Precise Photothermal Treatment of Methicillin-Resistant S. aureus Infection via Phage Lysin-Cell Binding Domain-Modified Gold Nanosheets.

The increasing spread of antibiotic resistance in bacterial pathogens poses a huge threat to global human health. Precise targeting of bacterial pathogens while avoiding collateral damage to healthy tissues has become the overriding goal for bacterial infection treatment. Inspired by the host specificity of bacteriophages, a biomimetic intelligent platform was designed for highly precise photothermal treatment herein. As proof-of-concept, the lysin cell-binding domain (CBD) from a newly discovered virulent methicillin-resistant S. aureus (MRSA) phage Z was applied to the functionalization of gold nanosheets. Our results demonstrated that the bionanocomposite gold particles (Au@PEG-CBDz) could be effectively delivered directly to MRSA and kill them effectively under near infrared irradiation in vitro, while displaying good in vivo biocompatibility. This work is the first to report the combination of phage lysin navigatory function with photothermal effect-induced bactericidal activity from Au nanosheets, providing a novel therapeutic mode for the precision treatment of antibiotic-resistant bacterial infections.

Reshaping the Binding Pocket of Cellobiose 2-Epimerase for Improved Substrate Affinity and Isomerization Activity for Enabling Green Synthesis of Lactulose.

Enzymatic isomerization of lactose into lactulose via cellobiose 2-epimerase (CE) could provide an eco-friendly route for the industrial production of lactulose, a valuable food prebiotic. However, poor substrate affinity for lactose and preference for epimerization over isomerization hinder this application. Previous studies on CE improvement have focused on random mutagenesis or active site rational design; little is known about the relationship between substrate binding and enzyme efficacy, which was hence the subject of this study. First, residues 372W and 308W were identified as key for disaccharide recognition in CEs based on crystal structure alignment of the N-acetyl-glucosamine 2-epimerase superfamily and site-directed mutation. This binding domain was then reshaped through site saturation mutagenesis, resulting in seven mutants with enhanced isomerization activity. The optimal mutant CsCE/Q371E had significantly enhanced substrate affinity (Km, 269.65 mM vs Km, 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original CsCE. Molecular dynamics simulation further revealed that substituting Gln-371 with Glu strengthened the hydrogen-bonding network and altered the active site-substrate interactions, increasing the substrate stability and shifting the catalytic direction. This study uncovered new information about the substrate binding region and its mechanisms and impact on CE catalytic performance, paving the way for potential commercial applications.

De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae.

Hydroxytyrosol is an olive-derived phenolic compound of increasing commercial interest due to its health-promoting properties. In this study, a high-yield hydroxytyrosol-producing Saccharomyces cerevisiae cell factory was established via a comprehensive metabolic engineering scheme. First, de novo biosynthetic pathway of hydroxytyrosol was constructed in yeast by gene screening and overexpression of different phenol hydroxylases, among which paHD (from Pseudomonas aeruginosa) displayed the best catalytic performance. Next, hydroxytyrosol precursor supply was enhanced via a multimodular engineering approach: elimination of tyrosine feedback inhibition through genomic integration of aro4K229L and aro7G141S, construction of an aromatic aldehyde synthase (AAS)-based tyrosine metabolic pathway, and redistribution of metabolic flux between glycolytic pathway and pentose phosphate pathway (PPP) by introducing the exogenous gene Bbxfpkopt. As a result, the titer of hydroxytyrosol was improved by 6.88-fold. Finally, a glucose-responsive dynamic regulation system based on GAL80 deletion was implemented, resulting in the final hydroxytyrosol yields of 308.65 mg/L and 167.98 mg/g cell mass, the highest known from de novo production in S. cerevisiae to date.

Clinically relevant materials & applications inspired by food technologies.

Food science and technology have a fundamental and considerable overlap with medicine, and many clinically important applications were borne out of translational food science research. Globally, the food industry - through various food processing technologies - generates huge quantities of agro-waste and food processing byproducts that retain a significant biochemical potential for upcycling into important medical applications. This review explores some distinct clinical applications that are fabricable from food-based biopolymers and substances, often originating from food manufacturing side streams. These include antibacterial wound dressings and tissue scaffolding from the biopolymers cellulose and chitosan and antimicrobial food phytochemicals for combating antibiotic-resistant nosocomial infections. Furthermore, fermentation is discussed as the epitome of a translational food technology that unlocks further therapeutic value from recalcitrant food-based substrates and enables sustainable large-scale production of high-value pharmaceuticals, including novel fermented food-derived bioactive peptides (BPs).

