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.