Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus

Shuang Deng; Joonhoon Kim; Kyle R Pomraning; Yuqian Gao; James E Evans; Beth A Hofstad; Ziyu Dai; Bobbie-Jo Webb-Robertson; Samantha M Powell; Irina V Novikova; Nathalie Munoz; Young-Mo Kim; Marie Swita; Ana L Robles; Teresa Lemmon; Rylan D Duong; Carrie Nicora; Kristin E Burnum-Johnson; Jon Magnuson

doi:10.1016/j.ymben.2023.09.011

Identification of a specific exporter that enables high production of aconitic acid in Aspergillus pseudoterreus

Metab Eng. 2023 Nov:80:163-172. doi: 10.1016/j.ymben.2023.09.011. Epub 2023 Sep 29.

Authors

Shuang Deng¹, Joonhoon Kim², Kyle R Pomraning³, Yuqian Gao⁴, James E Evans⁵, Beth A Hofstad⁶, Ziyu Dai⁷, Bobbie-Jo Webb-Robertson⁸, Samantha M Powell⁹, Irina V Novikova¹⁰, Nathalie Munoz¹¹, Young-Mo Kim¹², Marie Swita¹³, Ana L Robles¹⁴, Teresa Lemmon¹⁵, Rylan D Duong¹⁶, Carrie Nicora¹⁷, Kristin E Burnum-Johnson¹⁸, Jon Magnuson¹⁹

Affiliations

¹ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: shuang.deng@pnnl.gov.
² DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: joonhoon.kim@pnnl.gov.
³ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: kyle.pomraning@pnnl.gov.
⁴ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: Yuqian.gao@pnnl.gov.
⁵ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: James.Evans@pnnl.gov.
⁶ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: beth.hofstad@pnnl.gov.
⁷ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: ziyu.dai@pnnl.gov.
⁸ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: bobbie-jo.webb-robertson@pnnl.gov.
⁹ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: samantha.powell@pnnl.gov.
¹⁰ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: irina.novikova@pnnl.gov.
¹¹ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: nathalie.munozmunoz@pnnl.gov.
¹² DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: young-mo.kim@pnnl.gov.
¹³ Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: marie.swita@pnnl.gov.
¹⁴ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: ana.robles@pnnl.gov.
¹⁵ Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: Teresa.lemmon@pnnl.gov.
¹⁶ Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: rylan.duong@pnnl.gov.
¹⁷ Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: carrie.nicora@pnnl.gov.
¹⁸ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA. Electronic address: Kristin.burnum-johnson@pnnl.gov.
¹⁹ DOE Agile Biofoundry, Emeryville, CA, 94608, USA; Energy and Environment Directorate, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.

PMID: 37778408
DOI: 10.1016/j.ymben.2023.09.011

Abstract

Aconitic acid is an unsaturated tricarboxylic acid that is attractive for its potential use in manufacturing biodegradable and biocompatible polymers, plasticizers, and surfactants. Previously Aspergillus pseudoterreus was engineered as a platform to produce aconitic acid by deleting the cadA (cis-aconitic acid decarboxylase) gene in the itaconic acid biosynthetic pathway. In this study, the aconitic acid transporter gene (aexA) was identified using comparative global discovery proteomics analysis between the wild-type and cadA deletion strains. The protein AexA belongs to the Major Facilitator Superfamily (MFS). Deletion of aexA almost abolished aconitic acid secretion, while its overexpression led to a significant increase in aconitic acid production. Transportation of aconitic acid across the plasma membrane is a key limiting step in its production. In vitro, proteoliposome transport assay further validated AexA's function and substrate specificity. This research provides new approaches to efficiently pinpoint and characterize exporters of fungal organic acids and accelerate metabolic engineering to improve secretion capability and lower the cost of bioproduction.

Keywords: Aconitic acid (AA); Aconitic acid exporter (AexA); Aspergillus pseudoterreus; Comparative global discovery proteomics; Major facilitator superfamily (MFS); Proteoliposome.

MeSH terms

Aconitic Acid* / metabolism
Aspergillus* / genetics
Aspergillus* / metabolism
Membrane Transport Proteins / genetics
Metabolic Engineering
Succinates / metabolism

Substances

Aconitic Acid
Membrane Transport Proteins
Succinates

Supplementary concepts

Aspergillus pseudoterreus