Bacillus subtilis is extensively applied as a microorganism for the high-level production of heterologous proteins. Traditional strategies for increasing the productivity of this microbial cell factory generally focused on the targeted modification of rate-limiting components or steps. However, the longstanding problems of limited productivity of the expression host, metabolic burden and non-optimal nutrient intake, have not yet been completely solved to achieve significant production-strain improvements. To tackle this problem, we systematically rewired the regulatory networks of the global nitrogen and carbon metabolism by random mutagenesis of the pleiotropic transcriptional regulators CodY and CcpA, to allow for optimal nutrient intake, translating into significantly higher heterologous protein production yields. Using a β-galactosidase expression and screening system and consecutive rounds of mutagenesis, we identified mutant variants of both CodY and CcpA that in conjunction increased production levels up to 290%. RNA-Seq and electrophoretic mobility shift assay (EMSA) showed that amino acid substitutions within the DNA-binding domains altered the overall binding specificity and regulatory activity of the two transcription factors. Consequently, fine-tuning of the central metabolic pathways allowed for enhanced protein production levels. The improved cell factory capacity was further demonstrated by the successfully increased overexpression of GFP, xylanase and a peptidase in the double mutant strain.

The use of the lactic acid bacterium Lactococcus lactis, primarily used in food fermentations, as therapeutic agent is no longer speculative but an imminent reality. After the successful completion of Phase I and II clinical trials in humans for the treatment of inflammatory bowel disease, an ongoing clinical trial to alleviate oral mucositis as well as the development of a pneumococcal and a flu vaccine using genetically modified L. lactis, many exciting possibilities exist to develop novel therapeutic and prophylactic biopharmaceuticals to alleviate a wide range of diseases. Here, we discuss existing characteristics of the systems currently employed and the nature of the immune responses evoked. We also discuss the criteria that are fundamental to making the systems feasible and efficient which should ultimately translate into human therapies. Finally, we examine the prospects for L. lactis to become a commercially viable therapeutic agent.

Although current polyvalent vaccines can prevent development of cervical cancer, they cannot be used to treat patients who already have the disease. Adenovirus expressing calreticulin-E7 (Ad-CRT-E7) has shown promising results in the cervical cancer murine model. We also demonstrated that immunization with Lactococcus lactis encoding HPV-16 E7 (Ll-E7) anchored to its surface induces significant HPV-16 E7-specific immune response. Here, we assessed the combination of both approaches in the treatment of a cervical cancer animal model. Intranasal preimmunization of Ll-E7, followed by a single Ad-CRT/E7 application, induced ∼80% of tumor suppression in comparison with controls. Mice treated with a combination of Ll-E7 and Ad-CRT/E7 resulted in a 70% survival rate 300 days post-treatment, whereas 100% of the mice in the control groups died by 50 days. Significant CD8+ cytotoxic T-lymphocytes infiltration was detected in the tumors of mice treated with Ll-E7+Ad-CRT/E7. Tumors with regression showed a greater number of positive cells for in situ TUNEL staining than controls. Our results suggest that preimmunization with Ll-E7 enhances the Ad-CRT/E7-mediated antitumor effect. This treatment provides an enormous advantage over repeated applications of Ad-CRT/E7 by maintaining the effectiveness of the three-dose application of Ad-CRT/E7, but avoiding the high systemic toxicities associated with such repeat treatments.