(465g) (Keynote) Towards Efficient Bioproduction of Polymalic Acid and Malic Acid Production: System Metabolic and Process Engineering

Polymalic acid (PMA) is a novel polyester polymer that has been broadly used in the medical and food industries. Its monomer, L-malic acid (MA), is also a potential C4 platform chemical. Recently, great researches have been attempted to develop biotechnological process for PMA and L-malic acid production. Here, we integrated multi-omics, genome-scale metabolic model and genome-editing technique to engineer the metabolic nodes for efficient PMA production. Reductive TCA cycle and glyoxylate shunt were verified that could strengthen carbon flux for PMA and MA synthesis. Subsequently, some nodes, including pyruvate carboxylase gene (pyc), malate synthase gene (mls), isocitrate lyase gene (icl), and carbon response transcription factor cat8 gene, were overexpressed, respectively. Among of them, the OE::mls-icl-cat8 strain was showed the greatest PMA titer and yield (37.94g/L and 0.45g/g glucose) in shake-flask, and increased by 26% and 25% compared with the wild type strain, respectively. CRISPR/Cas9 system was successfully developed and applied as a simple and powerful tool for targeted gene knock-out and/or knock-in A. pullulans. Some renewable biomass feedstocks, i.e. sugarcane molasses and xylose mother liquor, were also presented.