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Article

Transcriptomic Analysis Revealed the Differences in Lipid Accumulation between Spores and Mycelia of Mucor circinelloides WJ11 under Solid–State Fermentation

by
Feifei Xin
1,
Wenrui Dang
1,
Yufei Chang
1,
Ruixue Wang
1,
Hongjuan Yuan
1,
Zhike Xie
1,
Chuanchuan Zhang
2,
Sha Li
2,
Hassan Mohamed
1,3,
Huaiyuan Zhang
1,* and
Yuanda Song
1,*
1
Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, 266 Xincun West Road, Zibo 255000, China
2
Shandong FullSail Biotechnology Co., Ltd., Gaoqing County Economic Development Zone, Zibo 256399, China
3
Department of Botany and Microbiology, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt
*
Authors to whom correspondence should be addressed.
Fermentation 2022, 8(12), 667; https://doi.org/10.3390/fermentation8120667
Submission received: 1 November 2022 / Revised: 18 November 2022 / Accepted: 19 November 2022 / Published: 23 November 2022
(This article belongs to the Section Fermentation Process Design)
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Abstract

:
The oleaginous fungus Mucor circinelloides has been studied for microbial oil production. Solid–state fermentation may be more suitable for lipid production than submerged fermentation due to its special filamentous structure and lower fermentation costs. M. circinelloides WJ11 under solid–state fermentation indicated that the total fatty acid content of mycelia was significantly higher than that of spores (15.0 and 10.4% in mycelia and spores after 192 h, respectively), while the biomass of the fungal mycelia was lower than that of the spores, reaching 78.2 and 86.9 mg/g, respectively. Transcriptomic studies showed that a total of 9069 genes were differentially expressed between spores and mycelia during solid–state fermentation, of which 4748 were up-regulated and 4321 were down-regulated. Among them, triglyceride-related synthases in M. circinelloides were significantly up-regulated in the mycelia. The mRNA expression level of ATP: citrate lyase was obviously increased to provide more acetyl-CoA for fatty acid synthesis in mycelia, moreover, the metabolism of leucine and isoleucine can also produce more acetyl-CoA for lipid accumulation in M. circinelloides. For NADPH supply, the expression of the pentose phosphate pathway was significantly up-regulated in mycelia, while NADP+-dependent malic enzyme was also increased by 9.5-fold under solid–state fermentation. Compared with gene expression in spores, the autophagy pathway was clearly up-regulated in mycelia to prove that autophagy was related to lipid accumulation in M. circinelloides.

1. Introduction

Due to the vital role of polyunsaturated fatty acids (PUFAs) in maintaining human health, people have increased their interest in their multiple nutritional effects [1]. In recent years, animal and vegetable oils are relatively restricted in the terms of season and climate, resulting in microbial oils, as a substitute for animal and vegetable oils, being used as alternative commercial sources of PUFAs [2]. Oleaginous microorganisms capable of producing oils including bacteria, fungi, yeasts, and microalgae, can accumulate up to 20% of their cell dry weight (CDW) [3]. In particular, lipid-producing fungi can accumulate large amounts of PUFAs, such as gamma-linolenic acid (GLA) in Mucor circinelloides and arachidonic acid (ARA) in Mortierella alpina [4,5]. To date, submerged fermentation is by far the most common cultivation process for microbial lipid production, as it can be mechanized and extended for production [6]. However, during submerged fermentation multiple factors are to be considered, such as expensive fermentation tanks, temperature, and stirring controls, in addition to enough energy supply system, resulting in the huge cost of microbial lipid production [7]. Compared with submerged fermentation, solid–state fermentation has several advantages in terms of cost control, including lower wastewater yield, less energy demand, a simple fermentation medium, easier aeration, and less bacterial pollution [8]. Moreover, solid–state fermentation is particularly associated with fungi, which are better suited to work on solid substrates where mycelia can grow and expand better, providing an environment with physical and chemical properties similar to the natural habitats of microbes [9]. Previous studies had shown that oleaginous fungi can produce abundant lipids under solid–state fermentation conditions. Lipids can be accumulated by the filamentous fungus Phanerochaete chrysosporium through solid–state culture, and the conversion efficiency of the substrate to lipid reached 0.277 g/g substrate [7]. Additionally, M. alpina CCF2861 effectively transformed exogenous fatty acids from the animal fat substrate to ARA; a maximum yield was achieved with 32.1 mg/g of bioproduct with cornmeal mixed with 5% (w/w) of an animal fat by-product as substrate [10].
M. circinelloides is a well-known and thoroughly described fungal species with a high capacity for lipid accumulation and a good ability to produce industrially relevant essential PUFAs, especially GLA in submerged fermentation [11]. A newly isolated oil-producing fungus, M. circinelloides Q531, can transform mulberry branches into lipids with the maximum yield and lipid content of fungal cells being 42.43 mg/g dry substrate and 28.8%, respectively [12]. Similarly, M. circinelloides WJ11 is not suitable for high-density fermentation, with its strong sporulation ability and specific filamentous structure. Hence, solid–state fermentation may be an alternative fermentation method for lipids accumulation in M. circinelloides WJ11. Furthermore, the previous study found that there was a significant difference in lipid accumulation between spores and mycelia in the case of solid–state fermentation of M. circinelloides, and the lipid content of mycelia was 27.7% higher than in spores under the same cultivation, indicating that there is a large difference between mycelia and spores in the lipid synthesis metabolic pathway [13]. Therefore, the transcriptome of the high-oil-producing strain M. circinelloides WJ11 was sequenced and a comparative analysis at the transcriptional levels between mycelia and spores was carried out to study their differences in lipid production modes. The transcriptome analysis in this study revealed the molecular basis of lipid accumulation and well-known genetic information for the development of M. circinelloides WJ11 as an important model for lipid accumulation of spores and mycelia studies under solid–state fermentation conditions.

