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. 2022 Jun 22:13:919364.
doi: 10.3389/fmicb.2022.919364. eCollection 2022.

Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11

Abu Bakr Ahmad Fazili  1 , Aabid Manzoor Shah  1 , Mohammed Fahad Albeshr  2 , Tahira Naz  1 , Mohammad Abass Dar  3 , Wu Yang  1 , Victoriano Garre  4 , Khalid Majid Fazili  3 , Eijaz Ahmed Bhat  5 , Yuanda Song  1
Affiliations

Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11

Abu Bakr Ahmad Fazili et al. Front Microbiol. .

Abstract

Mucor circinelloides serves as a model organism to investigate the lipid metabolism in oleaginous microorganisms. It is considered as an important producer of γ-linolenic acid (GLA) that has vital medicinal benefits. In this study, we used WJ11, a high lipid-producing strain of M. circinelloides (36% w/w lipid, cell dry weight, CDW), to examine the role in lipid accumulation of two mitochondrial malic enzyme (ME) genes malC and malD. The homologous overexpression of both malC and malD genes enhanced the total lipid content of WJ11 by 41.16 and 32.34%, respectively. In parallel, the total content of GLA was enhanced by 16.73 and 46.76% in malC and malD overexpressing strains, respectively, because of the elevation of total lipid content. The fact that GLA content was enhanced more in the strain with lower lipid content increase and vice versa, indicated that engineering of mitochondrial MEs altered the fatty acid profile. Our results reveal that mitochondrial ME plays an important role in lipid metabolism and suggest that future approaches may involve simultaneous overexpression of distinct ME genes to boost lipid accumulation even further.

Keywords: Mucor circinelloides; genetic engineering; health benefits; lipid accumulation; malic enzyme; oleaginous fungi; γ-linolenic acid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Chromatogram of Mc-mal- 2076 (control), Mc-malC-2, and Mc-malD-3 strains at 72 h.
FIGURE 2
FIGURE 2
The overexpression of malC and malD gene. (A) Structure of the relevant region of pMAT2075 plasmid. (B) Structure of pMAT2075-malC plasmid, showing primer positions, and expected size fragments after PCR. (C) Structure of pMAT2075-malD plasmid, showing primer positions, and expected size fragments after PCR.
FIGURE 3
FIGURE 3
Confirmation of the integration of overexpressing constructs by (A) the use of 1F and 1R primers to amplify pyrF and 5′ carRP region showing a band size of 1.5 kb in Mc-malC-2, and Mc-cmalD-3 strains. Lane M: Marker. Lane 1 designates the control. Lanes 2 and 3 illustrates pyrF gene presence in Mc-malC-2 and Mc-cmalD-3 strains, respectively. (B) The use of 2F and 2R primers to amplify 3′ carRP region and malC gene, showing a band size of 2.4 kb in Mc-malC-2 strain. Lane M: Marker, Lane 1 designates the control, Lane 2 shows PCR amplification of 3′ carRP region and malC gene. (C) The use of 3F and 3R primers to amplify 3′ carRP region and malD gene, showing a band size of 2.1 kb in Mc-malD-3 strain. Lane M: Marker, Lane 1 designates the control, Lane 2 shows PCR amplification of 3′ carRP region and malD gene.
FIGURE 4
FIGURE 4
Relative mRNA levels determined by reverse transcription-quantitative PCR (RT-qPCR) of malC and malD genes in Mc-malC-2 and Mc-malD-3, respectively, normalized against their corresponding levels in the control strain Mc-mal-2076. Three biological replicates were used to calculate the respective values. Standard error of the mean (SEM) is indicated by error bars. Significant differences are indicated by asterisks: *p < 0.05 and **p < 0.01.
FIGURE 5
FIGURE 5
Malic enzyme (ME) specific activity in Mc-mal-2076, Mc-malC-2, and Mc-cmalD-3 strains. Three biological replicates were used to calculate the respective values. SEM is indicated by error bars. Significant differences are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001.
FIGURE 6
FIGURE 6
Accumulation of lipids and cell growth in overexpressing transformants (Mc-malC-2 and Mc-cmalD-3) and control strain (Mc-mal-2076). (A) Ammonium consumption profile, (B) glucose consumption profile, (C) lipid accumulation profile, and (D) cell dry weight (CDW). Three biological replicates were used to calculate the respective values. SEM is indicated by error bars. Significant differences are indicated by asterisks: *p < 0.05 and **p < 0.01.
FIGURE 7
FIGURE 7
Cycling of malate via different pathways. ACL, ATP, citrate lyase; ME, Malic enzyme; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; AcCOA, Acetyl CoA; MDH, malate dehydrogenase; CSY, citrate synthase; ACO, aconitase; IDH, isocitrate dehydrogenase; αKGDHC, α-ketoglutarate dehydrogenase complex; SCoAS, succinyl-CoA synthetase; SDH, succinate dehydrogenase; FUM, fumarase.
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