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

Dai, Ziyu; Pomraning, Kyle R.; Deng, Shuang; Kim, Joonhoon; Campbell, Kristen B.; Robles, Ana L.; Hofstad, Beth A.; Munoz, Nathalie Munoz; Gao, Yuqian; Lemmon, Teresa; Swita, Marie S.; Zucker, Jeremy D.; Kim, Young-Mo; Burnum-Johnson, Kristin E.; Magnuson, Jon K.

doi:10.1186/s13068-023-02288-1

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

Journal Article · Wed Mar 29 00:00:00 EDT 2023 · Biotechnology for Biofuels and Bioproducts

DOI:https://doi.org/10.1186/s13068-023-02288-1· OSTI ID:1963950

Dai, Ziyu ^[1]; ^[1]; Deng, Shuang ^[2]; ^[1]; Campbell, Kristen B. ^[3]; Robles, Ana L. ^[1]; Hofstad, Beth A. ^[1]; Munoz, Nathalie Munoz ^[1]; Gao, Yuqian ^[1]; Lemmon, Teresa ^[3]; Swita, Marie S. ^[3]; ^[1]; ^[1]; ^[1]; ^[1]

USDOE Agile BioFoundry, Emeryville, CA (United States); Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
USDOE Agile BioFoundry, Emeryville, CA (United States)Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

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 base 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.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

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

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1963950

Report Number(s):: PNNL-SA-182738

Journal Information:: Biotechnology for Biofuels and Bioproducts, Journal Name: Biotechnology for Biofuels and Bioproducts Journal Issue: 1 Vol. 16; ISSN 2731-3654

Publisher:: BioMed CentralCopyright Statement

Country of Publication:: United States

Language:: English

References (61)

