# Engineering Kluyveromyces marxianus for 3-hydroxypropionic acid production at elevated temperature from Jerusalem artichoke tubers and crude glycerol

## TL;DR

Scientists engineered a heat-tolerant yeast to efficiently produce a key chemical for biodegradable plastics from plant material and waste glycerol at high temperatures.

## Contribution

The novel contribution is the first thermotolerant yeast-based bioprocess for 3-HP production from non-food feedstocks at elevated temperatures.

## Key findings

- Engineered yeast produced 32.31 g/L of 3-HP at 42°C from Jerusalem artichoke tubers.
- 3-HP titer reached 33.15 g/L using pure glycerol at 42°C with optimized metabolic pathways.
- The process reduced cooling water use by 60% and CO2 emissions by 27.1 tons annually per 1000-ton fermenter.

## Abstract

This study engineered the thermotolerant yeast Kluyveromyces marxianus to produce 3-hydroxypropionic acid (3-HP), a key precursor for biodegradable plastics, via the malonyl-CoA pathway using non-food feedstocks. The 3-HP titer was further increased through deleting Adh2A and Ach1, which prevents the synthesis of byproducts ethanol and acetic acid. Using Jerusalem artichoke tuber powder, engineered strain produced 27.32 and 32.31 g/L of 3-HP at 37 °C and 42 °C through fed-batch fermentation. Metabolic reconstruction replaced the native FADH2-dependent glycerol pathway (GUT1/GUT2) with an NADH-generating GDH1/DAK1 pathway, significantly enhancing glycerol utilization and increasing intracellular NADH supply by 62 %. Overexpression of Utr1 can further enhance the NADPH supply. Combined with heterologous expression of a codon-optimized, high-activity malonyl-CoA reductase (MCR) mutant (MCRN940V/K1106W/S1114R), the engineered strain achieved 3-HP titers of 33.15 g/L in fed-batch fermentation using pure glycerol at 42 °C. Crucially, it also produced 26.57 g/L 3-HP directly from crude glycerol at 42 °C. The thermotolerant fermentation at 42 °C, unprecedented for yeast-based 3-HP synthesis, reduces cooling water consumption by approximately 60 %, translating to an estimated annual CO2 reduction of 27.1 tons per 1000-ton fermenter. This work establishes a cost-effective, industrially scalable bioprocess for valorizing Jerusalem artichoke tubers and crude glycerol into a key platform chemical for biodegradable plastics and green chemicals, leveraging the strain's substrate flexibility, process robustness, and significant environmental advantages.