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Poly-γ-glutamic acid (γ-PGA) is an anionic polymer with wide-ranging applications in the areas of medicine, light chemical industry, wastewater treatment, and agriculture. However, the production cost of γ-PGA is high for the requirement of adding the expensive precursor L-glutamic acid during fermentation, which hinders its widespread application. In this study, in order to improve γ-PGA yield, central carbon metabolism was engineered to enhance the carbon flux of tricarboxylic acid (TCA) cycle and glutamic acid synthesis in a γ-PGA production strain Bacillus licheniformis WX-02. Firstly, pyruvate dehydrogenase (PdhABCD) and citrate synthase (CitA) were overexpressed to strengthen the flux of pyruvate into TCA cycle, resulting in 34.93% and 11.14% increase of γ-PGA yield in B. licheniformis WX-02, respectively. Secondly, the carbon flux to glyoxylate shunt was rewired via varying the expression of isocitrate lyase (AceA), and a 23.24% increase of γ-PGA yield was obtained in AceA down-regulated strain WXPbacAaceBA. Thirdly, deletion of pyruvate formate-lyase gene pflB led to a 30.70% increase of γ-PGA yield. Finally, combinatorial metabolic engineering was applied, and γ-PGA titer was enhanced to 12.02 g/L via overexpressing pdhABCD and citA, repressing aceA, and deleting pflB, with a 69.30% improvement compared to WX-02. Collectively, metabolic engineering of central carbon metabolism is an effective strategy for enhanced γ-PGA production in B. licheniformis, and this research provided a promising strain for industrial production of γ-PGA.

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91 g L−1, 85.22% higher than that of the wild type (7.51 g L−1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24 μmol gDCW−1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.

Abstract Biotransformation of wasted feathers via feather-degrading enzyme has gained immense popularity, low conversion efficiency hinders its scale application, and the main purpose of this study is to improve feather-degrading enzyme production in Bacillus licheniformis. Firstly, keratinase from Bacillus amyloliquefaciens K11 was attained with the best performance for feather hydrolysis, via screening several extracellular proteases from Bacillus; also, feather powder was proven as the most suitable substrate for determination of feather-degrading enzyme activity. Then, expression elements, including signal peptides and promoters, were optimized, and the combination of signal peptide SPSacC with promoter Pdual3 owned the best performance, keratinase activity aggrandized by 6.21-fold. According to amino acid compositions of keratinase and feeding assays, Ala, Val, and Ser were proven as critical precursors, and strengthening these precursors’ supplies via metabolic pathway optimization resulted in a 33.59% increase in the keratinase activity. Furthermore, keratinase activity reached 2210.66 U/mL, up to 56.74-fold from the original activity under the optimized fermentation condition in 3-L fermentor. Finally, the biotransformation process of discarded feathers by the fermented keratinase was optimized, and our results indicated that 90.94% of discarded feathers (16%, w/v) were decomposed in 12 h. Our results suggested that strengthening precursor amino acids’ supplies was an efficient strategy for enhanced production of keratinase, and this research provided an efficient strain as well as the biotransformation process for discarded feather re-utilization.

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Proline metabolism influences the formation of reactive oxygen species (ROS), and is involved in a wide range of cellular processes. However, the relation between proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results indicated that the deletion of Δ1-pyrroline-5-carboxylate dehydrogenase encoded gene ycgN resulted in 85.22% higher yield of γ-PGA in B. licheniformis WX-02. But the deletion of proline dehydrogenase encoded gene ycgM had no effect on γ-PGA synthesis. Meanwhile, a 2.92-fold higher level of P5C was detected in ycgN deficient strain WXΔycgN, while the P5C levels in WXΔycgM and double mutant strain WXΔycgMN remained the same, compared to WX-02. The ROS level of WXΔycgN was 1.18-fold higher than that of WX-02, and the addition of n-acetylcysteine (antioxidant) into medium could decrease its ROS level, further reduced the γ-PGA yield. Our results showed that proline catabolism played an important role in maintaining ROS homeostasis, and the deletion of ycgN caused P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis. γ-PGA is an anionic polyamide with various applications in biomedical and industrial fields. Proline metabolism influences the intracellular reactive oxygen species (ROS) and is involved in a wide range of cellular processes. Here, we report the effects of proline metabolism on γ-PGA synthesis. Our results indicated that deletion of ycgN promoted the synthesis of γ-PGA by increasing the intracellular levels of Δ1-pyrroline-5-carboxylate to generate a transient ROS signal in B. licheniformis WX-02. This study provides the valuable information that enhanced synthesis of γ-PGA by knocking out of ycgN.

Poly gamma glutamic acid ( -PGA) is an anionic polyamide with numerous applications. Proline metabolism influences the formation of reactive oxygen species (ROS), and is involved in a wide range of cellular processes. However, the relation between proline metabolism and -PGA synthesis has not yet been analyzed. In this study, our results indicated that the deletion of 1-pyrroline-5-carboxylate dehydrogenase encoded gene ycgN resulted in 85.22% higher yield of -PGA in B. licheniformis WX-02. But the deletion of proline dehydrogenase encoded gene ycgM had no effect on -PGA synthesis. Meanwhile, a 2.92-fold higher level of P5C was detected in ycgN deficient strain WX ycgN, while the P5C levels in WX ycgM and double mutant strain WX ycgMN remained the same, compared to WX-02. The ROS level of WX ycgN was 1.18-fold higher than that of WX-02, and the addition of n-acetylcysteine (antioxidant) into medium could decrease its ROS level, further reduced the -PGA yield. Our results showed that proline catabolism played an important role in maintaining ROS homeostasis, and the deletion of ycgN caused P5C accumulation, which induced a transient ROS signal to promote -PGA synthesis in B. licheniformis.

Application of long non-coding RNAs (lncRNAs) for modulation of breast cancer (BC) has attracted much attention. Here, we probed into the role and underlying mechanism of long intergenic non-coding RNA 01270 (LINC01270) in BC. With the help of bioinformatics tools, we identified laminin subunit alpha 2 ( LAMA2 ) as a BC-related differentially expressed gene to discern the effect of LAMA2 in BC cells. LAMA2 was initially poorly expressed while LINC01270 was highly expressed in BC. BC cells were subsequently treated with sh-LINC01270 or/and sh-LAMA2 for exploration of their regulatory mechanism in BC, which unfolded that LINC01270 inhibition up-regulated LAMA2 and inactivated the MAPK signaling pathway to suppress malignant characteristics of BC cells. Functional assays demonstrated that LINC01270 bound to DNMT1 , DNMT3a , and DNMT3b promoted the methylation of CpG islands in LAMA2 promoter and inhibited the LAMA2 expression. Moreover, our data suggested that LAMA2 suppressed MAPK signaling pathway to inhibit BC cell malignant characteristics. The in vitro results were re-produced with the help of the in vivo experimentations. In conclusion, LINC01270 silencing inhibited the methylation of LAMA2 promoter to suppress the activation of MAPK signaling pathway, which subsequently restrained the BC progression. Graphical abstract 1, Overexpression of LAMA2 inhibits malignant features of BC cells. 2, LINC01270 promotes LAMA2 promoter methylation by recruiting DNMTs to the LAMA2 promoter region. 3, 5-aza-dc reverses the promotion of LAMA2 promoter methylation by LINC01270. 4, LAMA2 inhibits malignant features of BC cells by suppressing the activation of MAPK signaling pathway.