Engineering Methylorubrum extorquens for the Biosynthesis of d-Lactic Acid from Methanol

d-lactic acid (d-LA) has garnered significant attention in the field of manufacturing thermostable poly(lactic acid) (PLA) due to its potential to enhance the thermal stability and performance of materials. However, conventional microbial fermentation for producing d-LA relying on sugar- and starch-based feedstocks may exacerbate food-security challenges. As a renewable and nonfood C1 substrate, methanol offers a sustainable alternative to address this limitation. In this study, we engineered Methylorubrum extorquens for efficient d-LA biosynthesis using methanol as the primary carbon and energy source. By combining multicopy chromosomal integration of Leuconostoc mesenteroides d-lactate dehydrogenase (d-LDH) with pyruvate flux enhancement and formate-boosted methanol metabolism, the engineered strain achieved a d-LA titer of 1.17 g/L in shake-flask cultivation. Following transcriptomic analysis revealed that the process of formaldehyde dissimilation, serine cycle, and phosphoketolase pathway plays a significant role in the high production of d-LA. Subsequent fed-batch fermentation with 0.34 g/L sodium formate supplied during the exponential growth period of the AD05 strain achieved a titer of 8.39 g/L, which represents the highest reported d-LA production from methanol feedstock. This study provides a potential route for the future biosynthesis of other pyruvate-derived biochemicals via methanol bioconversion in Methylorubrum extorquens.