- Pomraning, Kyle R;
- Dai, Ziyu;
- Munoz, Nathalie;
- Kim, Young-Mo;
- Gao, Yuqian;
- Deng, Shuang;
- Kim, Joonhoon;
- Hofstad, Beth A;
- Swita, Marie S;
- Lemmon, Teresa;
- Collett, James R;
- Panisko, Ellen A;
- Webb-Robertson, Bobbie-Jo M;
- Zucker, Jeremy D;
- Nicora, Carrie D;
- De Paoli, Henrique;
- Baker, Scott E;
- Burnum-Johnson, Kristin E;
- Hillson, Nathan J;
- Magnuson, Jon K
Biological engineering of microorganisms to produce value-added chemicals is a promising route to sustainable manufacturing. However, overproduction of metabolic intermediates at high titer, rate, and yield from inexpensive substrates is challenging in non-model systems where limited information is available regarding metabolic flux and its control in production conditions. Integrated multi-omic analyses of engineered strains offers an in-depth look at metabolites and proteins directly involved in growth and production of target and non-target bioproducts. Here we applied multi-omic analyses to overproduction of the polymer precursor 3-hydroxypropionic acid (3HP) in the filamentous fungus Aspergillus pseudoterreus. A synthetic pathway consisting of aspartate decarboxylase, beta-alanine pyruvate transaminase, and 3HP dehydrogenase was designed and built for A. pseudoterreus. Strains with single- and multi-copy integration events were isolated and multi-omics analysis consisting of intracellular and extracellular metabolomics and targeted and global proteomics was used to interrogate the strains in shake-flask and bioreactor conditions. Production of a variety of co-products (organic acids and glycerol) and oxidative degradation of 3HP were identified as metabolic pathways competing with 3HP production. Intracellular accumulation of nitrogen as 2,4-diaminobutanoate was identified as an off-target nitrogen sink that may also limit flux through the engineered 3HP pathway. Elimination of the high-expression oxidative 3HP degradation pathway by deletion of a putative malonate semialdehyde dehydrogenase improved the yield of 3HP by 3.4 × after 10 days in shake-flask culture. This is the first report of 3HP production in a filamentous fungus amenable to industrial scale biomanufacturing of organic acids at high titer and low pH.