(472d) Engineering Acid-Tolerant Issatchenkea Orientalis SD108 for High-Level 3-Hydroxypropionic Acid Production

3-Hydroxypropionic acid (3HP) has been identified as one of the top 12 sugar-based platform chemicals for biorefinery by the US Department of Energy. Despite numerous efforts aimed at enhancing the titers, yields and productivities of 3HP in fermentation, commercially viable biomanufacturing of 3HP has remained elusive partly due to the high cost of downstream processing associated with neutral pH fermentation. In this study, we engineered low-pH-tolerant yeast Issatchenkia orientalis SD108 for 3HP production. Through comprehensive approaches involving sequence similarity network analysis, in vitro enzymatic assays and in vivo production tests, we identified a novel set of pathway genes that demonstrated a notable 2.7-fold enhancement (i.e., from 0.9 g/L to 2.4 g/L). By exploring the copy number effect, aspartate decarboxylase (i.e., PAND) was found as the rate-limiting step. Leveraging the landing pad strain, two-round transformation enables us to get a strain with integration of 8 copies of PAND, which produced 8 g/L 3HP. Further engineering by push-pull-block strategies led to an increasement to 30 g/L 3-HP with a yield of 0.6 g-3HP/g-glucose in shake-flask fermentation. Through scaling up and optimization in fed-batch fermentation using low-cost corn steer liquor medium at pH4, we achieved 3HP production exceeding 100 g/L with a yield of 0.7 g/g. Techno-economic and life cycle analyses indicated the feasibility of our approach for achieving industrially relevant scale production of 3HP. This research represents a significant step towards the realization of large-scale industrial production of biobased 3HP.