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Modular Assembly of Ordered Hydrophilic Proteins Improve Salinity Tolerance in Escherichia coli.

ABSTRACT: Most late embryogenesis abundant group 3 (G3LEA) proteins are highly hydrophilic and disordered, which can be transformed into ordered α-helices to play an important role in responding to diverse stresses in numerous organisms. Unlike most G3LEA proteins, DosH derived from Dinococcus radiodurans is a naturally ordered G3LEA protein, and previous studies have found that the N-terminal domain (position 1-103) of DosH protein is the key region for its folding into an ordered secondary structure. Synthetic biology provides the possibility for artificial assembling ordered G3LEA proteins or their analogues. In this report, we used the N-terminal domain of DosH protein as module A (named DS) and the hydrophilic domains (DrHD, BnHD, CeHD, and YlHD) of G3LEA protein from different sources as module B, and artificially assembled four non-natural hydrophilic proteins, named DS + DrHD, DS + BnHD, DS + CeHD, and DS + YlHD, respectively. Circular dichroism showed that the four hydrophile proteins were highly ordered proteins, in which the α-helix contents were DS + DrHD (56.1%), DS + BnHD (53.7%), DS + CeHD (49.1%), and DS + YLHD (64.6%), respectively. Phenotypic analysis showed that the survival rate of recombinant Escherichia coli containing ordered hydrophilic protein was more than 10% after 4 h treatment with 1.5 M NaCl, which was much higher than that of the control group. Meanwhile, in vivo enzyme activity results showed that they had higher activities of superoxide dismutase, catalase, lactate dehydrogenase and less malondialdehyde production. Based on these results, the N-terminal domain of DosH protein can be applied in synthetic biology due to the fact that it can change the order of hydrophilic domains, thus increasing stress resistance.

SUBMITTER: Guo L

PROVIDER: S-EPMC8123400 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results:By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF ? factor/anti-? switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions:The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Project description:This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic Escherichia coli. In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in csfB abolished CsfA major subunit accumulation in the periplasm, which could be restored by trans-complementation with a complete copy of the csfB gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the csfE gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of csfE expression in trans did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the csfF gene also resulted in an elongated pilus morphology identical to that of the csfE deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a csfF deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the csfB and csfF genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was trans-complemented with a wild-type copy of csfB or csfF, respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations.

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Modular Assembly of Ordered Hydrophilic Proteins Improve Salinity Tolerance in Escherichia coli.

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