Enhanced extracellular invertase production in Bacillus subtilis by boosting synthesis and secretion in a suitable chassis cell

<title>Abstract</title> Background Soybeans are an important agricultural product, but they contain a significant amount of raffinose, which can cause flatulence when fermented by intestinal microorganisms. Invertase InvDz13 from <italic>Microbacterium trichothecenolyticum</italic> can effectively hydrolyze raffinose in soybean into the prebiotic melibiose, improving the nutritional value and economic benefits of soybean derivatives. However, its low yield limits industrial applications. Results This study produced InvDz13 in <italic>Bacillus subtilis</italic> , resulting in a recombinant enzyme with the natural enzyme's excellent properties. The extracellular InvDz13 production in <italic>B. subtilis</italic> was enhanced by boosting synthesis and secretion in a suitable chassis cell. Various extracellular protease-deficient chassis cells were constructed, and it was found that Vpr and WprA enhance the extracellular InvDz13 activity. The recombinant InvDz13 production was further enhanced by sequentially engineering the promoter, screening the signal peptide, and regulating key elements in the secretion pathway. The recombinant strain WBSS142I4PA, constructed using the promoter P _{<italic>spoVG</italic>} -P _{<italic>spoVG142</italic>} and signal peptide SP _YomL , co-expressing signal peptide peptidases SppA, exhibited a 12.79-fold increase in extracellular InvDz13 activity in shake flasks. Notably, the extracellular InvDz13 activity of WBSS142I4PA in a 3-L fermenter reached 568.86 U/mL. To our knowledge, this is the highest invertase activity reported in <italic>B. subtilis</italic> to date. Conclusions This is the first study to express InvDz13 in <italic>B. subtilis</italic> and develop a novel multidimensional combinatorial strategy to enhance its extracellular production. This study provides a theoretical foundation for the large-scale application of InvDz13 in soybean processing and paves novel avenues for protein production in <italic>B. subtilis</italic> .