Control of Cell Size and Nutrient Utilization by a Novel Regulator <i>mraZ</i> and Its Impact on Polyhydroxybutyrate (PHB) Synthesis in <i>Cupriavidus necator</i> H16

<i>Cupriavidus necator</i> is a well-established microbial platform for polyhydroxyalkanoate (PHA) production and numerous metabolic engineering strategies have been applied to improve its performance. However, little is known of the relationship between cell size and the production of the poly-3-hydroxybutyrate (PHB), with studies mainly focusing on cell division markers such as <i>ftsZ</i>, <i>mreB</i>, and <i>minCD</i>. These genes are influenced by multiple regulatory factors, though the specific regulators have not been characterized. This study investigated the role of a novel regulatory gene <i>mraZ</i>, which is located upstream of <i>ftsZ</i> and acts as a transcriptional repressor of cell growth and division, in PHA production by <i>C. necator</i>. We constructed an <i>mraZ-</i>deleted strain to assess the physiological and metabolic changes. qRT-PCR revealed the upregulated expression of <i>ftsZ</i> and PHA biosynthesis genes, along with ∼20% higher cell growth and PHA accumulation in the mutant across various culture conditions. Simultaneously, the cell length was reduced by >5 times that of the wild type, as confirmed by scanning and transmission electron microscopy. Although the smaller cell size increased the surface-to-volume ratio and enhanced the sugar utilization efficiency, it did not alter the molecular weight or mechanical properties of PHB. These findings highlight the value of targeting upstream regulators such as <i>mraZ</i> and provide new insights into the significance of reduced cell size in relation to PHB production. Considering that regulators constitute ∼12% of all genes in <i>C. necator</i>, our results highlight the untapped potential of regulator-based molecular engineering for optimizing PHA production.