Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis or Mycobacterium bovis and still remains one of the world’s biggest global health burdens. Recently, engineered polyhydroxyalkanoate (PHA) biobeads produced in both E. coli and Lactococcus lactis displaying mycobacterial antigens were found to induce significant cell mediated immune responses in mice. We observed that such PHA beads contained host cell proteins as impurities which we hypothesized to have the potential to induce immunity. In this study we aimed to develop PHA beads produced in mycobacteria (mycobacterial PHA biobeads, MBB) and test their potential as TB vaccine in a mouse model. As a model organism, nonpathogenic Mycobacterium smegmatis was engineered to produce MBB or MBB with immobilized mycobacterial antigens Ag85A and ESAT-6 on their surface (A:E- MBB). Three key enzymes involved in the poly(3-hydroxybutyric acid) pathway, namely β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC), were engineered into E. coli?mycobacteria shuttle plasmids and expressed in trans. Immobilization of specific antigens to the surface of the MBB was achieved by creating a fusion with the PHA synthase which remains covalently attached to the polyester core, resulting in PHA biobeads displaying covalently immobilized antigens. MBB, A:E-MBB and a mycobacterial vector control (MVC) were used in a mouse immunology trial, with comparison to PBS vaccinated and BCG vaccinated groups. We successfully produced MBB and A:E-MBB and used them as vaccines to induce a cellular immune response to mycobacterial antigens.