Zymomonas mobilis is a model bacterial ethanologen and has been engineered to produce lignocellulosic biofuels and biochemicals such as 2,3‐butanediol. We have previously identified promoters of different strengths using systems biology datasets and characterized them using the flow cytometry–based dual reporter–gene system. Here, we further demonstrated the capability of applying the dual reporter–gene system and omics datasets on discovering inducible promoters. Ten candidate ethanol‐inducible promoters were identified through omics datasets mining and clustering. Using the dual reporter–gene system, these promoters were characterized under natural growth, ethanol stress, and ethanol‐induced condition to investigate the transcriptional strength and ethanol inducibility. The results demonstrated that three promoters of P0405, P0435, and P0038 driving the expression of native genes of ZMO0405, ZMO0435, and ZMO0038, correspondingly, are potential ethanol‐responsive promoters and may be growth related. This study not only identified and verified three ethanol‐inducible promoters as biological parts, which can be used to synchronize the expression of heterologous pathway genes with the ethanol production process of Z. mobilis, but also demonstrated the power of combining omics datasets and dual reporter–gene system to identify biological parts for metabolic engineering and synthetic biology applications in Z. mobilis and related microorganisms.
A promoter
plays a crucial role in controlling the expression of
the target gene in cells, thus being one of the key biological parts
for synthetic biology practices. Although significant efforts have
been made to identify and characterize promoters with different strengths
in various microorganisms, the compatibility of promoters within different
hosts still lacks investigation. In this study, we chose the native
Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting
promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial
cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and
sorted in E. coli, with candidate promoters
exhibiting high fluorescence intensity collected. A total of 53 variants
were finally selected and sequenced by Sanger sequencing. The sequencing
results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter
strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify
key mutations within the Pgap promoter region that
affect the promoter compatibility. Finally, six important sites were
identified and confirmed to help increase Pgap strength
in E. coli while keeping similar strength
of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining
these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than
native Pgap in E. coli. This study thus not only determined six important sites affecting
promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate
and determine nucleotide sequences in promoter regions affecting promoter
compatibility, which can be applied in other microorganisms to help
reveal universal factors affecting promoter compatibility and design
promoters with desired strengths among different microbial cell factories.
SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly–Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly–Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2–P1↓P1’ (Gly–Gly↓Ala) position using the YESS–PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly–Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1–P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1’ position Ala of Smt3 was found to play a vital role in maintaining Ulp1’s precise cleavage after the Gly–Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.