Molecular farming utilizes plants as a platform for producing recombinant biopharmaceuticals. Duckweed, the smallest and fastest growing aquatic plant, is a promising candidate for molecular farming. However, the efficiency of current transformation methods is generally not high in duckweed. Here, we developed a fast and efficient transformation procedure in Lemna minor ZH0403, requiring 7–8 weeks from screening calluses to transgenic plants with a stable transformation efficiency of 88% at the DNA level and 86% at the protein level. We then used this transformation system to produce chicken interleukin-17B (chIL-17B). The plant-produced chIL-17B activated the NF-κB pathway, JAK-STAT pathway, and their downstream cytokines in DF-1 cells. Furthermore, we administrated chIL-17B transgenic duckweed orally as an immunoadjuvant with mucosal vaccine against infectious bronchitis virus (IBV) in chickens. Both IBV-specific antibody titer and the concentration of secretory immunoglobulin A (sIgA) were significantly higher in the group fed with chIL-17B transgenic plant. This indicates that the duckweed-produced chIL-17B enhanced the humoral and mucosal immune responses. Moreover, chickens fed with chIL-17B transgenic plant demonstrated the lowest viral loads in different tissues among all groups. Our work suggests that cytokines are a promising adjuvant for mucosal vaccination through the oral route. Our work also demonstrates the potential of duckweed in molecular farming.

Terrestrialization is an important evolutionary process that plants experienced. However, little is known about how land plants acquired aquatic growth behaviors. Here, we integrate multiproxy evidence to elucidate the evolution of the aquatic plant duckweed. Three genera of duckweeds show chronologically gradual degeneration in root structure and stomatal function and a decrease in lignocellulose content, accompanied by the contraction of relevant gene families and/or a decline in their transcription levels. The number of genes in main phytohormone pathways is also gradually decreased. The coordinated action of genes involved in auxin signaling and rhizoid development causes a gradual decrease in adventitious roots. Additionally, the significant expansion of the flavonoid pathway is related to the adaptation of duckweeds to floating growth. This study reconstructs the evolutionary history of duckweeds, tracing its journey from land back to water — a reverse trajectory of early land plants.

Duckweeds, recognized as the fastest growing aquatic flowering plants, exhibit substantial biomass production. Recently, they have been emerged as a potential industrial crop for efficient and eco-friendly protein production and nutrient recovery compared to conventional crops. The objective of the study was to determine the biomass accumulation rate, protein content and amino acid analysis of duckweeds (Lemna minor D0158).We cultured L. minor D0158 from its preserved state in the gene bank into both aseptic (in-vitro) and open condition at controlled room. The medium was Hoagland Solution (HS) mixed with sucrose in in-vitro condition while 1/5 concentration of HS (without sucrose) was in the open condition. Subsequently, we determined the dry biomass growth by calculating the weight difference adopting the Bergmann Method, and then calculated the protein percentage following the Kjeldahl Method. Moreover, the amino acid profiling was obtained using the Hydrolysis and Liquid Chromatography Technique. Later on, we compared the results with those of the other duckweeds and soybean together with the FAO requirements. We found that L. minor D0158 possessed the dry biomass growth rate of 6.72 g/m2/d with 33.13% protein content within 7-day cultivation period. Moreover, it exhibited significantly higher levels of branched chain amino acids (BCCAs) than soybean and met the FAO requirements. However, methionine (Met) content was found to be slightly low. The study suggested that L. minor D0158 might offer a sustainable solution for protein food security in future.

Despite urea being the most widely used nitrogen fertilizer, many plants show reduced growth and protein content when urea is the nitrogen source. The underlying causes of nitrogen metabolism imbalance in urea supply remain unclear. In this study, we discovered that plastidic glutamine synthetase (GS2) can improve the physiological traits and protein production of duckweed cultivated with urea. By comparing the urea utilization processes between two duckweed strains, LpZH0104 and Lp9595, which exhibited marked differences under the same urea treatment, we found that GS2 significantly impacts nitrogen metabolism. Overexpressing the GS2 gene in an efficient genetic transformation system of duckweed enhanced nitrogen‑carbon metabolism, leading to a 10 % increase in biomass and a 24 % increase in protein content in the overexpression lines (OE lines). Protein productivity and PPPN (protein production per unit of applied fertilizer N) increased by 47 % and 32 %, respectively. Conversely, silencing the GS2 gene reduced these traits. Additionally, transforming GS2 gene into rice, tobacco, and Arabidopsis also enhanced their biomass and protein content under urea cultivation conditions. This study highlights GS2's role in enhancing nitrogen metabolism and physiological traits in plants cultivated with urea, offering valuable insights for improving biomass and protein production in crops using urea fertilizer.