2. Materials and Methods

2.1. Preparation of Fungal Spore Suspension and Solid–State Fermentation Medium

The spores of M. circinelloides WJ11 were cultivated on potato dextrose agar (PDA) medium for 4 days at 28 ℃, and then spores were collected with distilled water. The spore suspension was diluted to the final concentration of 107 spores/mL. A sheet of cellophane was placed on the surface of the plate medium and 100 μL spore suspension of M. circinelloides WJ11 was inoculated onto plates including fermentation solid medium (approximately 25 mL/plate) and incubated at 28 °C for 4 days (as shown in Figure S1). The fermentation medium composition as follows: glucose, 60 g/L; MgSO4∙7H2O, 1.5 g/L; diammonium tartrate, 2.0 g/L; KH2PO4, 7.0 g/L; Na2HPO4, 2.0 g/L; yeast extract, 1.5 g/L; CaC12∙2H2O, 0.1 g/L; FeC13∙6H2O, 8.0 mg/L; ZnSO4∙7H2O, 1.0 mg/L; CuSO4∙5H2O, 0.1 mg/L; Co(NO3)2∙6H2O, 0.1 mg/L; MnSO4∙5H2O, 0.1 mg/L; agar, 20.0 g/L.

2.2. Separation of Spores and Mycelia under Solid–State Fermentation

The cellophane placed on the agar medium was peeled off and the solid–state fermentation products including spores and mycelia were collected in 50 mL centrifuge tubes containing distilled water, shaken vigorously through a vortex oscillator, and filtered through two layers of nylon gauze to separate the mycelia and spores from the products [14]. The filtered spore suspension and the isolated mycelia were washed more than three times with distilled water, the washed spore suspension was centrifuged, the supernatant was poured off, and the spores were retained in centrifuge tubes for subsequent experiments. Then, all samples of spores and mycelia after freeze-drying were subjected to cell growth and fatty acid analysis.

2.3. Lipid Extraction and Fatty Acid Analysis

The biomass of freeze-dried samples (spores and mycelia) from solid–state fermentation was determined gravimetrically [15]. Spores and mycelia of M. circinelloides WJ11 were lysed with hydrochloric acid (4 mol/L) and total fatty acid (TFA) was extracted with chloroform: methanol (2: 1, v/v) using pentadecanoic acid (C15:0) as an internal standard according to the method reported by Folch et al. [16]. After methyl esterification with 10% (w/w) methanol/hydrochloric acid, fatty acid methyl esters were determined by gas chromatography Agilent 123–3232 column (30 m × 320 μm × 0.25 μm), and the program was set as follows according to a previous study [17]: 120 °C for 3 min, ramped up to 200 °C at 5 °C/min, then to 220 °C at 4 °C/min and held for 2 min. Each sample was repeated three times.

2.4. RNA Sequencing and Analyzes

For transcriptomic analysis, the spores, and mycelia of M. circinelloides WJ11 were collected at 48 h, according to its growth curve under solid–state fermentation. Three biologically replicated samples were then frozen in liquid nitrogen and stored at −80 °C for further analysis. Total RNA was extracted by Trizol according to the manufacturer’s instructions (Sangon Biotech, Shanghai, China). The RNA concentration was determined by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and the integrity of RNA was detected by Agilent 2100 bioanalyzer (Agilent, Santa Clara, CA, USA).
After the cDNA library of each sample was established, low quality, splice contamination, and high nitrogen content of unknown bases in the raw reads obtained from the sequencing were removed to ensure reliable results. The clean reads were aligned to the genome sequence using Bowtie2, and gene expression levels were then calculated by RSEM (v1.2.12). The DEseq2 method was used to identify differentially expressed genes (DEGs) between the groups based on the principle of the negative binomial distribution, and the DEGs detection was performed according to the method described previously [18]. Based on the quantitative genes of the gene expression level and various analyses (principal components, correlation, differential gene screening, etc.), the deep mining analysis of gene ontology (GO) functional significance enrichment analysis, and pathway significance enrichment analysis, were conducted on the DEGs in the selected samples. A hypergeometric test was used to identify the GO terms with significant differences in DEGs between the two groups and analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

2.5. Statistical Analysis

All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant when p < 0.05 and very significant when p < 0.01.

3. Results and Discussion

3.1. Cell Growth and Lipid Content of M. circinelloides under Solid–State Fermentation

M. circinelloides WJ11 was cultured under solid–state fermentation with a nitrogen-limited medium, and both spores and mycelia demonstrated similar and typical growth patterns. Biomass initially increased rapidly during the stationary phase of growth from 0 to 120 h, and then basically stabilized (Figure 1A). There were no significant differences from 0 to 48 h during solid–state fermentation, while after 48 h the biomass of the fungal spores was higher than that of the mycelia, with maximum biomass of 86.9 mg/g for the spores and 78.2 mg/g for the mycelia, respectively.
Lipid accumulation in both spores and mycelia started from 24 h (Figure 1B), and the TFA content increased rapidly from 6.5% to 11.6% of their CDW in mycelia and from 4.2% to 6.6% in spores from 24 to 48 h, and the lipid accumulation slowed down after 48 h. The maximum concentration of TFA in mycelia was 15.0% of CDW after 196 h, which was 44.2% higher than that of spores (10.4%). The lower lipid content of spores than mycelia may be because the spores are located at the top of the mycelia during solid–state fermentation, and the transportation of nutrients from the bottom to the top consumes a lot of energy [19]. Other than that, the less efficient uptake of nutrients from the medium during solid–state fermentation cultures due to the slender mycelia also results in a lower rate of nutrient acquisition by the apically located spores, which leads to some differences in lipid accumulation [9]. As shown in Table 1, the results also showed no significant difference in fatty acid composition between spores and mycelia.

