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When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD, and a high titer of 371mM (32g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361mM (32g/L) R-BDO with a yield of 81% or 365mM (33g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production.

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Development of real-time and in situ analytical methods for determination of food-borne pathogens and toxins ingested into the human body would be a promising research direction in the food-safety area. The present perspective starts with summarization of the up-to-date progress of the nanomaterial-assisted in vitro detection methods for pathogens and toxins and finally focuses on application of animal bioimaging to in vivo study, including prospective strategies for in vivo quantification of target pathogens or toxins and in vivo investigation of their behaviors inside the living body, with the assistance of real-time and non-invasive optical bioimaging. This perspective provides the advisory direction for food-safety research, from in vitro to in vivo, along with a prospective discussion of the further development roadmap of the food-safety detection techniques, especially the bioimaging-guided methods for investigation and mediation of the food contamination effect to human health.

Corynebacterium glutamicum, a Gram-positive bacterium used for the production of various biochemicals, is naturally a biotin auxotroph. We introduced the biotin genes from Bacillus subtilis on a plasmid, pBIO, into a lysine-producing derivative (termed AHP-3) that has been described previously, and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain was cultivated without biotin, indicating a suboptimal intracellular concentration of biotin. In an attempt to locate the potential bottleneck, we added pimelic acid, an early biotin precursor, and found that growth rate could be restored fully, which demonstrates that the bottleneck is in pimeloylCoA (or pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes.

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4-Hydroxymandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122-126 were involved in its PobR ligand speci city. These results showed the effectiveness of directed evolution in switching the ligand speci city of a biosensor and improving HMA production.

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