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Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

3-Hydroxypropionic acid (3-HP) is an important platform chemical, and biological production of 3-HP from glycerol as a carbon source using glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) has been revealed to be effective because it involves a relatively simple metabolic pathway and exhibits higher yield and productivity than other biosynthetic pathways. Despite the successful attempts of 3-HP production from glycerol, the biological process suffers from problems arising from low activity and inactivation of the two enzymes. To apply the directed evolutionary approach to engineer the 3-HP production system, we constructed a synthetic selection device using a 3-HP-responsive transcription factor and developed a selection approach for screening 3-HP-producing microorganisms. The method was applied to an ALDH library, specifically aldehyde-binding site library of alpha-ketoglutaric semialdehyde dehydrogenase (KGSADH). Only two serial cultures resulted in enrichment of strains showing increased 3-HP production, and an isolated KGSADH variant enzyme exhibited a 2.79-fold higher catalytic efficiency toward its aldehyde substrate than the wild-type one. This approach will provide the simple and efficient tool to engineer the pathway enzymes in metabolic engineering.

The pseudotargeted lipidomics method integrates the advantages of untargeted andtargeted lipidomics methods as a novel emerging approach. In this study, a green andefficient pseudotargeted lipidomics method based on ultra-high performancesupercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) wasdeveloped. The tandem mass spectra of the analytes were obtained by using UHPSFCwith quadrupole-time of flight MS (Q-TOF MS) in MS E mode and the multiplereaction monitoring (MRM) transitions of the lipidome were defined. Then, thecandidate MRM transitions were verified by UHPSFC with triple quadrupole massspectrometry (QqQ MS) in the scheduled MRM mode. In total, 758 potential lipidscorresponding to 509 and 249 MRM transitions were detected within 8 min in positiveand negative modes, respectively. The established pseudotargeted lipidomics methodwas validated to have excellent analytical characteristics. Compared with thepseudotargeted method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), the UHPSFC-MS/MS-basedpseudotargeted method not only reduced the analytical time by half but also improvedthe sensitivity and resolution for most analytes, especially had better separation forlipid isomers. Besides, the UHPSFC-MS/MS-based pseudotargeted method showedhigher sensitivity and better repeatability for most analytes than the UHPSFC-MS/MS-based untargeted method. The established method was finally applied to investigatingthe lipid profiles of the plasma from the depressed rats and 33 differential variableswere screened, which related to three metabolic pathways. The results indicated thatthe UHPSFC-MS/MS-based pseudotargeted method is reliable and efficient and couldbe used in the lipidomics studies.

The engineering of biological systems to achieve specific purposes requires design tools that function in a predictable and quantitative manner. Recent advances in the field of synthetic biology, particularly in the programmable control of gene expression at multiple levels of regulation, have increased our ability to efficiently design and optimize biological systems to perform designed tasks. Furthermore, implementation of these designs in biological systems highlights the potential of using these tools to build microbial cell factories for the production of chemicals and fuels. In this paper, we review current developments in the design of tools for controlling gene expression at transcriptional, post-transcriptional and post-translational levels, and consider potential applications of these tools.