Nong-flavor (NF) Daqu, a critical fermentation starter for traditional Baijiu, harbors diverse starch-degrading enzymes with poorly characterized functional dynamics. This study transcended traditional quality assessments by developing molecular approaches to dissect starch-hydrolyzing enzyme genes. Specific and degenerate primers targeting glucoamylase, α-amylase, and α-glucosidase genes were designed, and key genes were qualitatively identified with distinct distributions among NF Daqus and unique presences between JXL and HB Daqu. Quantitative PCR revealed six genes with elevated expression in JXL Daqu versus HB Daqu, and which peaked during late fermentation in both Daqus. Metagenomics identified greater enzymatic diversity in HB Daqu. Phylogenetic clustering confirmed evolutionary conservation (GH13/GH15/GH31 families) and specificity of core enzyme genes across both Daqus. Enzymatic assays demonstrated the dominance of saccharification over α-glucosidase activity in both Daqus, with significantly higher α-glucosidase activity in JXL than HB Daqu. Divergent starch degradation strategies emerged: JXL prioritized high enzyme expression/activity, while HB utilized broader gene abundance. Based on Pearson correlation analysis, the saccharification activity showed the highest but weak correlation with α-glucosidase gene_15963 (r = 0.26), and was also positively correlated with the expression of all other enzyme genes except one glucoamylase gene. Meanwhile, α-glucosidase activity was most strongly linked to glucoamylase gene_22243 (r = 0.76), with additional correlations with two α-glucosidase genes being observed. This establishes RNA-based biomarkers for real-time quality control. Our findings decode divergent microbial strategies (JXL: high-expression/high-activity vs. HB: high-diversity) and provide a molecular framework for optimizing starch utilization in Baijiu fermentation. This technology holds potential to enable precision-driven standardization of traditional food production, which would reduce processing waste and enhance resource efficiency.

To explore microbial resources for ester production in sub-high-temperature Daqu, this study first established that the esterifying enzyme activity in Daqu predominantly originated from fungi, with Rhizopus being the dominant fungal genus. Six Rhizopus strains capable of decomposing esters were isolated and purified from Daqu. Following secondary screening, strain M1 exhibited the highest esterification activity (40.26 U/mL) and was identified as Rhizopus oryzae based on morphological characteristics and molecular biological analyses. This strain was subsequently designated as Rhizopus oryzae M1 (R. oryzae M1). Using mycelial powder of strain M1 as the inoculum and sterilized wheat bran as the substrate, a pure-culture Fuqu was prepared. Orthogonal array design experiments were conducted to optimize the preparation process of this Fuqu, using esterifying enzyme activity as the evaluation index. Under the optimal conditions, the spore count and esterification activity of the pure-culture Fuqu reached 1.73 × 10⁹ CFU/g and 80.13 U/g, respectively. This pure-culture Fuqu was subsequently used as an inoculum to produce fortified Daqu. Following orthogonal optimization of the Daqu preparation process, the esterification activity of the fortified Daqu reached 103.22 U/g, and its key physicochemical indices met the requirements for high-quality sub-high-temperature Daqu. Analysis of the microbial community structure revealed that Rhizopus was the dominant fungal genus in the fortified Daqu, with its relative abundance increased by 35% compared to the non-fortified Daqu. Consistent with this, the esterifying enzyme activity of the fortified Daqu was 51.79% higher, suggesting that Rhizopus may have been largely responsible for the increase in esterification capacity. In laboratory-scale Baijiu brewing trials, this fortified Daqu produced a base Baijiu with a total ester content of 2.74 g/L, representing a 40.5% increase over the non-fortified Daqu and further confirming the pivotal role of Rhizopus in driving the esterifying enzyme activity. This study successfully screened a high esterifying enzyme-producing strain, R. oryzae M1, systematically optimized its enzyme production and Qu-making processes, and provides an excellent microbial strain and process reference for the preparation of fortified Daqu and the enhancement of Baijiu flavor.