Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral therapeutic applications, e.g., production of vaccine components. In the bacterial genus Sphingomonas from the Alphaproteobacteria class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of sphingomonas as a safe expression system for production of proteins for therapeutic applications, in this study, Sphingobium japonicum (SJ) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in SJ identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines. KEY POINTS: • Novel molecular tools for protein expression in non-model bacteria. • Bacteria with GSL instead of LPS as a potential vector for protein delivery.