3.2. RNA Sequencing and Analysis in Spores and Mycelia of M. circinelloides

Total RNA was extracted from the spores and mycelia of M. circinelloides WJ11 and then constructed into the RNA-seq libraries, which were sequenced on the mgiseq 2000. After data cleaning and quality control, the clean reads were processed to the Q20 range of 96.78–96.93% for each sample as shown in Table 2, indicating the transcriptomics data were valid and further experimental analysis can be carried out.
To find the potential mechanism underlying the different capacities of lipid accumulation between spores and mycelia in M. circinelloides WJ11, the DEGs potentially involved in the biochemistry pathway of lipid accumulation was calculated. The gene sets in spores and mycelia have the same biological functions, with 11,124 genes in spores and 11,388 genes in mycelia (Figure S2), and the unique genes in spore accounted for 1.6%, mycelia unique genes, 451. By comparing the corresponding mRNA levels in spores and mycelia, the up-regulated or down-regulated genes were determined. Compared with spores, the number of up-regulated and down-regulated genes was 4748 and 4321, respectively (Figure 2A), indicating a clear difference between the spore- and mycelia-associated enzymes of M. circinelloides WJ11 during solid–state fermentation. The DEGs detected in this study provide a global perspective for the transcriptome of M. circinelloides under solid–state fermentation conditions.
According to the GO annotation results and official classification, the DEGs were classified by function, and the enrichment analysis was carried out by using the phyper function in R software. A total of 6913 genes were significantly enriched (Figure S3), and most of the unique genes were categorized as “intracellular” (31.1%). The unique genes mapped into GO terms were further clustered in the KEGG database, and a total of 3735 unique genes, were assigned to 32 KEGG pathways (Figure 2B). The most abundant group containing 1401 (37.5% of 3735) genes is related to “Global and overview maps” in “Metabolism”. The following is 17.2% (644/3735) of the mapped genes related to “Translation” and “Carbohydrate metabolism”, 15.6% (582/3735) related to “Transport and catabolism”, 13.4% (500/3735) related to “Signal transduction”, and so on. A total of 303 genes (8.1%) were classified as the “Lipid metabolism”, which we considered most important under solid–state fermentation.

3.3. The Expression Difference of Glycerolipid Biosynthesis between Spores and Mycelia of M. circinelloides

Triglycerides (TAG) are the main oils accumulated by oil-producing fungi, which are a suitable storage form for fatty acids due to their low biotoxicity, and are produced through the Kennedy pathway [20]. There are significant changes in the gene expression related to the TAG synthesis pathway (Figure 3), and the data (in the Table S1) showed that most of the genes from TAG synthesis pathway are significantly upregulated, while the degradation pathway is significantly downregulated in mycelia compared with spores.
Among the genes encoding the main enzymes in the TAG-synthesis, glycerol-3-phosphate acyltransferase (GPAT) catalyzing the initial step in the assembly of glycerolipids was significantly up-regulated in mycelia mostly with a 36.5-fold increase max compared with spores under solid–state fermentation. More importantly, there is one expressed elevated GPAT (Gene ID: HMPREF1544_03730) not expressed in spores and only in mycelia under solid–state fermentation. As shown in Table 3, the GPAT in Metarhizium robertsii is located in the endoplasmic reticulum and is directly related to the formation of lipid droplets in fungal cells. Compared with wild type and gene-rescue mutant, which was amplified together with its promoter and the 3′-untranslated region, the GPAT deletion mutant showed a reduced ability to produce conidia and total lipids [21]. In Arabidopsis thaliana, the overexpression of JcGPAT1 and JcGPAT2 from Jatropha curcas under constitutive and seed-specific promoters increased total oil content. Among them, the transgenic seeds of JcGPAT2-OE lines contained 43–60% more oil than the seeds as control, indicating that these homologs are involved in oil biosynthesis [22].
The last step of TAG synthesis is catalyzed by diacylglycerol acyltransferase (DGAT), transcriptome data showed that one type I DGAT (DGAT1) and two type II DGATs (DGAT2-1 and DGAT2-2) identified in M. circinelloides WJ11 were all up-regulated in mycelia compared with spores under solid–state fermentation. Among them, DGAT2-2 was significantly increased by 11.7-fold, resulting in the synthesis of more lipids in mycelia in the form of TAG than spores. This result coincides with previous studies demonstrating that DGAT2B overexpression significantly increased the lipid content of M. circinelloides under solid–state fermentation conditions [13], further demonstrating the important role of DGAT2B in lipid accumulation during solid–state fermentation. Furthermore, the expression of triglyceride lipase (TLP), which degrades most of the TAG into free fatty acids, was higher in mycelia than in spores, which may have led to the oxidation of some TAG into fatty acids.
Lysophosphatidic acid acyltransferase (LPAT) in the Kennedy pathway was also mostly up-regulated in mycelia. Furthermore, one of the 11 genes encoding LPAT was expressed 138.0-fold higher in mycelia compared to spores and may be a key gene in lipid accumulation, and the other three genes encoding LPAT were also significantly increased in mycelia under solid–state fermentation, 13.5-, 7.2-, and 6.1-fold, respectively. The sn-2 acyltransferase gene from yeast Saccharomyces cerevisiae exhibits LPAT activity [37], was introduced into Brassica napus cv Hero, and the seed oil content of the transgenic plants was significantly increased from 6.1 to 8.2% (based on seed dry weight), while the total long chain fatty acids proportion and content in seed TAG were increased [23], suggesting that LPAT is also a key gene in oil drop formation. From phosphatidate to diacylglycerol, two of the six genes encoding phosphatidate phosphatase (PAP) were also significantly changed between mycelia and spores, with a 55.6- and 3.5-fold increase in mycelia, respectively, indicating PAP may also contribute to the lipid enhancement of M. circinelloides WJ11.

