Balancing dual-enzyme expression and synergistically engineering of 4-hydroxyphenylpyruvate dioxygenase for efficient 3-hydroxy-3-methylbutyrate biosynthesis by whole-cell catalysis

3-Hydroxy-3-methylbutyrate (HMB), a key L-leucine metabolite, alters protein metabolic balance during the growth of new muscle tissue and exhibits anti-catabolic effects, garnering widespread attention as a dietary supplement for sarcopenia treatment and sports training. Here, we identified an efficient 4-hydroxyphenylpyruvate dioxygenase from Allokutzneria albata ( Aa HPPD) via molecular docking and whole-cell reaction yield verification. By coupling this enzyme with an efficient L-amino acid deaminase derived from Proteus vulgaris ( Pv LAAD) for whole-cell biocatalysis, 8.66 mM HMB was produced from 25 mM L-leucine, achieving a molar conversion of 34.6%. To coordinate the expression of pathway enzymes, ribosome binding site combinations were optimized, increasing the molar conversion to 56.5%. Additionally, for the rate-limiting enzyme Aa HPPD, distal mutations enhancing thermostability were identified using the Protein Repair One Stop Shop server and Disulfide by Design 2. The co-expression of the double mutant Aa HPPD Q65L/E99D and Pv LAAD resulted in a 61.24% conversion rate. To further improve catalytic activity, four sites identified via alanine scanning were subjected to grouped saturation mutagenesis based on spatial location. The final strain, co-expressing Aa HPPD Q65L/E99D/L260I/V306I and Pv LAAD, achieved a molar conversion of 73.73% for HMB production using 25 mM L-Leu as substrates within 10 h under optimal whole-cell conditions. This study established an efficient method for HMB production via whole-cell catalysis and provided a feasible strategy for engineering the thermostability and activity of the rate-limiting enzyme, as well as synergistically optimizing the expression levels of cascade enzymes.