3-Hydroxypropionic acid is an important malonyl-CoA-derived platform chemical whose efficient biosynthesis is constrained by limited utilization of malonyl-CoA across subcellular compartments. Using a biosensor-guided transcription factor mutagenesis screen combined with transcriptomic and functional validation, we identify four positive mutants NRG1_R224H, STB3_L52Y, PDR1_T820C and PGD1_V243D that increase cytosolic malonyl-CoA accumulation by globally reprogramming transcription to reinforce central carbon flux and acetyl-CoA precursor supply, and remodel amino acid, redox, and lipid metabolism to favor malonyl-CoA accumulation. We further utilize mitochondrial malonyl-CoA for 3-HP production through dynamic control of HFA1 and optimized POS5 expression, and further develop a dual-compartment coordination strategy to efficiently exploit cytosolic and mitochondrial malonyl-CoA pools. Integration of optimized pathways in diploid strains enables coordinated precursor utilization, achieving 81.8 g/L 3-HP in 5-L fed-batch fermentation, the highest titer reported to date in Saccharomyces cerevisiae. This work establishes a generalizable framework for multi-compartment malonyl-CoA utilization in eukaryotic cell factories.
Jing Liu, Huiping Tan, Xi Deng et al.· Metabolic Engineering· 0 citations
Abstract Base editing enables precise genome modification without double-strand breaks but remains limited by narrow editing windows, DNA repair pathway biases, and restricted nucleotide diversity. Here, we report MUTATOR, a MUlTiplexAble and self-iTerative ORthogonal base-editing platform that enables N-to-N diversification in Escherichia coli. MUTATOR combines CWBE and ABE with iterative editing on two complementary DNA strands, thereby overcoming endogenous DNA repair constraints and expanding A-to-N and C-to-N editing outcomes across both strands. This strategy substantially expands accessible nucleotide outcomes, codon variants, and amino-acid diversity within existing editing windows relative to conventional editors. Using four gRNAs, MUTATOR facilitated four-site editing of ompR, generating 84 distinct amino-acid combinations and 252 codon combinations, with the synonymous OmpR_P160P variant increasing isobutanol production by up to 56.2%. We further applied MUTATOR to a 151-gene library encompassing transcriptional regulators, translation factors, DNA repair proteins, ribosomal components, and NAD(P)H-associated metabolic genes, identifying single and combinatorial mutations that markedly enhanced cell growth and ethanol utilization when ethanol was used as the sole carbon source. Together, these results establish MUTATOR as a broadly applicable platform for genome-wide diversification, functional dissection, and rapid engineering of industrial microbial chassis.
Xiangrui Fan, Liya Liang, Hongle Wang et al.· Nucleic Acids Research· 0 citations
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