3.4. Multiple Acetyl-CoA Sources for Fatty Acid Synthesis in Spores and Mycelia of M. circinelloides

Substrate acetyl-CoA in the fatty acid synthesis pathway of oil-producing fungi is mainly produced by ATP: citrate lyase (ACL) based on previous studies [38]. Research has shown a strong correlation between ACL activity and lipid accumulation in oil-producing yeasts [39]. Compared with the wild type, the fatty acid content of ACL mutant in marine-derived yeast Yarrowia lipolytica decreased by 36% [24], while the overexpression of ACL from Mus musculus with low Km value increased the lipid content from 7% to 23% [25]. In addition, the ACL promoter of Aspergillus oryzae was modified to increase its fatty acid content by 1.7-fold [26]. In this experiment, compared with spores, the gene expressions encoding ACL in mycelia were decreased by 45.5% and increased by 2.9-fold (Figure 4), respectively, and the lipid content in mycelia was significantly higher than that in spores under solid–state fermentation, suggesting that the ACL with gene ID HMPREF1544_03574 maybe the key provider of acetyl-CoA for lipid accumulation in oleaginous fungi M. circinelloides WJ11. Cytoplasmic acetyl-CoA synthase (ACS) can also produce acetyl-CoA from acetate [15], and the two of three genes encoding ACS in mycelia were both significantly up-regulated by 9.4- and 1.6-fold, respectively. Moreover, eight of the nine genes encoding acetaldehyde dehydrogenase (ALD) in the synthesis of acetate from acetaldehyde in mycelia were higher compared with spores, indicating that acetate was also another important source of acetyl-CoA under solid–state fermentation.
The degradation of branched-chain amino acids (BCAAs) can also produce acetyl-CoA as a substrate for lipid synthesis [40]. BCAAs, including leucine, isoleucine, and valine, were involved in lipid biosynthesis in triacylglycerol-rich Dunaliella tertiolecta, wherein their function in refilling the acetyl-CoA pool [41]. In this experiment, no additional amino acids were added, most of the genes expression related to the leucine synthesis pathway were higher in mycelia, and the gene encoding 3-isopropyl malate dehydrogenase (LEUB) was increased by 40.0-fold compared with spores (Figure 4), and it was also proven that LEUB was a vital target for the lipid synthesis pathway of M. circinelloides WJ11 in previous studies [42], indicating that leucine synthesis was improved in mycelia compared with spores under solid–state fermentation leading to lipid synthesis. In the degradation pathway of leucine to acetyl-CoA, the related expression genes were significantly up-regulated in mycelia compared with spores. In particular, 3-methylcrotonyl-CoA carboxylase (MCL), methylglutaconyl-CoA hydratase (AUH) and hydroxymethylglutaryl-CoA lyase (HMGCL) were all significantly higher in mycelia than in spores, with the three genes encoding AUH had a 71.8-, 4.5- and 3.2-fold increase respectively, while HMGCL was only expressed in the mycelia under solid–state fermentation and the expression was 34.1, indicating that the leucine metabolism pathway to supply acetyl-CoA was boosted, providing more substrates for lipid synthesis. As for the isoleucine synthesis pathway, some genes were significantly lower in mycelia than in spores, however, the isoleucine degradation pathway-related expression genes were significantly up-regulated, as two genes encoding 2-oxoisovalerate dehydrogenase E2 (DBT), which were also involved in the degradation of leucine to acetyl-CoA, were significantly increased by 9.3- and 7.1-fold and genes encoding enoyl-CoA hydratase (ECH) and 3-hydroxy acyl-CoA dehydrogenase (HCD) were significantly up-regulated by up to 5.9-, 3.2-,3.4-, and 23.6-fold in mycelia; furthermore, a gene encoding ECH expressed in mycelia only with 13.6 under solid–state fermentation. In summary, ACL is not the only source of acetyl-CoA during microbial lipid accumulation, and the synthesis and degradation of BCAAs is also a major provider of substrates for lipid synthesis.

3.5. The Pentose Phosphate Pathway, Not Only a Malic Enzyme, Contributes the Main NADPH for Lipid Accumulation in M. circinelloides

Reducing power in the form of NADPH is another pivotal substrate in fatty acid biosynthesis [4]. Previous studies have suggested that malic enzyme (ME) is the main NADPH provider for fatty acid synthesis in oil-producing fungi, and also found that lipid content of Aspergillus nidulans lacking ME activity mutant reduced to half of the original [27]. Endogenous overexpression of the ME1 gene in Phaeodactylum tricornutum increased the transformants’ neutral and total lipid content [43]. What is more, the gene encoding ME isoform E from M. alpina was homologously expressed and the ME overexpression increased the fatty acid content by 30% compared to that of the control [28]. In this study, the mRNA relative expression of three of the four genes encoding ME in mycelia was decreased under solid–state fermentation, by 75.7%, 15.2%, and 13.7%, respectively, and the relative expression value of another gene was 9.5-fold compared with spores. Through BLAST analysis of the increasing gene in mycelia, it was found that overexpression of the gene in submerged fermentation of fungi can increase the lipid content of cells from 12% to 30% of biomass [29]. However, the genetically modified strain (ME-overexpression) showed a lipid content similar to that of a prototrophic non-overexpressing control strain, indicating that ME activity is not the only bottleneck for lipid accumulation in the oil-producing fungus M. circinelloides [44], Likewise, the ME gene (mce2) of an oil-producing fungus, M. alpina, was expressed in Y. lipolytica, and there were no significant changes in lipid content, suggesting that ME is not a major source of NADPH for lipid accumulation in Y. lipolytica [30]. All of the above results indicate that ME can provide NADPH for microbial lipid accumulation in some species and not in others, and unfortunately, the role of genes encoding ME in M. circinelloides under solid–state fermentation conditions is still controversial and needs further study, suggesting that there are other sources of NADPH in this species.
According to the previous study, the pentose phosphate pathway may be another important source of NADPH in the process of fatty acid synthesis [2], and the addition of glutamic acid during the culture of M. alpina enhanced the activity of the pentose phosphate pathway and thus increased lipid production [45]. The overexpression of g6pdh1 and g6pdh2 genes encoding glucose-6-phosphate dehydrogenase (G6PDH) in M. circinelloides WJ11 increased the fatty acid content of CDW by 23–38% and 41–47%, respectively, indicating that G6PDH in pentose phosphate pathway plays an important role in lipid synthesis [31]. Gene expression level of G6PDH in Rhodosporidium toruloides was up-regulated by 1.9-fold during the lipid accumulation period under nitrogen limitation [46], and the protein expression level of 6PGDH was also significantly increased [47]. The gene expression level of 6PGDH in Y. lipolytica was significantly higher during lipid accumulation than during cell growth [48]. There were three genes encoding G6PDH in M. circinelloides WJ11 in our transcriptome experiment, two of which were significantly up-regulated in mycelia, and the relative expression levels were 5.2-fold and 22.9-fold, respectively, indicating that the increased expression of genes encoding G6PDH in mycelia would provide more NADPH for lipid accumulation under solid–state fermentation. Not only that, the two genes encoding 6PGDH were significantly decreased by 57.6% and increased by 1.9-fold, respectively. Thus, the pentose phosphate pathway plays an important role as the NADPH provider for lipid accumulation in the solid–state fermentation of M. circinelloides WJ11.

