather waste.

Engineering flexible loops to enhance thermal stability of keratinase for efficient keratin degradation.

Keratinase-catalyzed degradation of keratin waste has been shown to be a promising recycling method. Although the recombinant KerZ1 derived from Bacillus subtilis has shown the highest activity among the keratinases reported so far, the low thermal stability caused by the unstable flexible loops limited its keratin-degrading ability. To this end, the flexible loops of KerZ1 were engineered to be more hydrophobic and rigid through B-factor calculations, molecular dynamics simulations, and ß-turn redesign. We developed several highly thermostable keratinase variants and showed enhanced keratin degradation activity. In particular, the loop regions of the variants KerZ1A128D/L240N, KerZ1T77E/L240N and KerZ1T77C/A128D were designed to be more stable, with Tm values increased by 8 °C, 6 °C and 5 °C, and corresponding t1/2 increased by 2.3, 3.3 and 5.0 times. The keratin degradation activity of the variant KerZ1T77C/A128D at 60 °C was enhanced by 46 % compared with KerZ1WT. The strategy of this research and the obtained keratinase variants will be a significant improvement in the complete degradation of keratin.

Improved acid-stress tolerance of Lactococcus lactis NZ9000 and Escherichia coli BL21 by overexpression of the anti-acid component recT.

Acid accumulation caused by carbon metabolism severely affects the fermentation performance of microbial cells. Here, different sources of the recT gene involved in homologous recombination were functionally overexpressed in Lactococcus lactis NZ9000 and Escherichia coli BL21, and their acid-stress tolerances were investigated. Our results showed that L. lactis NZ9000 (ERecT and LRecT) strains showed 1.4- and 10.4-fold higher survival rates against lactic acid (pH 4.0), respectively, and that E. coli BL21 (ERecT) showed 16.7- and 9.4-fold higher survival rates than the control strain against lactic acid (pH 3.8) for 40 and 60 min, respectively. Additionally, we found that recT overexpression in L. lactis NZ9000 improved their growth under acid-stress conditions, as well as increased salt- and ethanol-stress tolerance and intracellular ATP concentrations in L. lactis NZ9000. These findings demonstrated the efficacy of recT overexpression for enhancing acid-stress tolerance and provided a promising strategy for insertion of anti-acid components in different hosts.

Evolutionary engineering of industrial microorganisms-strategies and applications.

Microbial cells have been widely used in the industry to obtain various biochemical products, and evolutionary engineering is a common method in biological research to improve their traits, such as high environmental tolerance and improvement of product yield. To obtain better integrate functions of microbial cells, evolutionary engineering combined with other biotechnologies have attracted more attention in recent years. Classical laboratory evolution has been proven effective to letting more beneficial mutations occur in different genes but also has some inherent limitations such as a long evolutionary period and uncontrolled mutation frequencies. However, recent studies showed that some new strategies may gradually overcome these limitations. In this review, we summarize the evolutionary strategies commonly used in industrial microorganisms and discuss the combination of evolutionary engineering with other biotechnologies such as systems biology and inverse metabolic engineering. Finally, we prospect the importance and application prospect of evolutionary engineering as a powerful tool especially in optimization of industrial microbial cell factories.

[Effect of xiaoke granule on blood glucose, urinary protein and glomerular morphology in rats with diabetic nephropathy].

OBJECTIVE: To observe the changes of blood glucose, urinary protein and renal glomerular morphology in rats with diabetic nephropathy, and the effect of xiaoke granule (XKG) on them. METHODS: The diabetic nephropathy model was established by 3/4 nephrectomy and once intraperitoneal injection of streptozotocin (STZ). The experimental rats were divided into the model group, the XKG group, the positive control group and the sham operation group. Blood was taken from rat's caudal vein to test the fasting blood glucose (FBG) once every week after STZ injection and at the same time, urinary protein in 24 hrs (UP/24h) was investigated. All the rats were sacrificed 2, 6 weeks after STZ injection and morphological examination on their kidney was performed. RESULTS: Six weeks after STZ injection, glomerular sclerosis in various degrees was seen in the model group, but the pathological change was significantly milder in the treated groups. FBG in the model group was higher than that in the sham operation group at all time points respectively (P<0.05), while in the XKG group and the positive control group, the change was improved significantly. UP/24h in the model group was higher than that in the sham operation group at all time points respectively, and that in the XKG group at 1, 2 and 3 weeks after STZ injection was significantly lower than that in the model group. FBG and UP/24h showed a positive correlation. CONCLUSION: A rat model of diabetic nephropathy was duplicated successfully. The elevated blood glucose level in diabetic nephropathy model could induce proteinuria. One of the routes of treatment of diabetic nephropathy by XKG is to reduce the blood glucose, eliminate the proteinuria, and thus to improve the pathological change in renal glomeruli.