Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus

Abstract

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.

The survivor strain: isolation and characterization of Phormidium yuhuli AB48, a filamentous phototactic cyanobacterium with biotechnological potential

“…Metabolomics : The metabolite fraction was dried under a vacuum to remove moisture and was chemically derivatized and analyzed as previously reported ( Kim et al, 2015 ; Pomraning et al, 2021 ). Briefly, the extracted metabolites were derivatized by methoxyamination and trimethylsilyation (TMS), and then the samples were analyzed by GC–MS.…”

Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production

“…A second set of extracellular metabolites were quantified from 20 μl of spent media that were dried under vacuum. Chemical derivatization, analysis by GC-MS (same instrument as citation), and data processing was done as previously described ( Pomraning et al, 2021 ). Glucose content was determined via enzymatic kit (R-Biopharm, Darmstadt, Germany) per manufacturer's instructions.…”

Itaconic acid production is regulated by LaeA in Aspergillus pseudoterreus

“…The alignments were sorted, converted to bam format, and quantified with Samtools v1.3.1 ( Li, 2011 ). For global extracellular metabolomics analysis, supernatants were dried, chemically derivatized, acquired and analyzed as previously reported ( Pomraning et al, 2021 ). Specific metabolites were also quantified using a GC-MS with external calibration curves corresponding to authentic chemical standards as described previously ( Pomraning et al, 2021 ).…”

“…The refractive index at 45C and the UV absorption at 210 nm were measured. Targeted and global proteomics was performed as previously described ( Pomraning et al, 2021 ).…”