3.6. Autophagy Was Associated with Lipid Accumulation in Spores and Mycelia of M. circinelloides

Autophagy is a catabolic process by which damaged or unnecessary cytoplasmic material is engulfed in bulk by double-membrane vesicles called autophagosomes [49]. Following nutrient stress, cell autophagy as a primary catabolic pathway is induced to deliver superfluous or damaged cytoplasmic material and organelles to the lysosomal/vacuole for degradation and recycling for cellular survival [50]. Previous studies have shown that there is a certain relationship between autophagy and lipid metabolism in various species, and the regulation of autophagy in lipid biosynthesis and degradation has been shown to differ among various cells and species [51]. Basic autophagy in Arabidopsis thaliana contributes to TAG synthesis, while induced autophagy contributes to lipid droplet degradation [51]. Under nutritional constraints, autophagy is required for the synthesis of TAG and the recycling of ribosomal protein in Chlamydomonas [52]. Nitrogen limitation for lipid accumulation also led to cell autophagy in the oil-producing fungus M. circinelloides WJ11 under solid–state fermentation in this experiment. There was a significant difference in autophagy-related expression of genes between mycelia and spores (Figure 5). Carbon catabolite-derepressing protein kinase 1 (SNF1) was the catalytic subunit of the heterotrimeric complex, which is required for growth in the absence of glucose and regulates global changes in gene expression to utilize alternative carbon sources. There were 17 genes encoding SNF1 during solid–state fermentation and three genes were significantly down-regulated, by 83.1%, 61.0%, and 41.8%. Previous studies have clearly shown that SNF1 plays a central role in lipid metabolism, nitrogen fixation, and carbon incorporation [53]. The destruction of the SNF1 gene was observed in Y. lipolytica to promote cell growth and lipid accumulation and significantly reduce cellular saturated fatty acid levels [54]. SNF-β, which encodes for the β subunit of the adenosine 5′-monophosphate-activated protein kinase (AMPK) complex, plays an important role in lipid accumulation in the M. circinelloides WJ11 strain. The lipid content of CDW in the SNF-β knockout strain increased by 32% (from 19.0% to 25.0%), however, in the SNF-β overexpressing strain, the lipid content of CDW decreased by about 25% (from 19.0% to 14.2%) compared to the control strain [32]. Thus, data from previous studies suggest that the genes encoding SNF1, which were significantly down-regulated in mycelia in this experiment, have an important regulatory role in the lipid synthesis pathway. Following the order, the genes encoding protein kinase autophagy-related 1 (ATG1) are the key regulators of autophagy and coordinates a complex signaling program to orchestrate the formation of autophagosomes, whose relative expressions were changed by 0.1-, 11.0-, and 0.3-fold, respectively. ATG1 was also activated by protein kinase autophagy-related 13 (ATG13), and the expression of genes encoding ATG13 was significantly up-regulated in the mycelia under solid–state fermentation by 1.6-, 6.5- and 2.3-fold, respectively, while the gene encoding ATG13 (gene ID HMPREF1544_06371) was only expressed in the mycelia and the expression levels were 15.2, but not expressed in the spores. The ATG1 and ATG13 complex plays a vital role in initiating autophagy, sensing nutritional status signals, recruiting downstream ATG proteins to autophagosome formation sites, and controlling autophagosome formation [55]. Thus, the ATG1 and ATG13 genes play an important role in lipid-rich mycelia under solid–state fermentation.
The rapamycin (TOR) complex’s target downstream of SNF1 is a conserved multifunctional serine/threonine protein kinase found in all eukaryotes that control several important signaling pathways related to growth and development [33]. TOR complex included threonine-protein kinase TOR (TOR), the target of rapamycin complex subunit (LST8) and regulatory associated protein of TOR (Raptor), and three of four genes encoding TOR were significantly down-regulated (32.0%, 43.5% and 1.6% lower, respectively), however, LST8 was significantly up-regulated by 1.5-fold. Early data suggested that baicalein inhibits lipid accumulation by controlling the cell cycle of 3T3-L1 cells and the TOR signaling pathway [56]. Furthermore, the results of stress experiments also confirm that TOR overexpression in Arabidopsis thaliana negatively regulates lipid peroxidation by controlling reactive oxygen species levels under oxidative stress conditions [33]. Our transcriptomic data are also consistent with previous studies showing that TOR expression is negatively correlated with lipid synthesis in fermentation products. In addition, the activity of the TOR complex was also inhibited by serine palmitoyl transferase (LSB1/2), and most of the relatively expressed genes encoding LSB1/2 were significantly down-regulated in mycelia in the present study, by 15.7%, 65.2%, and 56.6%, respectively. The budding yeast threonine protein kinase (SCH9), a functional homolog of mammalian S6 kinase, is a major effector of the TOR complex in the regulation of cell growth in response to nutrient utilization and stress. One study revealed a key role in the SCH9 N-terminal structural domain and provided a new mechanism for the regulation of the main signaling branch of the TOR complex [57]. Nitrogen starvation also directly promoted the activity of SCH9, and its relative expression in mycelia was mostly significantly higher than that in spores under solid–state fermentation of M. circinelloides WJ11. Two genes located downstream of SCH9 encoding the nutrient-sensitive kinase (RIM15) were identified in previous studies as key biomarkers for predicting cell fate before acute glucose removal stress [58], with significant changes of 10.4% decreasing and 1.8-fold increasing, respectively.
Transcriptional regulatory protein (UME6) and the multicopy suppressor of SNF1 mutation proteins 2 and 4 (MSN2/4) are located downstream of RIM15, and the gene expression levels have significant changes, while MSN2/4 and protein kinase autophagy-related 8 (ATG8) located downstream of UME6 can regulate the lipid accumulation of M. circinelloides WJ11. It is worth noting that the yeast S. cerevisiae with MSN2/4 double mutant (msn2Δmsn4Δ) showed severe growth defects when using oleic acid as the sole carbon source and reduced the transcription level of the main β-oxidation genes. What is more, lipid analysis showed that the levels of triacylglycerol and steroidal esters were increased in the msn2Δmsn4Δ strain, manifesting that MSN2/4 plays a key role in the regulation of fatty acid oxidation in oleaginous fungi [34]. However, only one of the three genes encoding MSN2/4 was significantly lower by 6.1%, and the gene with down-regulated expression may be a key regulator of the fatty acid synthesis pathway in M. circinelloides.
Studies in recent years have noted that autophagy is highly conserved in eukaryotic cells and recognized as a conjugation pathway that attaches ATG8 to phosphatidylethanolamine (PE), which coats emerging autophagic membranes and assists with cargo recruitment, vesicle enclosure, and subsequent vesicle docking with the tonoplast [59]. PE-lipidated ATG8 has also been shown to localize to lipid droplets and contribute to their formation [60]. Rhodnius prolixus silenced two essential genes of the autophagy pathway, Atg6/Beclin1 (RpAtg6) and Atg8/LC3 (RpAtg8), and monitored lipid storage during starvation, resulting in higher levels of TAG in the fat body and the flight muscle. Data suggest that autophagy in fat bodies is important for allowing insects to mobilize energy from lipid storage [35]. In addition, ATG8 overexpression strain in M. alpina with an external supply of ethanolamine significantly increased arachidonic acid-rich triacylglycerol and biomass synthesis, and the final fatty acid content increased by approximately 110% compared with that in the wildtype strain [36]. Compared with spores with low fatty acid content, in the autophagy regulation system, the expression of the ATG8 gene in mycelia was regulated by a 3.7-fold increase and 36.2% decrease, respectively, and which gene involved in autophagy regulating fatty acid synthesis needs further study.

