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Bioelectrochemical systems (BESs) based on Shewanella oneidensis MR-1 offer great promise for sustainable energy/chemical production, but the low rate of electron generation remains a crucial bottleneck preventing their industrial application. Here, we reconstructed a genome-scale metabolic model of MR-1 to provide a strong theoretical basis for novel BES applications. The model iLJ1162, comprising 1,162 genes, 1,818 metabolites and 2,084 reactions, accurately predicted cellular growth using a variety of substrates with 86.9% agreement with experimental results, which is significantly higher than the previously published models iMR1_799 and iSO783. The simulation of microbial fuel cells indicated that expanding the substrate spectrum of MR-1 to highly reduced feedstocks, such as glucose and glycerol, would be beneficial for electron generation. In addition, 31 metabolic engineering targets were predicted to improve electricity production, three of which have been experimentally demonstrated, while the remainder are potential targets for modification. Two potential electron transfer pathways were identified, which could be new engineering targets for increasing the electricity production capacity of MR-1. Finally, the iLJ1162 model was used to simulate the optimal biosynthetic pathways for six platform chemicals based on the MR-1 chassis in microbial electrosynthesis systems. These results offer guidance for rational design of novel BESs.

5-Aminolevulinic acid (5-ALA) is the key precursor for the biosynthesis of tetrapyrrole compounds, with wide applications in medicine, agriculture and other burgeoning fields. Because of its potential applications and disadvantages of chemical synthesis, alternative biotechnological methods have drawn increasing attention. In this review, the recent progress in biosynthetic pathways and regulatory mechanisms of 5-ALA synthesis in biological hosts are summarized. The research progress on 5-ALA biosynthesis via the C4/C5 pathway in microbial cells is emphasized, and the corresponding biotechnological design strategies are highlighted and discussed in detail. In addition, the detection methods and applications of 5-ALA are also reviewed. Finally, perspectives on potential strategies for improving the biosynthesis of 5-ALA and understanding the related mechanisms to further promote its industrial application are conceived and proposed.

Acetate is an economical and environmental-friendly alternative carbon source. Herein, the potential of harnessing Corynebacterium glutamicum as a host to produce 3-hydroxypropionic acid (3-HP) from acetate was explored. First, the expression level of malonyl-CoA reductase from Chloroflexus aurantiacus was optimized through several strategies, strain Cgz2/sod-N-C* showed an MCR enzyme activity of 63 nmol/mg/min and a 3-HP titer of 0.66 g/L in flasks. Next, the expression of citrate synthase in Cgz2/sod-N-C* was weakened to reduce the acetyl-CoA consumption in the TCA cycle, and the resulting strain Cgz12/sod-N-C* produced 2.39 g/L 3-HP from 9.32 g/L acetate. However, the subsequent deregulation of the expression of acetyl-CoA carboxylase genes in Cgz12/sod-N-C* resulted in an increased accumulation of intracellular fatty acids, instead of 3-HP. Accordingly, cerulenin was used to inhibit fatty acid synthesis in Cgz14/sod-N-C*, and its 3-HP titer was further increased to 4.26 g/L, with a yield of 0.50 g 3-HP/g-acetate. Finally, the engineered strain accumulated 17.1 g/L 3-HP in a bioreactor without cerulenin addition, representing the highest titer achieved using acetate as substrate. The results demonstrated that Corynebacterium glutamicum is a promising host for 3-HP production from acetate.

Advances in robotic system-assisted genome editing techniques and computer-aided design tools have significantly facilitated the development of microbial cell factories. Although multiple separate software solutions are available for vector DNA assembly, genome editing, and verification, by far there is still a lack of complete tool which can provide a one-stop service for the entire genome modification process. This makes the design of numerous genetic modifications, especially the construction of mutations that require strictly precise genetic manipulation, a laborious, time-consuming and error-prone process. Here, we developed a free online tool called GEDpm-cg for the design of genomic point mutations in C. glutamicum. The suicide plasmid-mediated counter-selection point mutation editing method and the overlap-based DNA assembly method were selected to ensure the editability of any single nucleotide at any locus in the C. glutamicum chromosome. Primers required for both DNA assembly of the vector for genetic modification and sequencing verification were provided as design results to meet all the experimental needs. An in-silico design task of over 10,000 single point mutations can be completed in 5 min. Finally, three independent point mutations were successfully constructed in C. glutamicum guided by GEDpm-cg, which confirms that the in-silico design results could accurately and seamlessly be bridged with in vivo or in vitro experiments. We believe this platform will provide a user-friendly, powerful and flexible tool for large-scale mutation analysis in the industrial workhorse C. glutamicum via robotic/software-assisted systems.