Title: Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Abstract

Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol^-1 glucose in the basemore » strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol^-1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol^-1 sugars and resulted in a final titer of 36.0 g/L 3-HP. The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.« less

Authors:

Dai, Ziyu ^[1]; Pomraning, Kyle R.  ^[2];

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• Search DOE PAGES for ORCID "0000-0003-2324-2881"

Deng, Shuang ^[3]; Kim, Joonhoon  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-7425-1828"

Campbell, Kristen B. ^[4]; Robles, Ana L. ^[1]; Hofstad, Beth A. ^[1]; Munoz, Nathalie Munoz ^[1]; Gao, Yuqian ^[1]; Lemmon, Teresa ^[4]; Swita, Marie S. ^[4]; Zucker, Jeremy D.  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-7276-9009"

Kim, Young-Mo  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-8972-7593"

Burnum-Johnson, Kristin E.  ^[1];

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• Search DOE PAGES for ORCID "0000-0002-2722-4149"

Magnuson, Jon K.  ^[1]

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• Search DOE PAGES for ORCID "0000-0001-7712-7024"

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+ Show Author Affiliations

1. USDOE Agile BioFoundry, Emeryville, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
2. USDOE Agile BioFoundry, Emeryville, CA (United States);Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
3. USDOE Agile BioFoundry, Emeryville, CA (United States)Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
4. Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Publication Date:

Wed Mar 29 00:00:00 EDT 2023

Research Org.:

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

Sponsoring Org.:

USDOE Office of Energy Efficiency and Renewable Energy (EERE), Office of Sustainable Transportation. Bioenergy Technologies Office (BETO)

OSTI Identifier:

1963950

Report Number(s):

PNNL-SA-182738
Journal ID: ISSN 2731-3654

Grant/Contract Number:  

AC05-76RL01830; NL0030038

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology for Biofuels and Bioproducts

Additional Journal Information:

Journal Volume: 16; Journal Issue: 1; Journal ID: ISSN 2731-3654

Publisher:

BioMed Central

Country of Publication:

United States

Language:

English

Subject:

09 BIOMASS FUELS; Beta-alanine pathway, 3-hydroxypropionic acid, Aspergillus niger, Aspergillus pseudoterreus