Zhang Yuyang,Chen Hongping,Zhang Yao,Yin Huifang,Zhou Chenyan,Wang Yan
Background Violaceins have attracted much attention as potential targets used in medicines, food additives, insecticides, cosmetics and textiles, but low productivity was the key factor to limit their large-scale applications. This work put forward a direct RBS engineering strategy to engineer the violacein biosynthetic gene cluster cloned from Chromobacterium violaceum ATCC 12,472 to efficiently improve the fermentation titers.Results Through four-rounds of engineering of the native RBSs within the violaceins biosynthetic operon vioABCDE, this work apparently broke through the rate-limiting steps of intermediates conversion, resulting in 2.41-fold improvement of violaceins production compared to the titers of the starting strain Escherichia coli BL21(DE3) (Vio12472). Furthermore, by optimizing the batch-fermentation parameters including temperature, concentration of IPTG inducer and fermentation time, the maximum yield of violaceins from (BCDE)m (tnaA−) reached 3269.7 µM at 2 mM tryptophan in the medium. Interestingly, rather than previous reported low temperature (20 ℃), we for the first time found the RBS engineered Escherichia coli strain (BCDE)m worked better at higher temperature (30 ℃ and 37 ℃), leading to a higher-level production of violaceins.Conclusion sTo our knowledge, this is the first time that a direct RBS engineering strategy is used for the biosynthesis of natural products, having the potential for a greater improvement of the product yields within tryptophan hyperproducers and simultaneously avoiding the costly low temperature cultivation for large-scale industrial production of violaciens. This direct RBS engineering strategy could also be easily and helpfully used in engineering the native RBSs of other larger and value-added natural product biosynthetic gene clusters by widely used site-specific mutagenesis methods represented by inverse PCR or CRISPR-Cas9 techniques to increase their fermentation titers in the future.
Md Ramim Tanver Rahman,Zaixiang Lou,Fuhao Yu,Peng Wang,Hongxin Wang
Cell-to-cell communication or quorum sensing (QS) leads to biofilm formation and causing other virulence factors which are extreme problems for food safety, biofilm related infectious diseases etc. This study evaluated the anti-QS activity of the Amomum tsaoko extract (0.5–4 mg/ml) by using Chromobacterium violaceum a biosensor strain and biofilm formation by crystal violate assay. Experimental results demonstrated that the overall yield of Amomum tsao-ko extract was 11.33 ± 0.3% (w/w). MIC for Staphylococcus aureus (Gram positive), Salmonella Typhimurium and Pseudomonas aeruginosa (Gram negative) was 1, 2 and 2 mg/ml, respectively. A concentration of 4 mg/ml extract showed highest biofilm inhibition 51.96% on S. Typhimurium when 47.06%, 45.28% were shown by S. aureus , P. aeruginosa respectively . The damage of biofilm architecture was observed by Confocal Laser Scanning Microscopy (CLSM). A level of 44.59% inhibition of violacein production was demonstrated when the dose was 4 mg/ml. Swarming motility inhibition was observed in a dose dependent manner. Taken together, the treatment of A. tsaoko extract can deliver value to food product and medicine by controlling pathogenesis.
Cheng Qu,Yuchuang Geng,Zhongxiang Ding,Yuzhu Li,Hao Jiang,Mengke Su,Honglin Liu
A feedback inhibition effect of high autoinducer levels on metabolite secretion in Chromobacterium subtsugae (C. subtsugae) was evidenced by in situ spatiotemporal surface-enhanced Raman spectroscopy (SERS) profiling. The hierarchical hydrophobic plasmonic array in agar medium is structured by oil/water/oil (OL/W/OH) triphasic interfacial self-assembly. The hydrophobic layer acts as a “door curtain” to selectively permit adsorption of a quorum sensing (QS)-regulated fat-soluble metabolite, i.e., violacein (Vio), and significantly blocks nonspecific adsorption of water-soluble proteins, etc. The SERS profiling clearly evidences that the diffusion of N-hexanoyl-l-homoserine lactone (C6-HSL) in agar medium quickly triggers the initial synthesis of Vio in C. subtsugae CV026 but surprisingly inhibits the intrinsic synthesis of Vio in C. subtsugae ATCC31532. The latter negative response might be related to the VioS repressor of ATCC31532, which negatively controls violacein production without influencing the expression of the CviI/R QS system. Moreover, two sender–receiver systems are constructed by separately coculturing CV026 or ATCC31532 with Hafnia alvei H4 that secretes large amounts of C6-HSL. Expectedly, the cocultivation similarly triggers the initial synthesis of Vio in CV026 but seems to have a quite weak negative effect on the intrinsic synthesis in ATCC31532. In fact, the negative regulation in ATCC31532 might be affected by a diffusion-dependent concentration effect. The H4 growth and its secretion of C6-HSL are a slow and continuous process, thereby avoiding the gathering of local high concentrations. Overall, our study put forward an in situ SERS strategy as an alternative to traditional bioluminescent tools for highly sensitively analyzing the spatiotemporal communication and cooperation in live microbial colonies.
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