4. Conclusions

In the present study, the biomass and lipid content between mycelia and spores in M. circinelloides WJ11 were significantly different under the solid–state fermentation strategy, and the lipid content of mycelia was higher than that of spores. The spores and mycelia of WJ11 were deeply sequenced in transcriptome to identify the DEGs between spores and mycelia. The results showed that the mRNA level of related enzymes was significantly up-regulated in the TAG synthesis pathway. Acetyl-CoA is provided by multiple pathways, not only ACL, but also amino acid and acetic acid metabolism may play a very important role in lipid accumulation, and the pentose phosphate pathway may be the main NADPH provider. In addition, cell autophagy is also related to lipid synthesis in M. circinelloides WJ11, which provides a new strategy for future lipid research.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation8120667/s1, Table S1: Differential expression of lipid synthesis related genes in Mucor circinelloides WJ11 under solid–state fermentation conditions. Figure S1: Establishment of Mucor circinelloides WJ11 under solid–state fermentation condition. Figure S2: Wayne plot of DEGs between spores and mycelia under solid–state fermentation. Figure S3: Differential gene GO enrichment histogram between spores and mycelia of Mucor circinelloides WJ11 under solid–state fermentation.

Author Contributions

Data curation, F.X.; formal analysis, F.X. and R.W.; investigation, Z.X. and H.Y.; resources, C.Z. and S.L.; software, Y.C.; supervision, H.M.; validation, W.D.; writing—original draft, F.X.; writing—review and editing, H.Z. and Y.S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Natural Science Foundation of Shandong Province (ZR2022MC108) and the Research project of the Yiyuan Industrial Technology Research Institute (platform).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained in the main article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Biomass and TFA content of M. circinelloides WJ11 under solid–state fermentation in spores and mycelia. (A) CDW determination; (B) TFA content.
Figure 1. Biomass and TFA content of M. circinelloides WJ11 under solid–state fermentation in spores and mycelia. (A) CDW determination; (B) TFA content.
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Figure 2. Differences between groups and categorization of the KEGG function for unigenes in M. circinelloides WJ11 under solid–state fermentation conditions. (A) Scattering plot of DEGs between spores and mycelia; (B) KEGG classification for unigenes of spores and mycelia.
Figure 2. Differences between groups and categorization of the KEGG function for unigenes in M. circinelloides WJ11 under solid–state fermentation conditions. (A) Scattering plot of DEGs between spores and mycelia; (B) KEGG classification for unigenes of spores and mycelia.
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Figure 3. Genes expression changes of TAG synthesis between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, in green were downregulated, and those in grey were no significant change. Enzymes note: CPT, diacylglycerol cholinephosphotransferase; DGAT1, diacylglycerol acyltransferase 1; DGAT2, diacylglycerol acyltransferase 2; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lysophospholipid acyltransferase; PAP, phosphatidic acid phosphatase; PGAT, phospholipid: diacylglycerol acyltransferase; PLC, phospholipase C; TLP, triglyceride lipase.
Figure 3. Genes expression changes of TAG synthesis between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, in green were downregulated, and those in grey were no significant change. Enzymes note: CPT, diacylglycerol cholinephosphotransferase; DGAT1, diacylglycerol acyltransferase 1; DGAT2, diacylglycerol acyltransferase 2; GPAT, glycerol-3-phosphate acyltransferase; LPAT, lysophospholipid acyltransferase; PAP, phosphatidic acid phosphatase; PGAT, phospholipid: diacylglycerol acyltransferase; PLC, phospholipase C; TLP, triglyceride lipase.