Metabolic engineering of 3-hydroxypropionic acid biosynthesis in Escherichia coli : Biosynthesis of 3-Hydroxypropionic Acid Chu, Hun Su; Kim, Young Soo; Lee, Chan Mu Biotechnology and Bioengineering, Vol. 112, Issue 2 https://doi.org/10.1002/bit.25444	journal	October 2014
The Renewable Chemicals Industry Christensen, Claus Hviid; Rass-Hansen, Jeppe; Marsden, Charlotte C. ChemSusChem, Vol. 1, Issue 4 https://doi.org/10.1002/cssc.200700168	journal	April 2008
Extremely rapid acclimation of Escherichia coli to high temperature over a few generations of a fed‐batch culture during slow warming Guyot, Stéphane; Pottier, Laurence; Hartmann, Alain MicrobiologyOpen, Vol. 3, Issue 1 https://doi.org/10.1002/mbo3.146	journal	December 2013
Chloroflexus aurantiacus secretes 3-hydroxypropionate, a possible intermediate in the assimilation of CO₂ and acetate Holo, Helge Archives of Microbiology, Vol. 151, Issue 3, p. 252-256 https://doi.org/10.1007/BF00413138	journal	February 1989
Aspergillus niger citric acid accumulation: do we understand this well working black box? Karaffa, Levente; Kubicek, Christian P. Applied Microbiology and Biotechnology, Vol. 61, Issue 3 https://doi.org/10.1007/s00253-002-1201-7	journal	January 2003
Biosynthetic pathways for 3-hydroxypropionic acid production Jiang, Xinglin; Meng, Xin; Xian, Mo Applied Microbiology and Biotechnology, Vol. 82, Issue 6 https://doi.org/10.1007/s00253-009-1898-7	journal	February 2009
Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terreus Okabe, Mitsuyasu; Lies, Dwiarti; Kanamasa, Shin Applied Microbiology and Biotechnology, Vol. 84, Issue 4 https://doi.org/10.1007/s00253-009-2132-3	journal	July 2009
A deficiency of manganese ions in the presence of high sugar concentrations is the critical parameter for achieving high yields of itaconic acid by Aspergillus terreus Karaffa, Levente; Díaz, Rafael; Papp, Benedek Applied Microbiology and Biotechnology, Vol. 99, Issue 19 https://doi.org/10.1007/s00253-015-6735-6	journal	June 2015
Deletion analysis of the itaconic acid biosynthesis gene cluster components in Aspergillus pseudoterreus ATCC32359 Deng, Shuang; Dai, Ziyu; Swita, Marie Applied Microbiology and Biotechnology, Vol. 104, Issue 9 https://doi.org/10.1007/s00253-020-10418-0	journal	March 2020
Microbial production of 1,3-propanediol Biebl, H.; Menzel, K.; Zeng, A. -P. Applied Microbiology and Biotechnology, Vol. 52, Issue 3 https://doi.org/10.1007/s002530051523	journal	September 1999
Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA Kobayashi, Keiichi; Hattori, Takasumi; Honda, Yuki Journal of Industrial Microbiology and Biotechnology, Vol. 41, Issue 5 https://doi.org/10.1007/s10295-014-1419-2	journal	May 2014
Enhanced production of β-alanine through co-expressing two different subtypes of l-aspartate-α-decarboxylase Wang, Lei; Piao, Xiaoyu; Cui, Shumei Journal of Industrial Microbiology and Biotechnology, Vol. 47, Issue 6-7 https://doi.org/10.1007/s10295-020-02285-5	journal	July 2020
A Novel NAD+-dependent aldehyde dehydrogenase encoded by the puuC gene of Klebsiella pneumoniae DSM 2026 that utilizes 3-hydroxypropionaldehyde as a substrate Raj, Subramanian Mohan; Rathnasingh, Chelladurai; Jung, Woo-Chel Biotechnology and Bioprocess Engineering, Vol. 15, Issue 1 https://doi.org/10.1007/s12257-010-0030-2	journal	February 2010
Breeding of Aspergillus terreus mutant TN-484 for itaconic acid production with high yield Yahiro, Kazutoyo; Takahama, Tetsushi; Park, Young Soo Journal of Fermentation and Bioengineering, Vol. 79, Issue 5 https://doi.org/10.1016/0922-338X(95)91272-7	journal	January 1995
Growth Media Bennett, J. W.; Lasure, Linda L. More Gene Manipulations in Fungi, 441-458 https://doi.org/10.1016/B978-0-12-088642-5.50027-2	book	January 1991
Effect of pH and stirring rate on itaconate production by Aspergillus terreus Riscaldati, Emanuele; Moresi, Mauro; Petruccioli, Maurizio Journal of Biotechnology, Vol. 83, Issue 3 https://doi.org/10.1016/S0168-1656(00)00322-9	journal	October 2000
