The increase of O-acetylation and N-deacetylation in cell wall promotes acid resistance and nisin production through improving cell wall integrity in Lactococcus lactis.

Cell wall is closely related to bacterial robustness and adsorption capacity, playing crucial roles in nisin production in Lactococcus lactis. Peptidoglycan (PG), the essential component of cell wall, is usually modified with MurNAc O-acetylation and GlcNAc N-deacetylation, catalyzed by YvhB and XynD, respectively. In this study, increasing the two modifications in L. lactis F44 improved autolysis resistance by decreasing the susceptibility to PG hydrolases. Furthermore, both modifications were positively associated with overall cross-linkage, contributing to cell wall integrity. The robust cell wall rendered the yvhB/xynD-overexpression strains more acid resistant, leading to the increase of nisin production in fed-batch fermentations by 63.7 and 62.9%, respectively. Importantly, the structural alterations also reduced nisin adsorption capacity, resulting in reduction of nisin loss. More strikingly, the co-overexpression strain displayed the highest nisin production (76.3% higher than F44). Our work provides a novel approach for achieving nisin overproduction via extensive cell wall remodeling.

Promoting acid resistance and nisin yield of Lactococcus lactis F44 by genetically increasing D-Asp amidation level inside cell wall.

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H+ due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44.

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 µg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms.

DNA-assisted upconversion nanoplatform for imaging-guided synergistic therapy and laser-switchable drug detoxification.

The side effects of chemotherapy bring significant physical and psychological suffering to patients. To solve this urgent medical problem, Yb3+ and Er3+ co-doped NaLuF4 upconversion nanoparticles (UCNPs) were constructed for upconversion luminescence (UCL)-labeled diagnosis under 980 nm laser irradiation. The UCNPs were then modified layer by layer with polypyrrole and a special programming DNA segment as photothermal conversion agents and controllable drug carriers, respectively. The nanoplatform was successfully used for imaging-guided synergistic therapy (photothermal therapy and chemotherapy) at a safe power density (300 mW cm-2), and DNA-assisted detoxification at lower temperature in cancer cells when the laser off. The synergistic therapy of the nanoplatform achieved a higher therapeutic index (∼85%) than chemotherapy only (∼44%) and photothermal therapy only (∼25%) in vitro. In vivo experiments also suggested that the nanoplatform had a higher therapeutic effect and lower side effects. The toxicity study was also evaluated, indicating the nanoplatform is low toxic to living system. This multifunctional upconversion nanoplatform provided an innovative method for imaging-guided photothermal-chemotherapy and laser-switchable drug detoxification.

PEDOT nanocomposites mediated dual-modal photodynamic and photothermal targeted sterilization in both NIR I and II window.

PEDOT nanoparticles with a suitable nanosize of 17.2 nm, broad adsorption from 700 to 1250 nm, and photothermal conversion efficiency (η) of 71.1%, were synthesized using an environmentally friendly hydrothermal method. Due to the electrostatic attraction between indocyanine green (ICG) and PEDOT, the stability of ICG in aqueous solution was effectively improved. The PEDOT nanoparticles modified with glutaraldehyde (GTA) targeted bacteria directly, and MTT experiments demonstrated the low toxicity of PEDOT:ICG@PEG-GTA in different bacteria and cells. Pathogenic bacteria were effectively killed by photodynamic therapy (PDT) and photothermal therapy (PTT) with PEDOT:ICG@PEG-GTA in the presence of near-infrared (NIR) irradiation (808 nm for PDT, and 1064 nm for PTT). The combination of the two different bacteriostatic methods was significantly more effective than PTT or PDT alone. The obtained PEDOT:ICG@PEG-GTA may be used as a novel synergistic agent in combination photodynamic and photothermal therapy to inactivate pathogenic bacteria in both the NIR I and II window.