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Figure 4. Genes expression changes of acetyl-CoA synthesis, leucine and isoleucine synthesis and degradation between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, in green were downregulated, and those in grey were no significant change. Enzymes note: ACA, acetyl-CoA acyltransferase; ACD, acyl-CoA dehydrogenase; ACL, ATP: citrate lyase; ACS, acetyl-CoA synthetase; ALD, aldehyde dehydrogenase (NAD+); AUH, methylglutaconyl-CoA hydratase; BCD, butyryl-CoA dehydrogenase; BCK, 2-oxoisovalerate dehydrogenase E1; CIMA, (R)-citramalate synthase; CS, citrate synthase; DBT, 2-oxoisovalerate dehydrogenase E2; DLAT, dihydrolipoamide acetyltransferase; ECH, enoyl-CoA hydratase; HCD, 3-hydroxyacyl-CoA dehydrogenase; HMGCL, hydroxymethylglutaryl-CoA lyase; ILVA, threonine dehydratase; ILVC, ketol-acid reductoisomerase; ILVD, dihydroxy-acid dehydratase; ILVE, branched-chain amino acid aminotransferase; ILVH, acetolactate synthase I/III; IVD, isovaleryl-CoA dehydrogenase; LEUA, 2-isopropyl malate synthase; LEUB, 3-isopropyl malate dehydrogenase; LEUC, 3-isopropyl malate; LEU1, 3-isopropyl malate dehydratase; LPD, dihydrolipoamide dehydrogenase; MCL, 3-methylcrotonyl-CoA carboxylase; PC, pyruvate carboxylase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase.
Figure 4. Genes expression changes of acetyl-CoA synthesis, leucine and isoleucine synthesis and degradation between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, in green were downregulated, and those in grey were no significant change. Enzymes note: ACA, acetyl-CoA acyltransferase; ACD, acyl-CoA dehydrogenase; ACL, ATP: citrate lyase; ACS, acetyl-CoA synthetase; ALD, aldehyde dehydrogenase (NAD+); AUH, methylglutaconyl-CoA hydratase; BCD, butyryl-CoA dehydrogenase; BCK, 2-oxoisovalerate dehydrogenase E1; CIMA, (R)-citramalate synthase; CS, citrate synthase; DBT, 2-oxoisovalerate dehydrogenase E2; DLAT, dihydrolipoamide acetyltransferase; ECH, enoyl-CoA hydratase; HCD, 3-hydroxyacyl-CoA dehydrogenase; HMGCL, hydroxymethylglutaryl-CoA lyase; ILVA, threonine dehydratase; ILVC, ketol-acid reductoisomerase; ILVD, dihydroxy-acid dehydratase; ILVE, branched-chain amino acid aminotransferase; ILVH, acetolactate synthase I/III; IVD, isovaleryl-CoA dehydrogenase; LEUA, 2-isopropyl malate synthase; LEUB, 3-isopropyl malate dehydrogenase; LEUC, 3-isopropyl malate; LEU1, 3-isopropyl malate dehydratase; LPD, dihydrolipoamide dehydrogenase; MCL, 3-methylcrotonyl-CoA carboxylase; PC, pyruvate carboxylase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase.
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Figure 5. Genes expression changes of autophagy pathway between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, and those in green were downregulated. Enzymes note: ATG1, serine/threonine-protein kinase ULK2; ATG8, GABA(A) receptor-associated protein; ATG13, autophagy-related protein 13; LSB1/2, serine palmitoyltransferase; LST8, the target of rapamycin complex subunit LST8; MSN2/4, zinc finger protein MSN2/4; PKA, protein kinase A; RIM15, serine/threonine-protein kinase RIM15; SCH9, serine/threonine protein kinase SCH9; SNF1, carbon catabolite-derepressing protein kinase; TOR, serine/threonine-protein kinase mTOR; UME6, transcriptional regulatory protein UME6.
Figure 5. Genes expression changes of autophagy pathway between mycelia and spores in M. circinelloides WJ11 under solid–state fermentation. Genes in red were upregulated, and those in green were downregulated. Enzymes note: ATG1, serine/threonine-protein kinase ULK2; ATG8, GABA(A) receptor-associated protein; ATG13, autophagy-related protein 13; LSB1/2, serine palmitoyltransferase; LST8, the target of rapamycin complex subunit LST8; MSN2/4, zinc finger protein MSN2/4; PKA, protein kinase A; RIM15, serine/threonine-protein kinase RIM15; SCH9, serine/threonine protein kinase SCH9; SNF1, carbon catabolite-derepressing protein kinase; TOR, serine/threonine-protein kinase mTOR; UME6, transcriptional regulatory protein UME6.
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Table
Table 1. Fatty acid composition of spores and mycelia from 24 h to 192 h under solid–state fermentation of M. circinelloides WJ11.
Table 1. Fatty acid composition of spores and mycelia from 24 h to 192 h under solid–state fermentation of M. circinelloides WJ11.
ComponentTime (h)Fatty Acid Composition (%)
C14:0C16:0C16:1C18:0C18:1C18:2C18:3
Spores24 1.5 ± 0.317.1 ± 0.42.1 ± 0.35.0 ± 0.433.5 ± 0.517.8 ± 0.122.9 ± 0.3
481.9 ± 0.317.9 ± 0.23.1 ± 0.52.4 ± 0.242.5 ± 0.413.5 ± 0.218.7 ± 0.4