Gene Source Screening as a Tool for Naringenin Production in Engineered Saccharomyces cerevisiae.

Flavonoids are plant secondary metabolites with great potential in the food industry. Metabolic engineering of Saccharomyces cerevisiae is a sustainable production technique. However, the current naringenin production yield is low because of inefficient enzymatic activity. Hence, this study uses gene source screening as a tool to identify the best gene source for enzymes such as 4-coumarate: coenzyme ligase (4CL) and chalcone synthase (CHS). For the first time, the 4CL gene from Medicago truncatula and the CHS gene from Vitis vinifera were expressed in S. cerevisiae, and this combination provided the highest yield of naringenin, which was 28-fold higher as compared to the reference strain. The combinations obtained similar performance in the Y-28 strains, where the highest production was 28.68 mg/L. Our results demonstrated that the selection and combination of enzymes from the correct gene source could greatly improve naringenin production. For the future, this could help commercialize flavonoid production, which would result in natural food preservatives and additives.

Metabolic Engineering of Saccharomyces cerevisiae for De Novo Production of Kaempferol.

Kaempferol is a polyphenolic compound with various reported health benefits and thus harbors considerable potential for food-engineering applications. In this study, a high-yield kaempferol-producing cell factory was constructed by multiple strategies, including gene screening, elimination of the phenylethanol biosynthetic branch, optimizing the core flavonoid synthetic pathway, supplementation of precursor PEP/E4P, and mitochondrial engineering of F3H and FLS. A total of 86 mg/L of kaempferol was achieved in strain YL-4, to date the highest production titer in yeast. Furthermore, a coculture system and supplementation of surfactants were investigated, to relieve the metabolic burden as well as the low solubility/possible transport limitations of flavonoids, respectively. In the coculture system, the whole pathway was divided across two strains, resulting in 50% increased cell growth. Meanwhile, supplementation of Tween 80 in our engineered strains yielded 220 mg/L of naringenin and 200 mg/L of mixed flavonoids-among the highest production titer reported via de novo production in yeast.

Antimicrobial and antioxidant activities of phenolic metabolites from flavonoid-producing yeast: Potential as natural food preservatives.

We analysed the antimicrobial and antioxidant activities of phenolic metabolites secreted from a naringenin-producing Saccharomyces cerevisiae strain (a GRAS organism), against the pure flavonoid naringenin and its prenylated derivatives, to assess their potential as natural food preservatives. Agar disc diffusion assay was used to analyse the antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, while DMPD chemiluminescence assay was used to analyse antioxidant activity, based on DMPD+-scavenging activity. Our results showed that the engineered yeast metabolites exhibited both strong antimicrobial and DMPD+-scavenging activity, particularly the metabolite phenylacetaldehyde. Pure naringenin had poor antimicrobial and DMPD+-scavenging effects. Prenylated varieties, 6-prenylnaringenin and 8-prenylnaringenin, inhibited only S. aureus, while only 8-prenylnaringenin exhibited moderate DMPD+-scavenging activity. Our results suggested that phenolic metabolites secreted from naringenin-producing yeast would be a sustainable source of natural food preservatives.

Enhancement of Naringenin Biosynthesis from Tyrosine by Metabolic Engineering of Saccharomyces cerevisiae.

Flavonoids are an important class of plant polyphenols that possess a variety of health benefits. In this work, S. cerevisiae was metabolically engineered to produce the flavonoid naringenin, using tyrosine as the precursor. Our strategy to improve naringenin production comprised three modules. In module 1, we employed a modified GAL system to overexpress the genes of the naringenin biosynthesis pathway and investigated their synergistic action. In module 2, we simultaneously up-regulated acetyl-CoA production and down-regulated fatty acid biosynthesis in order to increase the precursor supply, malonyl-CoA. In module 3, we engineered the tyrosine biosynthetic pathway to eliminate the feedback inhibition of tyrosine and also down-regulated competing pathways. It was found that modules 1 and 3 played important roles in improving naringenin production. We succeeded in producing up to ∼90 mg/L of naringenin in our final strain, which is a 20-fold increase as compared to the parental strain.