Abstract

Due to their small sizes, bacterial intracellular PHA accumulation is limited. The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod bacteria. Inhibition of MreB resulted in a change in shape from rod to sphere. Yet, the bacterial growth rate was slowed down. In this study, a plasmid containing complemented mreB gene was overexpressed in mreB mutated E. coli JM109SG, leading to a restored bacterial growth. The plasmid based mreB expression resulted in elastic and enlarged cell sizes, leading to a 162% increase on PHA yield compared to wild type JM109SG. In minimal medium supplemented with 20 g L-1 glucose, accumulating about 7.5 g L-1 P3HB after 48 hr at 30°C, resulting in an 3HB yield of 0.375 g/g glucose (7.5 g/L 3HB produced/20 g/L glucose consumed), which is 78% of the theoretical maximum from glucose. Meanwhile, E. coli JM109SG (∆mreB/pTK-mreB-PBAD:sulA/pBHR68) was further enlarged by over-expression of sulA gene to inhibit cell division FtsZ ring assembly, generating filamentary E. coli with larger internal space for PHA accumulation. As a result, more than 200% increases on poly (3-hydroxybutyrate) (PHB) contents and cell dry weights (CDW) were achieved. As observed, elongated cells of E. coli JM109SG (∆mreB/pTK-mreB-PBAD:sulA/pBHR68) precipitated to the bottom of the test tube after only 30 minutes compared with the control of E. coli JM109SG (pBHR68) induced with arabinose. MreB is the actin-like MreB cytoskeleton and most of the studies on this protein refer to cell morphology. This is the first example on PHB production related to cell shapes, prompting a fundamental rethink of the relationship between cell shape and inclusion body production. Because intracellular PHA accumulation increased in mreB mutant E. coli with a larger shape, it is likely that our findings are also valid for many other cellular inclusions.