Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Di Liu; Hee Jin Hwang; Peter B Otoupal; Gina M Geiselman; Joonhoon Kim; Kyle R Pomraning; Young-Mo Kim; Nathalie Munoz; Carrie D Nicora; Yuqian Gao; Kristin E Burnum-Johnson; Oslo Jacobson; Samuel Coradetti; Jinho Kim; Shuang Deng; Ziyu Dai; Jan-Philip Prahl; Deepti Tanjore; Taek Soon Lee; Jon K Magnuson; John M Gladden

doi:10.1016/j.ymben.2023.05.001

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Metab Eng. 2023 Jul:78:72-83. doi: 10.1016/j.ymben.2023.05.001. Epub 2023 May 16.

Authors

Di Liu¹, Hee Jin Hwang², Peter B Otoupal³, Gina M Geiselman³, Joonhoon Kim⁴, Kyle R Pomraning⁴, Young-Mo Kim⁵, Nathalie Munoz⁵, Carrie D Nicora⁶, Yuqian Gao⁵, Kristin E Burnum-Johnson⁵, Oslo Jacobson⁷, Samuel Coradetti², Jinho Kim⁸, Shuang Deng⁴, Ziyu Dai⁴, Jan-Philip Prahl⁷, Deepti Tanjore⁷, Taek Soon Lee⁸, Jon K Magnuson⁹, John M Gladden¹⁰

Affiliations

¹ Biomanufacturing and Biomaterials Department, Sandia National Laboratories, Livermore, CA, USA; Agile BioFoundry, Department of Energy, Emeryville, CA, USA. Electronic address: diliu@lbl.gov.
² Biomanufacturing and Biomaterials Department, Sandia National Laboratories, Livermore, CA, USA; Agile BioFoundry, Department of Energy, Emeryville, CA, USA.
³ Biomanufacturing and Biomaterials Department, Sandia National Laboratories, Livermore, CA, USA; Agile BioFoundry, Department of Energy, Emeryville, CA, USA; DOE Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA.
⁴ Agile BioFoundry, Department of Energy, Emeryville, CA, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
⁵ Agile BioFoundry, Department of Energy, Emeryville, CA, USA; Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
⁶ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
⁷ Agile BioFoundry, Department of Energy, Emeryville, CA, USA; Advanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁸ DOE Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; Biological Systems and Engineering, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁹ Agile BioFoundry, Department of Energy, Emeryville, CA, USA; DOE Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, USA.
¹⁰ Biomanufacturing and Biomaterials Department, Sandia National Laboratories, Livermore, CA, USA; Agile BioFoundry, Department of Energy, Emeryville, CA, USA; DOE Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA. Electronic address: jmgladden@lbl.gov.

PMID: 37201565
DOI: 10.1016/j.ymben.2023.05.001

Abstract

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4 g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future.

Keywords: 3-Hydroxypropionic acid; 3-Hydroxypropionic acid transporter; Lignocellulosic hydrolysate; Malonyl-CoA reductase; R. toruloides.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Lignin* / metabolism
Metabolic Engineering* / methods

Substances

lignocellulose
hydracrylic acid
Lignin

Supplementary concepts

Rhodotorula toruloides