Multistrategic Engineering of <i>Escherichia coli</i> for High-Level Production of 3,4-Dihydroxybutyric Acid

3,4-Dihydroxybutyric acid (3,4-DHBA) is an important precursor for the synthesis of high-value fine chemicals; however, its sustainable production in microbial hosts is limited by plasmid instability and low titers. In this study, we aimed to construct a chromosome-integrated <i>E. coli</i> platform for plasmid-free and inducer-free biosynthesis of 3,4-DHBA from xylose and glycerol. First, preliminary pathway reconstruction through elimination of competing routes and genomic integration of key xylose dehydrogenase increased 3,4-DHBA accumulation to 1.30 g/L. Subsequently, systematic enzyme screening identified optimal aldehyde dehydrogenase and xylonate dehydratase, and an NAD⁺ regeneration module was introduced to strengthen redox cofactor cycling for enhanced 3,4-DHBA production. The successful plasmid- and inducer-free biosynthesis of 3,4-DHBA was accomplished by integrating all pathway genes into the genome. Furthermore, transcriptome analysis revealed that xylose transporters were unrecognized metabolic bottlenecks, and their targeted overexpression significantly improved xylose uptake and 3,4-DHBA flux. The final strain achieved a titer of 3.08 g/L in shake flask cultivation and 46.10 g/L in fed-batch fermentation with a yield of 0.49 g/g xylose and a productivity of 0.92 g/(L·h), which are the highest values reported to date. This integrated strategy establishes a scalable and cost-effective route for 3,4-DHBA production, highlighting the value of combining pathway engineering with transportation optimization in biocatalyst design and enabling lignocellulosic biomass hydrolyzates as substrates for 3,4-DHBA production.