The growth and EPA synthesis ofShewanella oneidensis MR-1 and expectation of EPA biosynthetic pathway

Shewanella oneidensis MR-1 has the ability to inhale certain metals and chemical compounds and exhale these materials in an altered state; as a result, this microorganism has been widely applied in bioremediation protocols. However, the relevant characteristics of cell growth and biosynthesis of PuFAs have yet to be thoroughly investigated. Therefore, in this study, we have attempted to characterize the growth and fatty acid profiles ofS. oneidensis MR-1 under a variety of temperature conditions. The fastest growth ofS. oneidensis MR-1 was observed at 30°C, with a specific growth rate and doubling time of 0.6885 h⁻¹ and 1.007 h. The maximum cell mass of this microorganism was elicited at a temperature of 4°C. The eicosapentaenoic acid (EPA) synthesis ofS. oneidensis MR-1 was evaluated under these different culture temperatures.S. oneidensis MR-1 was found not to synthesize EPA at temperatures in excess of 30°C, but was shown to synthesize EPA at temperatures below 30°C. The EPA content was found to increase with decreases in temperature. We then evaluated the EPA biosynthetic pathway, using a phylogenetic tree predicted on 16s rRNA sequences, and the homology of ORFs betweenS. oneidensis MR-1 andShewanella putrefaciens SCRC-2738, which is known to harbor a polyketide synthase (PKS)-like module. The phylogenetic tree revealed that MR-1 was very closely related to bothMoritella sp., which is known to synthesize DHA via a PKS-like pathway, andS. putrefaciens, which has been reported to synthesize EPA via an identical pathway. The homology between the PKS-like module ofS. putrefaciens SCRC-2738 and the entire genome ofS. oneidensis MR-1 was also analyzed, in order to mine the genes associated with the PKS-like pathway inS. oneidensis MR-1. A putative PKS-like module for EPA biosynthesis was verified by this analysis, and was also corroborated by the experimental finding thatS. oneidensis MR-1 was able to synthesize EPA without the expression of dihomo-γ-linoleic acid (DGLA) and arachidonic acid (AA) formed during EPA synthesis via the FAS pathway.     

Uridine phosphorylase from Shewanella oneidensis MR-1: Heterological expression, regulation, transcription, and properties

A DNA fragment containing a promoter-operator and structural parts of the uridine phosphorylase gene from Shewanella oneidensis MR-1 was cloned. Cross-heterological expression of the udp genes from Sh. oneidensis MR-1 and Escherichia coli under the control of authentic regulatory regions is shown. The UDP protein accumulates in an active form in the cytoplasmic fraction of cells. The recombinant UDP protein from Sh. oneidensis MR-1 obtained by heterological expression was isolated and characterized. E. coli udp gene promoter activity was observed during heterological expression in Sh. oneidensis MR-1 cells under both aerobic and anaerobic conditions.     

Developing a base-editing system to expand the carbon source utilization spectra of Shewanella oneidensis MR-1 for enhanced pollutant degradation

Shewanella oneidensis MR-1, a model strain of exoelectrogenic bacteria (EEB), plays a key role in environmental bioremediation and bioelectrochemical systems because of its unique respiration capacity. However, only a narrow range of substrates can be utilized by S. oneidensis MR-1 as carbon sources, resulting in its limited applications. In this study, a rapid, highly efficient, and easily manipulated base-editing system pCBEso was developed by fusing a Cas9 nickase (Cas9n (D10A)) with the cytidine deaminase rAPOBEC1 in S. oneidensis MR-1. The C-to-T conversion of suitable C within the base-editing window could be readily and efficiently achieved by the pCBEso system without requiring double-strand break or repair templates. Moreover, double-locus simultaneous editing was successfully accomplished with an efficiency of 87.5%. With this tool, the key genes involving in N-acetylglucosamine (GlcNAc) or glucose metabolism in S. oneidensis MR-1 were identified. Furthermore, an engineered strain with expanded carbon source utilization spectra was constructed and exhibited a higher degradation rate for multiple organic pollutants (i.e., azo dyes and organoarsenic compounds) than the wild-type when glucose or GlcNAc was used as the sole carbon source. Such a base-editing system could be readily applied to other EEB. This study not only enhances the substrate utilization and pollutant degradation capacities of S. oneidensis MR-1 but also accelerates the robust construction of engineered strains for environmental bioremediation.     

Unexpected role of electron-transfer hub in direct degradation of pollutants by exoelectrogenic bacteria

Exoelectrogenic bacteria (EEB) are capable of anaerobic respiration with diverse extracellular electron acceptors including insoluble minerals, electrodes and flavins, but the detailed electron transfer pathways and reaction mechanisms remain elusive. Here, we discover that CymA, which is usually considered to solely serve as an inner-membrane electron transfer hub in Shewanella oneidensis MR-1 (a model EEB), might also function as a reductase for direct reducing diverse nitroaromatic compounds (e.g. 2,4-dichloronitrobenzene) and azo dyes. Such a process can be accelerated by dosing anthraquinone-2,6-disulfonate. The CymA-based reduction pathways in S. oneidensis MR-1 for different contaminants could be functionally reconstructed and strengthened in Escherichia coli. The direct reduction of lowly polar contaminants by quinol oxidases like CymA homologues might be universal in diverse microbes. This work offers new insights into the pollutant reduction mechanisms of EEB and unveils a new function of CymA to act as a terminal reductase.     

Current Production and Metal Oxide Reduction by Shewanella oneidensis MR-1 Wild Type and Mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

Intensification of bioelectricity generation in microbial fuel cells using Shewanella oneidensis MR-1 mutants with increased reducing activity

Electrogenicity of Shewanella oneidensis MR-1 mutants FRS1 and FRB1 with reducing activity 30–40% higher than in the original strain was studied in various microbial fuel cells (MFC) developed in the course of the work. The voltage and current density developed by the mutants were 1.7 times higher than in the case of S. oneidensis MR-1. A correlation was found between reducing activity of the cells and the voltage and current density developed in MFC. The possibility for enhanced bioelectricity production in MFC by genetic modification of S. oneidensis MR-1 was demonstrated.     

Expression of a Tetraheme Protein, Desulfovibrio vulgaris Miyazaki F Cytochrome c3, in Shewanella oneidensis MR-1

Cytochrome c₃ from Desulfovibrio vulgaris Miyazaki F was successfully expressed in the facultative aerobe Shewanella oneidensis MR-1 under anaerobic, microaerophilic, and aerobic conditions, with yields of 0.3 to 0.5 mg of cytochrome/g of cells. A derivative of the broad-host-range plasmid pRK415 containing the cytochrome c₃ gene from D. vulgaris Miyazaki F was used for transformation of S. oneidensis MR-1, resulting in the production of protein product that was indistinguishable from that produced by D. vulgaris Miyazaki F, except for the presence of one extra alanine residue at the N terminus.