961.9 ± 0.419.4 ± 0.52.8 ± 0.32.4 ± 0.339.8 ± 0.113.5 ± 0.220.5 ± 0.1
1441.9 ± 0.118.0 ± 0.43.0 ± 0.22.4 ± 0.142.5 ± 0.513.5 ± 0.318.5 ± 0.1
1921.8 ± 0.217.7 ± 0.33.0 ± 0.32.3 ± 0.343.9 ± 0.413.0 ± 0.518.1 ± 0.4
Mycelia241.8 ± 0.312.5 ± 0.13.6 ± 0.15.6 ± 0.241.9 ± 0.617.4 ± 0.317.1 ± 0.4
481.4 ± 0.219.6 ± 0.52.8 ± 0.34.8 ± 0.241.9 ± 0.312.4 ± 0.417.1 ± 0.2
961.3 ± 0.120.0 ± 0.32.4 ± 0.34.8 ± 0.241.1 ± 0.212.6 ± 0.217.6 ± 0.5
1441.4 ± 0.119.6 ± 0.52.8 ± 0.14.6 ± 0.542.0 ± 0.312.4 ± 0.517.2 ± 0.2
1921.4 ± 0.319.6 ± 0.22.7 ± 0.24.3 ± 0.342.7 ± 0.611.8 ± 0.217.6 ± 0.5
Table
Table 2. Basic data of six transcriptome samples before analysis.
Table 2. Basic data of six transcriptome samples before analysis.
SampleTotal Raw Reads (M)Total Clean Reads (M)Total Clean Bases (Gb)Clean Reads Q20 (%)Clean Reads Q30 (%)Clean Reads Ratio (%)
MWJ11_145.5743.576.5496.9392.2395.6
MWJ11_243.8242.016.396.8792.0895.86
MWJ11_343.8242.046.3196.992.1395.93
SWJ11_145.5742.976.4596.7891.8894.28
SWJ11_245.5742.876.4396.8992.1994.07
SWJ11_345.5743.176.4896.9192.1994.73
Table
Table 3. Gene manipulation for increasing microbial oil content in different species.
Table 3. Gene manipulation for increasing microbial oil content in different species.
StrainGene Name and Modification MethodLipid Content ChangesReference
Mucor circinelloideshomologously overexpressed DGAT2Bthe TFA content in mycelia increased by 68.0% (from 13.6 to 22.8%) [13]
Arabidopsis thalianaoverexpressed GPAT2 from Jatropha curcas (Acc. No. NBRI-UA-Alm-0406)the oil content was enhanced by 60% (from 33.3 to 53.5 mg/100 mg)[22]
Brassica napus cv Herooverexpressed LPAT from yeast Saccharomyces cerevisiaethe seed oil content increased from 6.1 to 8.2%[23]
Yarrowia lipolyticaACL mutantdecreased by 36%[24]
Yarrowia lipolyticaoverexpressed ACL from Mus musculusthe lipid content increased from 7% to 23%[25]
Aspergillus oryzaemodified the ACL promoterthe fatty acid content increased by 1.7-fold[26]
Aspergillus nidulanslacked ME activity mutant the lipid content was reduced to half of the original[27]
Mortierella alpinahomologously expressed the gene encoding ME isoform Ethe fatty acid content increased by 30% compared to that of the control[28]
Mucor circinelloideshomologously expressed the MEthe lipid content of cells increased from 12% to 30% of the biomass[29]
Yarrowia lipolyticaoverexpressed the ME gene (mce2) from Mortierella alpinathe lipid content was no significant changes[30]
Mucor circinelloideshomologously overexpressed g6pdh1 and g6pdh2 genes encoding G6PDHthe fatty acid content of CDW increased by 23–38% and 41–47%, respectively[31]
Mucor circinelloidesknockout SNF-βthe lipid content increased by 32% (from 19.0% to 25.0%)[32]
Arabidopsis thalianahomologously overexpressed TOR negatively regulated lipid peroxidation[33]
Yarrowia lipolyticaMSN2/4 double mutanttriacylglycerol and steroidal esters were increased[34]
Rhodnius prolixusSilenced Atg6/Beclin1 and Atg8/LC3accumulated higher levels of triglycerides[35]
Mortierella alpinahomologously overexpressed ATG8the fatty acid content increased by approximately 110% compared with the wildtype strain [36]
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Xin, F.; Dang, W.; Chang, Y.; Wang, R.; Yuan, H.; Xie, Z.; Zhang, C.; Li, S.; Mohamed, H.; Zhang, H.; et al. Transcriptomic Analysis Revealed the Differences in Lipid Accumulation between Spores and Mycelia of Mucor circinelloides WJ11 under Solid–State Fermentation. Fermentation 2022, 8, 667. https://doi.org/10.3390/fermentation8120667

AMA Style

Xin F, Dang W, Chang Y, Wang R, Yuan H, Xie Z, Zhang C, Li S, Mohamed H, Zhang H, et al. Transcriptomic Analysis Revealed the Differences in Lipid Accumulation between Spores and Mycelia of Mucor circinelloides WJ11 under Solid–State Fermentation. Fermentation. 2022; 8(12):667. https://doi.org/10.3390/fermentation8120667

Chicago/Turabian Style

Xin, Feifei, Wenrui Dang, Yufei Chang, Ruixue Wang, Hongjuan Yuan, Zhike Xie, Chuanchuan Zhang, Sha Li, Hassan Mohamed, Huaiyuan Zhang, and et al. 2022. "Transcriptomic Analysis Revealed the Differences in Lipid Accumulation between Spores and Mycelia of Mucor circinelloides WJ11 under Solid–State Fermentation" Fermentation 8, no. 12: 667. https://doi.org/10.3390/fermentation8120667

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