Ting Ma

Currently, the production of high‐value chemicals from natural lignocellulose is of great significance for a circular economy. In this study, a robust platform was developed based on Sphingomonas sanxanigenens NXdP, which co‐utilizes glucose and xylose to synthesize polysaccharide sanxan, for efficient bioconversion of lignocellulose‐derived sugars. Sanxan is a promising high‐value product due to its high viscosity at low concentrations, excellent acid‐gelling capabilities, and unique rheological characteristics. Through a cell growth‐based screening strategy and gene manipulation, a key transporter (0233) required for the uptake of both glucose and xylose was identified, clarifying the co‐utilization mechanism. Additionally, the metabolic pathways of glucose and xylose were individually blocked to assess their contributions and activities. Furthermore, the metabolic network of strain NXdP was reprogrammed via a “flux‐redirection” strategy to develop a potential microbial host with improved sanxan production on mixed sugars. The final engineered strain S09 achieved a sanxan titer of 20.25 ± 0.78 g/kg under mixed‐sugar fermentation, representing a 13.51% ± 4.38% increase compared with the original strain NXdP. The mechanism underlying this enhanced production was revealed by central carbon metabolism (CCM) analysis. Overall, this study provides insights into the efficient conversion of lignocellulose‐derived sugars and offers genetic components for the green production of other high‐value‐added products.

Cyclic di-GMP (c-di-GMP) acts as a bacterial second messenger that regulates various physiological processes, including biofilm formation, motility, and virulence. It is synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs), with precise regulatory effects typically requiring the coordinated action of multiple DGCs and PDEs. Kosakonia oryzendophytica FY-07 (FY-07) exhibits high bacterial cellulose (BC) yield, rapid growth, and robustness and can produce BC under varying oxygen conditions; however, the underlying mechanism driving the phenotypic transition from cellulose-producing to motile states during fermentation has yet to be elucidated. In this study, we aimed to identify two DGCs, KoBdcA and KoBdcB, and one PDE, KoBpdA, whose encoding genes are located adjacent to the cellulose synthesis operon in FY-07. KoBdcA and KoBpdA form an antagonistic DGC-PDE pair that maintains intracellular c-di-GMP homeostasis. Deletion of either gene alters BC production and motility. In contrast, KoBdcB acts as an auxiliary component, and its deletion alone causes no phenotypic changes. KoBdcA regulates BC synthesis via an indirect interaction with BcsA mediated by KoBdcB. The Per-Arnt-Sim (PAS) domains of KoBdcA and KoBpdA bind hemin, enabling them to sense oxygen and modulate their enzymatic activities. Elevated DGC activity during early fermentation promotes BC production, while increased PDE activity later suppresses BC synthesis, shifting cells to a motile state. IMPORTANCE: In this study, we provide novel mechanistic insights into how synergistic interactions among multiple DGCs and PDEs, in response to oxygen sensing, control the dynamic switch between the sessile and motile states of biofilm-producing bacteria. The study findings provide theoretical insights into the localized regulation of c-di-GMP signaling in K. oryzendophytica and have significant implications for elucidating the metabolic regulatory network of c-di-GMP. These findings also provide valuable insights into the molecular interactions and mechanisms governing the association between Kosakonia species and their host plants.

The development of robust and efficient β-mannanases is key to advancing environmentally friendly industrial processes, such as guar gum fracturing fluid gel-breaking. Here, we report the identification and characterization of MG4, a novel thermotolerant and alkaliphilic β-mannanase mined from the Earth’s Microbiome database. The recombinant enzyme has a molecular weight of 63 kDa. MG4 displayed maximum activity at 65 °C and pH 9.0, and exhibited remarkable stability across a broad pH range (7.0–10.0). It retained over 80% of its activity after incubation at 50 °C for 1 h, and its activity was enhanced more than 40% by Mg²⁺ or Ca²⁺. Moreover, MG4 (20 mg/L) reduced the viscosity of guar gum fracturing fluid to <5 m·PaS within 30 min, outperforming ammonium persulfate (APS, 500 mg/L) which required 1 h, and produced 64.5% less insoluble residue. TEM imaging directly visualized the disruption of the guar gum polymer network by MG4, explaining its efficacy and suggesting reduced formation damage risk compared to chemical breakers. This work characterizes a highly promising biocatalyst whose thermostability, alkaliphily, efficient gel-breaking, low residue yield, and minimal formation damage potential position it as a superior, eco-friendly alternative for petroleum industry applications.