Simultaneous conversion of glucose and xylose to 3-hydroxypropionic acid in engineered Escherichia coli by modulation of sugar transport and glycerol synthesis Jung, In-Young; Lee, Jong-Won; Min, Won-Ki Bioresource Technology, Vol. 198 https://doi.org/10.1016/j.biortech.2015.09.079	journal	December 2015
Enhanced production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway by engineered Escherichia coli Cheng, Zhuan; Jiang, Jiaqi; Wu, Hui Bioresource Technology, Vol. 200 https://doi.org/10.1016/j.biortech.2015.10.107	journal	January 2016
Enhanced production of 3-hydroxypropionic acid from glucose and xylose by alleviation of metabolic congestion due to glycerol flux in engineered Escherichia coli Heo, Woong; Kim, Jun Hee; Kim, Sooah Bioresource Technology, Vol. 285 https://doi.org/10.1016/j.biortech.2019.121320	journal	August 2019
Improved production of 3-hydroxypropionic acid in engineered Escherichia coli by rebalancing heterologous and endogenous synthetic pathways Lee, Tae-Young; Min, Won-Ki; Kim, Hyo Jin Bioresource Technology, Vol. 299 https://doi.org/10.1016/j.biortech.2019.122600	journal	March 2020
Recent advances in biological production of 3-hydroxypropionic acid Kumar, Vinod; Ashok, Somasundar; Park, Sunghoon Biotechnology Advances, Vol. 31, Issue 6 https://doi.org/10.1016/j.biotechadv.2013.02.008	journal	November 2013
Identification of the gene encoding hydroxyacid-oxoacid transhydrogenase, an enzyme that metabolizes 4-hydroxybutyrate Kardon, Tamas; Noël, Gaëtane; Vertommen, Didier FEBS Letters, Vol. 580, Issue 9 https://doi.org/10.1016/j.febslet.2006.02.082	journal	March 2006
Impact of alg3 gene deletion on growth, development, pigment production, protein secretion, and functions of recombinant Trichoderma reesei cellobiohydrolases in Aspergillus niger Dai, Ziyu; Aryal, Uma K.; Shukla, Anil Fungal Genetics and Biology, Vol. 61 https://doi.org/10.1016/j.fgb.2013.09.004	journal	December 2013
Production of 3-hydroxypropionic acid via the malonyl-CoA pathway using recombinant fission yeast strains Suyama, Akiko; Higuchi, Yujiro; Urushihara, Masahiro Journal of Bioscience and Bioengineering, Vol. 124, Issue 4 https://doi.org/10.1016/j.jbiosc.2017.04.015	journal	October 2017
Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains Rathnasingh, Chelladurai; Raj, Subramanian Mohan; Lee, Youjin Journal of Biotechnology, Vol. 157, Issue 4, p. 633-640 https://doi.org/10.1016/j.jbiotec.2011.06.008	journal	February 2012
1,2 Propanediol utilization by Lactobacillus reuteri DSM 20016, role in bioconversion of glycerol to 1,3 propanediol, 3-hydroxypropionaldehyde and 3-hydroxypropionic acid Amin, Heba M.; Hashem, Abdelgawad M.; Ashour, Mohamed S. Journal of Genetic Engineering and Biotechnology, Vol. 11, Issue 1 https://doi.org/10.1016/j.jgeb.2012.12.002	journal	June 2013
A Second Pathway to Degrade Pyrimidine Nucleic Acid Precursors in Eukaryotes Andersen, Gorm; Björnberg, Olof; Polakova, Silvia Journal of Molecular Biology, Vol. 380, Issue 4 https://doi.org/10.1016/j.jmb.2008.05.029	journal	July 2008
Production of 3-hydroxypropionic acid from glucose and xylose by metabolically engineered Saccharomyces cerevisiae Kildegaard, Kanchana R.; Wang, Zheng; Chen, Yun Metabolic Engineering Communications, Vol. 2 https://doi.org/10.1016/j.meteno.2015.10.001	journal	December 2015
Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine Borodina, Irina; Kildegaard, Kanchana R.; Jensen, Niels B. Metabolic Engineering, Vol. 27 https://doi.org/10.1016/j.ymben.2014.10.003	journal	January 2015
Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803 Wang, Yunpeng; Sun, Tao; Gao, Xingyan Metabolic Engineering, Vol. 34 https://doi.org/10.1016/j.ymben.2015.10.008	journal	March 2016
Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose Chen, Zhen; Huang, Jinhai; Wu, Yao Metabolic Engineering, Vol. 39 https://doi.org/10.1016/j.ymben.2016.11.009	journal	January 2017
Sustainable Production of Acrylic Acid via 3-Hydroxypropionic Acid from Lignocellulosic Biomass Bhagwat, Sarang S.; Li, Yalin; Cortés-Peña, Yoel R. ACS Sustainable Chemistry & Engineering, Vol. 9, Issue 49 https://doi.org/10.1021/acssuschemeng.1c05441	journal	November 2021
Double-slit photoelectron interference in strong-field ionization of the neon dimer Kunitski, Maksim; Eicke, Nicolas; Huber, Pia Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-018-07882-8	journal	January 2019
Dynamic ice loss from the Greenland Ice Sheet driven by sustained glacier retreat King, Michalea D.; Howat, Ian M.; Candela, Salvatore G. Communications Earth & Environment, Vol. 1, Issue 1 https://doi.org/10.1038/s43247-020-0001-2	journal	August 2020
High Production of 3-Hydroxypropionic Acid in Klebsiella pneumoniae by Systematic Optimization of Glycerol Metabolism Li, Ying; Wang, Xi; Ge, Xizhen Scientific Reports, Vol. 6, Issue 1 https://doi.org/10.1038/srep26932	journal	May 2016
DMR (deacetylation and mechanical refining) processing of corn stover achieves high monomeric sugar concentrations (230 g L ⁻¹ ) during enzymatic hydrolysis and high ethanol concentrations (>10% v/v) during fermentation without hydrolysate purification or concentration Chen, Xiaowen; Kuhn, Erik; Jennings, Edward W. Energy & Environmental Science, Vol. 9, Issue 4 https://doi.org/10.1039/C5EE03718B	journal	January 2016
A previously undescribed pathway for pyrimidine catabolism Loh, K. D.; Gyaneshwar, P.; Markenscoff Papadimitriou, E. Proceedings of the National Academy of Sciences, Vol. 103, Issue 13 https://doi.org/10.1073/pnas.0600521103	journal	March 2006
Candida albicans Utilizes a Modified β-Oxidation Pathway for the Degradation of Toxic Propionyl-CoA* Otzen, Christian; Bardl, Bettina; Jacobsen, Ilse D. Journal of Biological Chemistry, Vol. 289, Issue 12 https://doi.org/10.1074/jbc.M113.517672	journal	March 2014
The Sequence Alignment/Map format and SAMtools Li, H.; Handsaker, B.; Wysoker, A. Bioinformatics, Vol. 25, Issue 16 https://doi.org/10.1093/bioinformatics/btp352	journal	June 2009
CNVnator: An approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing Abyzov, A.; Urban, A. E.; Snyder, M. Genome Research, Vol. 21, Issue 6 https://doi.org/10.1101/gr.114876.110	journal	February 2011
Poly(3-Hydroxypropionate): a Promising Alternative to Fossil Fuel-Based Materials Andreeßen, Björn; Taylor, Nicolas; Steinbüchel, Alexander Applied and Environmental Microbiology, Vol. 80, Issue 21 https://doi.org/10.1128/AEM.02361-14	journal	August 2014
Identification of Genes Associated with Morphology in Aspergillus niger by Using Suppression Subtractive Hybridization Dai, Z.; Mao, X.; Magnuson, J. K. Applied and Environmental Microbiology, Vol. 70, Issue 4 https://doi.org/10.1128/AEM.70.4.2474-2485.2004	journal	April 2004
Utilization of Glycerol as a Hydrogen Acceptor by Lactobacillus reuteri: Purification of 1,3-Propanediol:NAD+ Oxidoreductase † Talarico, Todd L.; Axelsson, Lars T.; Novotny, James Applied and Environmental Microbiology, Vol. 56, Issue 4 https://doi.org/10.1128/aem.56.4.943-948.1990	journal	January 1990
MPLEx: a Robust and Universal Protocol for Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses Nakayasu, Ernesto S.; Nicora, Carrie D.; Sims, Amy C. mSystems, Vol. 1, Issue 3 https://doi.org/10.1128/mSystems.00043-16	journal	May 2016
A highly efficient dilute alkali deacetylation and mechanical (disc) refining process for the conversion of renewable biomass to lower cost sugars Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-98	journal	January 2014
Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway Kildegaard, Kanchana R.; Jensen, Niels B.; Schneider, Konstantin Microbial Cell Factories, Vol. 15, Issue 1 https://doi.org/10.1186/s12934-016-0451-5	journal	March 2016
Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe Takayama, Seiya; Ozaki, Aiko; Konishi, Rie Microbial Cell Factories, Vol. 17, Issue 1 https://doi.org/10.1186/s12934-018-1025-5	journal	November 2018
Techno-economic analysis of the deacetylation and disk refining process: characterizing the effect of refining energy and enzyme usage on minimum sugar selling price and minimum ethanol selling price Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas Biotechnology for Biofuels, Vol. 8, Issue 1 https://doi.org/10.1186/s13068-015-0358-0	journal	October 2015
A kinetic model of the central carbon metabolism for acrylic acid production in Escherichia coli Oliveira, Alexandre; Rodrigues, Joana; Ferreira, Eugénio Campos PLOS Computational Biology, Vol. 17, Issue 3 https://doi.org/10.1371/journal.pcbi.1008704	journal	March 2021
Exploring Lactobacillus reuteri DSM20016 as a biocatalyst for transformation of longer chain 1,2-diols: Limits with microcompartment Chen, Lu; Hatti-Kaul, Rajni PLOS ONE, Vol. 12, Issue 9 https://doi.org/10.1371/journal.pone.0185734	journal	September 2017
Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system Wu, Yu-Wei; Yang, Shih-Hung; Hwangbo, Myung PLOS ONE, Vol. 14, Issue 9 https://doi.org/10.1371/journal.pone.0222143	journal	September 2019
Integration of Proteomics and Metabolomics Into the Design, Build, Test, Learn Cycle to Improve 3-Hydroxypropionic Acid Production in Aspergillus pseudoterreus Pomraning, Kyle R.; Dai, Ziyu; Munoz, Nathalie Frontiers in Bioengineering and Biotechnology, Vol. 9 https://doi.org/10.3389/fbioe.2021.603832	journal	April 2021
Diel metabolomics analysis of a hot spring chlorophototrophic microbial mat leads to new hypotheses of community member metabolisms Kim, Young-Mo; Nowack, Shane; Olsen, Millie T. Frontiers in Microbiology, Vol. 6 https://doi.org/10.3389/fmicb.2015.00209	journal	April 2015
Metabolic Engineering of Yeast for the Production of 3-Hydroxypropionic Acid Ji, Rong-Yu; Ding, Ying; Shi, Tian-Qiong Frontiers in Microbiology, Vol. 9 https://doi.org/10.3389/fmicb.2018.02185	journal	September 2018
Biological Production of 3-Hydroxypropionic Acid: An Update on the Current Status Matsakas, Leonidas; Hrůzová, Kateřina; Rova, Ulrika Fermentation, Vol. 4, Issue 1 https://doi.org/10.3390/fermentation4010013	journal	February 2018
The Role of Metal Ions in Fungal Organic Acid Accumulation Karaffa, Levente; Fekete, Erzsébet; Kubicek, Christian P. Microorganisms, Vol. 9, Issue 6 https://doi.org/10.3390/microorganisms9061267	journal	June 2021
Production of 3-Hydroxypropionic Acid from Acrylic Acid by Newly Isolated Rhodococcus erythropolis LG12 Lee, Sang-Hyun, Journal of Microbiology and Biotechnology, Vol. 19, Issue 5 https://doi.org/10.4014/jmb.0808.473	journal	May 2009
Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM Li, Heng arXiv https://doi.org/10.48550/arXiv.1303.3997	preprint	January 2013
Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway Kildegaard, Kanchana; Jensen, Niels; Schneider, Konstantin figshare https://doi.org/10.6084/m9.figshare.c.3629507	collection	January 2016
Techno-economic analysis of the deacetylation and disk refining process: characterizing the effect of refining energy and enzyme usage on minimum sugar selling price and minimum ethanol selling price [Supplementary Data] Chen, Xiaowen; Shekiro, Joseph; Pschorn, Thomas Figshare-Supplementary information for journal article at DOI: 10.1186/s13068-015-0358-0, 1 docx file (37.57 kB) https://doi.org/10.6084/m9.figshare.c.3629957	dataset	October 2015
Enhancing 3-hydroxypropionic acid production in combination with sugar supply engineering by cell surface-display and metabolic engineering of Schizosaccharomyces pombe Takayama, Seiya; Ozaki, Aiko; Konishi, Rie Figshare https://doi.org/10.6084/m9.figshare.c.4300535.v1	collection	January 2018