Heterologous integration-assisted metabolic engineering in Escherichia coli for elevated D-pantothenic acid production

Abstract

D-pantothenic acid (D-PA) is a vital water-soluble vitamin with diverse industrial applications, driving the demand for efficient microbial production. Here, we rationally engineered an Escherichia coli strain to enhance D-PA production through metabolic engineering. First, to enhance carbon utilization efficiency, competing byproduct pathways were deleted and the pentose phosphate pathway was downregulated. Next, the glucose and β-alanine transport systems were strategically enhanced, and cofactor availability was improved through engineering NADPH regeneration and ATP recycling pathways. Subsequently, pathway engineering was applied to fine-tune the expression of heterologous enzymes, thereby enhancing the metabolic pull toward D-PA biosynthesis. To enhance the supply of one-carbon donor required by the rate-limiting enzyme ketopantoate hydroxymethyltransferase (KPHMT), a heterologous 5,10-methylenetetrahydrofolate biosynthesis module was introduced. Finally, dynamic regulation of isocitrate synthase and pantothenate kinase was implemented to balance cell growth and D-PA production. As a result of the integrated metabolic engineering strategies, the final strain DPZ28/P31 achieved a D-PA titer of 98.6 g/L and a yield of 0.44 g/g glucose in a two-stage fed-batch fermentation. These findings provide valuable insights for industrial-scale production of D-PA and related compounds.

Keywords: Cofactor regeneration; D-pantothenic acid; Dynamic regulation; Escherichia coli; One-carbon donor; Substrate transport.