Boosting heterologous protein production yield by adjusting global nitrogen and carbon metabolic regulatory networks in Bacillus subtilis

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

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by Lactococcus lactis

<p>Abstract</p> <p>Background</p> <p>Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. <it>Lactococcus lactis </it>has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of <it>L. lactis </it>genetically modified to produce and secrete biologically active IP-10.</p> <p>Results</p> <p>The IP-10 coding region was isolated from human cDNA and cloned into an <it>L. lactis </it>expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of <it>L. lactis </it>secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of <it>L. lactis</it>. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by <it>L. lactis </it>was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes.</p> <p>Conclusion</p> <p>Expression and secretion of mature IP-10 was efficiently achieved by <it>L. lactis </it>forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant <it>L. lactis </it>represents a potentially useful tool to be used as a live vaccine <it>in vivo</it>.</p

Targeting diseases with genetically engineered Lactococcus lactis and its course towards medical translation

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.

Recombinant Adenovirus Delivery of Calreticulin-ESAT-6 Produces an Antigen-Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

Bacillus CalmetteGuerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M. tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M. tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRTESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-gamma and tumour necrosis factor-a in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRTESAT-6CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M. tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis

Abstract Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8? T-cells and directs migration of CD4? and CD8? lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by -4

Em carried by (pSEC:huIP-10). The diagram shows the nisin-inducible promoter PnisA, the ribosome binding-site of (RBS), the usp45 signal peptide of the gene (SPusp), and the coding region for the mature moiety of IP-10. The open circle represents a rho-independent transcription terminator fused just downstream to the IP-10 gene (not to scale).<p><b>Copyright information:</b></p><p>Taken from "Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by "</p><p>http://www.microbialcellfactories.com/content/7/1/22</p><p>Microbial Cell Factories 2008;7():22-22.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2503953.</p><p></p

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by -3

Motaxis. T lymphocytes obtained directly from human peripheral blood were stimulated with human IL-2 for 12 days. Chemotaxis assays using Boyden chambers were made with supernatants sterilized by filtration of both recombinant and wild-type . To determine the number of chemoattracted cells, the membranes were stained with hematoxylin and the cells counted by using light microscopy at 1000 × (A). Cells in the lower chamber were incubated with specific anti-CD3+ and counted by FACS analysis (B). Supernatants of wild-type (WT) and PBS were used as negative controls and Zymosan-activated serum (S+) as a positive control.<p><b>Copyright information:</b></p><p>Taken from "Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by "</p><p>http://www.microbialcellfactories.com/content/7/1/22</p><p>Microbial Cell Factories 2008;7():22-22.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2503953.</p><p></p

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by -0

Em carried by (pSEC:huIP-10). The diagram shows the nisin-inducible promoter PnisA, the ribosome binding-site of (RBS), the usp45 signal peptide of the gene (SPusp), and the coding region for the mature moiety of IP-10. The open circle represents a rho-independent transcription terminator fused just downstream to the IP-10 gene (not to scale).<p><b>Copyright information:</b></p><p>Taken from "Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by "</p><p>http://www.microbialcellfactories.com/content/7/1/22</p><p>Microbial Cell Factories 2008;7():22-22.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2503953.</p><p></p

Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by -1

were prepared from cell-free samples and analyzed by Western blotting using anti-IP-10. Mature IP-10 was detected in all the induced (+) cultures in the 10 kDa range as expected. No signal was found for noninduced cultures (-) of recombinant NZpSEC:huIP-10. No immature or incomplete forms of IP-10 were detected. M, protein molecular marker.<p><b>Copyright information:</b></p><p>Taken from "Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by "</p><p>http://www.microbialcellfactories.com/content/7/1/22</p><p>Microbial Cell Factories 2008;7():22-22.</p><p>Published online 28 Jul 2008</p><p>PMCID:PMC2503953.</p><p></p