Background: The structural diversity of extracellular polymeric substances produced by microorganisms is attracting particular attention. Poly-gamma-glutamic acid (γ-PGA) is a widely studied extracellular polymeric substance from Bacillus species. The function of γ-PGA varies with its molecular weight (Mw). Results: Herein, different endogenous promoters in Bacillus licheniformis were selected to regulate the expression levels of pgdS, resulting in the formation of γ-PGA with Mw values ranging from 1.61 × 10³ to 2.03 × 10⁴ kDa. The yields of γ-PGA and exopolysaccharides (EPS) both increased in the pgdS engineered strain with the lowest Mw and viscosity, in which the EPS content was almost tenfold higher than that of the wild-type strain. Subsequently, the compositions of EPS from the pgdS engineered strain also changed. Metabolomics and RT-qPCR further revealed that improving the transportation efficiency of EPS and the regulation of carbon flow of monosaccharide synthesis could affect the EPS yield. Conclusions: Here, we present a novel insight that increased pgdS expression led to the degradation of γ-PGA Mw and changes in EPS composition, thereby stimulating EPS and γ-PGA production. The results indicated a close relationship between γ-PGA and EPS in B. licheniformis and provided an effective strategy for the controlled synthesis of extracellular polymeric substances.

The diverse metabolic mechanisms underlying bacterial extracellular polymeric substances give rise to a wide array of components with distinct functionalities, including exopolysaccharides (EPS) and poly-γ-glutamic acid (γ-PGA). The coordinated synthesis of various types of extracellular polymeric substances necessitates comprehensive investigation from a global regulatory perspective. In this study, we examined the impact of multiple environmental stressors on Bacillus species, revealing that the EPS and γ-PGA produced respond to stress through metabolic and cellular process reorganization. The overexpression of global transcriptional regulators influenced the production of EPS and γ-PGA differently. Specifically, quorum sensing-related global regulators such as rsbRA, rapA, and the carbon utilization regulator ccpA-2 were found to enhance EPS synthesis. Conversely, positive global transcriptional regulators associated with γ-PGA synthesis included carbon and nitrogen utilization-related regulators ccpA-2, cggR, and nrgB. Notably, the global regulators nrgB and cggR increased γ-PGA production by 33.64% and 44.14%, respectively, while this enhancement was accompanied by a concomitant reduction in EPS production. In B. licheniformis, omics analyses have elucidated critical pathways and metabolites implicated in stress response mechanisms that induce alterations in amino acid metabolism, carbon source utilization, alongside the activation of global regulatory elements. These studies indicated that nrgB predominantly governs downstream genes associated with carbon metabolism, energy metabolism, signal transduction, and membrane transport processes. This work combines stress induction strategies and global transcription machinery engineering for investigating the coordinated synthesis of various types of extracellular polymeric substances, which has not been explored before. The insights gained from our research contribute to a deeper understanding of the regulatory networks governing the competition between γ-PGA and EPS, thereby providing a theoretical basis for the engineered modification of Bacillus licheniformis aimed at optimizing the production of extracellular polymeric substances.

The increasing global demand for protein underscores the necessity for sustainable alternatives to soybean-based animal feed, which poses a challenge to human food security. Thus, the search for sustainable, alternative protein sources is transforming the feed industry in its effort to sustainable operations. In this study, a microbial consortium was subjected to adaptive laboratory evolution using non-protein nitrogen (NPN) and wheat straw as the sole carbon source. The evolved microbial consortium was subsequently utilized to perform solid-state fermentation on wheat straw and NPN to produce feed protein. After 20 generations, the microbial consortium demonstrated tolerance to 5 g/L NPN, including ammonium sulfate, ammonium chloride, and urea, which represents a fivefold increase compared to the original microbial consortium. Among the three NPNs tested, the evolved microbial consortium exhibited optimal growth performance with ammonium sulfate. Subsequently, the evolved microbial consortium was employed for the solid-state fermentation (SSF) of wheat straw, and the fermentation conditions were optimized. It was found that the true protein content of wheat straw could be increased from 2.74% to 10.42% under specific conditions: ammoniated wheat straw (15% w/w), non-sterilization of the substrate, an inoculation amount of 15% (v/w), nitrogen addition amount of 0.5% (w/w), an initial moisture content of 70%, a fermentation temperature of 30 °C, and a fermentation duration of 10 days. Finally, the SSF process for wheat straw was successfully scaled up from 0.04 to 2.5 kg, resulting in an increased true protein content of 9.84%. This study provides a promising approach for the production of feed protein from straw and NPN through microbial fermentation, addressing protein resource shortages in animal feed and improving the value of waste straw.

This study explored the enhancement of exopolysaccharide (EPS) production by Ganoderma lucidum through bidirectional liquid fermentation, employing Clinacanthus nutans (Burm. f.) Lindau leaves as a medicinal substrate. The optimal concentration of C. nutans leaf powder was determined to be 6 g/L, resulting in a significant increase in both mycelial biomass (61.78%) and EPS yield (116.6%). Structural analyses indicated that the EPS supplemented with C. nutans underwent notable modifications. Fourier-transform infrared spectroscopy suggested the introduction of potential carbonyl groups and a shift in glycosidic linkage configuration. Monosaccharide composition analysis revealed a significant transition from a glucose-dominated profile in the control to a galactose-rich, more diverse profile, including uronic acids and amino sugars, in the experimental group. High-performance gel permeation chromatography demonstrated a transformation from a low, homogeneous molecular weight (4.7 kDa) to a heterogeneous, bimodal distribution featuring a prominent high-molecular-weight fraction (38.5 kDa). Consequently, the modified EPS exhibited significantly enhanced antioxidant activities, with scavenging rates for DPPH, hydroxyl, and ABTS radicals increasing to 55.5%, 35.1%, and 88.0%, respectively, at a concentration of 2 mg/mL. These findings demonstrate that C. nutans is an effective supplement for modulating the fermentation process of G. lucidum, not only boosting EPS production but also tailoring its structural characteristics to obtain polysaccharides with superior bioactivities, highlighting its potential in functional food and nutraceutical applications.