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. 2022 Oct 19;8(1):33-45.
doi: 10.1016/j.synbio.2022.10.004. eCollection 2023 Mar.

The ornithine-urea cycle involves fumaric acid biosynthesis in Aureobasidium pullulans var. aubasidani, a green and eco-friendly process for fumaric acid production

Xin Wei  1 , Miao Zhang  1 , Guang-Yuan Wang  2 , Guang-Lei Liu  1   3 , Zhen-Ming Chi  1   3 , Zhe Chi  1   3
Affiliations

The ornithine-urea cycle involves fumaric acid biosynthesis in Aureobasidium pullulans var. aubasidani, a green and eco-friendly process for fumaric acid production

Xin Wei et al. Synth Syst Biotechnol. .

Abstract

The current petroleum chemical methods for fumaric acid production can cause heavy pollution and global warming. In this study, the engineered strains of A. pullulans var. aubasidani were found to be suitable for green fumaric acid producer. Removal and complementation of the relevant genes showed only the ornithine-urea cycle (OUC) was involved in high level fumarate biosynthesis which was controlled by the Ca2+ signaling pathway. Removal of both the GOX gene encoding glucose oxidase and the PKS1 gene encoding the polyketide synthase for 3,5-dihydroxydecanoic acid biosynthesis and overexpression of the PYC gene encoding pyruvate carboxylase made the strain e-PYC produce 88.1 ± 4.3 g/L of fumarate at flask level and 93.9 ± 0.8 g/L of fumarate during the fed-batch fermentation. As a yeast-like fungal strain, it was very easy to cultivate A. pullulans var. aubasidani DH177 and their mutants in the bioreactor and to edit its genomic DNAs to enhance fumarate production. It was found that 2 mol of CO2 could be fixed during a maximal theoretical yield of 2 mol of fumarate per mole of glucose consumed in the OUC. Therefore, the OUC-mediated fumarate biosynthesis pathway in A. pullulans var. aubasidani was a green and eco-friendly process for the global sustainable development and carbon neutrality.

Keywords: A. pullulans var. aubasidani; AG, N-acetyl glutamate; Arg, arginase; Ass, argininosuccinate synthase; Ast, argininosuccinate lyase; Ca2+signaling pathway; Cps, carbamoyl phosphate synthase; FA, Fumaric acid; Fumaric acid; OUC, ornithine-urea cycle; Ornithine-urea cycle; Otc, ornithine transcarbamoylase.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Fig. 1
Fig. 1
Fumaric acid biosynthesis from the OUC and TCA cycle Pyc: Pyruvate carboxylase; At: Aspatrate aminotransferase; Cps: Carbamoyl phosphate synthase; Otc: Ornithine transcarbamoylase; Ass: Argininosuccinate synthase; Asl: Argininosuccinate lyase; Arg: Arginase; Sfc: Succinate-fumarate carrier; Icl: Isocitrate lyase: Fum: Fumarase.
Fig. 2
Fig. 2
The phylogenetical tree of the whole-genomic DNA sequences from the DH177 strain, A. pullulans var. aubasidani CBS 100524, A. pullulans P25 and any other Aureobasidium spp. and other fungal strains.
Fig. 3
Fig. 3
HPLC analysis of the fermentation products produced by the strain DH177 (A), the Δgox mutant (B), the standard gluconic acid (C) and the standard fumaric acid (D) and production of fumarate by the strain DH177 and the Δgox mutant (E). Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4
Recrystallization (A1-A6), purification and analysis of fermentation products of the mutant Δgox. A1-A6: the process of recrystallization (A1, the supernatant with fumarate; A2, the calcium sulfate from fumarate was precipitated by the added sulfuric acid; A3, the supernatant without calcium sulfate; A4, the recrystallized products at 4 °C; A5, the purified crystals; A6, the purified crystals observed under microscope). B: HPLC analysis of the products produced by the mutant Δgox; C: HPLC analysis of the recrystallized product; D: HPLC analysis of the standard fumaric acid; E: GC-MS analysis of the recrystallized product.
Fig. 5
Fig. 5
Fumarate titer and cell growth by the Δgox mutant and ΔgoxΔfum mutants (A), ΔgoxΔadsl mutants (B), ΔgoxΔicl1 mutants (C), ΔgoxΔicl2 mutants (D) and ΔgoxΔfum mutants (E). Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
Fig. 6
Fig. 6
Fumarate titer and cell growth by the mutant Δgox and ΔgoxΔasl mutants (A) and their complementing strains (B); ΔgoxΔcps1 mutants (C) and their complementing strains (D); ΔgoxΔcps2l mutants (E) and their complementing strains (F); ΔgoxΔcps2s mutants (G) and their complementing strains (H); ΔgoxΔsfc mutants (I) and their complementing strains (J). Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
Fig. 7
Fig. 7
Effects of Ca2+ signaling pathway on fumarate biosynthesis with and without CaCO3 (A), effects of deletion of the CRZ1 gene on fumarate biosynthesis (B) and effects of complementation of the CRZ1 gene on fumarate biosynthesis (C). Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
Fig. 8
Fig. 8
The Crz1 subcellular localization in the cells of the transformants carrying the CRZ1-GFP gene grown in the medium without CaCO3 (A) and with CaCO3 (B).
Fig. 9
Fig. 9
Fumarate titer and cell growth by the Δgox mutant, ΔgoxΔpks1 mutants (A) and strain ΔgoxΔpks1, PYC gene over-expressed strains (B). Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
Fig. 10
Fig. 10
Time course of fumarate titer, cell growth of the strain e-PYC and residual glucose concentration in the 10-L fermentation. During the fed batch fermentation, additional 200 g of sterile glucose was added into the fermenter at 120 h. Data are given as mean ± SD, n = 3, *P < 0.05, **P < 0.01.
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Further reading

    1. Du J., Cao N., Gong C.S., Tsao G.T., Yuan N. Fumaric acid production in airlift loop reactor with porous sparger. Appl Biochem Biotechnol Part A Enzyme Eng Biotechnol. 1997;65:541–556. - PubMed
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    1. Naude A., Nicol W. Fumaric acid fermentation with immobilized Rhizopus oryzae: quantifying time-dependent variations in catabolic flux. Process Biochem (Amsterdam, Neth) 2017;56:8–20.