As for the de novo lipid accumulation, concentrations of nitrogen and carbon sources respectively determine the biomass content and the quantity of lipids in general (36). Accordingly, the C/N ratio is significant for the accumulated lipid content and the oleaginous microbial biomass (36–39). Previous studies demonstrate that the lipid accumulation is boosted at a C/N molar ratio of greater than 20. It is worth noting that the lipid production declines instead at the C/N ratio higher than 70 in some cases (40). Therefore, to achieve a high-level lipid accumulation, the initial C/N molar ratio should be optimized (41–43). For the lipid fermentation of the Thraustochytridae sp. PKU#Sw8, the increase in DHA production coincided with the up-regulation of gene expression under nitrogen-deficient culture conditions (44). Chen et al. (45) optimized the culture of Thraustochytrid sp. PKU#SW8 under optimal culture conditions (glycerol, 20 g/L; peptone, 2.5 g/L; 80% seawater; pH 4.0; 28°C), the cell mass, DHA concentration and yield of PKU#SW8 were increased to 7.5 ± 0.05 g/L, 2.14 ± 0.03 g/L and 282.9 ± 3.0 mg/g, respectively, on a 5-L scale fermentation.

Cellular lipid content and lipid yield were 62.2% and 0.205 g/g glucose, respectively, using a medium with a carbon to nitrogen (C/N) molar ratio of 6.1 and a C/P molar ratio of 9,552 (46), which means that the accumulation of lipid can also be regulated by limiting phosphorus in the medium. As a consequence, the regulation of phosphorus and sulfur limitation is of great significance for the production of lipids from nitrogen-rich crude materials (41, 45, 47, 48).

Some small molecules can also regulate the accumulation of lipids (37, 48, 49). Li et al. (50) cultured Thraustochytriidae sp., a kind of marine oleaginous protists, by addition of different levels of sodium nitrate (1-50 mM) or urea (1-50 mM) in fermentation culture has a significant effect on fatty acid synthesis. They found that urine (50 mM) culture the cells accumulated 1.16 times the ω-3 PUFAs, of which DHA accounted for 49.49% and docosapentaenoic acid (DPA) was 5.28% compared with the original culture conditions. To sum up, it is easy to control lipid accumulation by small molecules, which is of great significance for the optimization of lipid production conditions (48, 51).

The accumulation of biomass and lipid-synthesizing fungi in any oily substance is highly affected by factors such as pH, temperature, light, and ventilation. Temperature change is also one of the factors affecting lipid accumulation (52, 53). They found that the low temperature has a significant impact on the formation of DHA, which can increase the DHA content from 43 to 65% of the total fatty acids. Low temperature may increase DHA content by facilitating a relatively large amount of substrates to enter the polyketide synthase (PKS) pathway (52).

Generally speaking, there are three ways of microbial fermentation: batch culture, fed-batch culture and continuous culture (38, 54–57). The batch culture is the most widely used for lipid fermentation. Wang et al. (58) studied Schizochytrium sp. PKU#Mn4 and Thraustochytrid sp. PKU#Mn16, found that the largest DHA yields were 21% and 18.9%, and the yields were 27.6 mg/L-h and 31.9 mg/L-h, respectively in in 5-L bioreactor fermentation operated with optimal conditions and dual oxygen control strategy. The production of DHA increased by 3.4 times and 2.8 times (g/L) respectively. Rhizopus sp. using solid-state fermentation and submerged fermentation can produce valuable alternative feed ingredient due to their high protein and the well-balanced lipid content and amino acid profile (59).

In addition, electro-fermentation is a promising technology that can improve the performance of biological processes. When lipids are produced yeast R. toruloides under electro-fermentation conditions, the proportion of saturated FAs increases significantly from 37 to 50% (60).

Take DHA as an example for cost estimation (72). If all the carbon sources needed to produce DHA were glucose, the amount of glucose required to produce 1 ton of biomass would be 2.78 tons. At the 2021 glucose price of us $903.9 per ton, it would cost US $2,512.84 to produce one ton of biomass. If there is only 50% lipid content in 1 ton of biomass, 40% of the lipid content is DHA (73). That's 0.2 tons of DHA. If one ton of DHA is produced, the calculated cost of glucose is $12,564.2. All this takes into account only glucose substrates, but if you add in other cost factors, including water and electricity, publicity, equipment, and so on, the cost of DHA increases further (72).

Recovery processes downstream of the fermenter typically contribute 60–80% to the cost of production of a fermentation product, therefore the fermentation step contributes only around 20–40% to the total production cost. The above analysis leads to the conclusion that the Schizochytrium limacinum grown on glucose cannot provide DHA cheaper than fish oil at present. If the biomass was used simply as an aquaculture feed additive, the downstream processing requirements would mostly disappear, although on the basis of equal DHA content, the biomass would still be more expensive than fish oil. Hence the need for cheaper nutrients for growing thraustochytrids (72).

In the future development, it is very important to improve production efficiency and reduce cost. First of all, in terms of carbon sources, the focus should be on replacing glucose while maintaining high biomass and lipid yields. In addition, lignocellulose hydrolysate may prove to be an inexpensive source of carbon for biomass production, which can efficiently metabolize xylose, and xylose metabolic engineering may help reduce fermentation costs (74). Metabolic engineering is also very important (75, 76). Therefore, strains should be improved, complete metabolic flux analysis should be carried out, and the protein engineering field should be evaluated with the goal of metabolic engineering, etc., in order to maximize the fatty acid production in the biomass.

Taking into consideration from another angle, developing new valuable products such as enzymes, and cell wall polysaccharides, during fungal fermentation besides fatty acids, would effectively reduce the cost. This “fungal-based biorefinery” strategy has not been applied in the studies, it may be the another choice to make the production of fatty acids more feasible (77).

Lignocellulose is constituted by hemicellulose, cellulose and lignin (91). In recent years, lignocellulosic biomass has been recognized as a potential alternative feedstock to produce biofuels (9). The fatty acid production with lignocellulosic biomass includes the following two steps: (1) the degradation of biomass to corresponding monosaccharides by heat-acid treatment or enzyme hydrolysis, and (2) the biodegradable sugar fermentation by promising oleaginous microorganisms (92–95). Lignocellulose cannot be directly utilized, but must be hydrolyzed, which produces compounds that inhibit the growth of fungi, such as furan aldehydes, weak acids, and aromatic compounds, during the pretreatment process (96). The cumulative deleterious effects of some inhibitors (such as furfural, formic acid, acetic acid, and vanillin) on fatty acid accumulation in oleaginous fungi have been investigated (96, 97). Intasit et al. (98) used an integrated biotechnology, fungi and yeast to bioconvert lignocellulosic biomass into biodiesel, first pretreatment of the fungus, the fungus Aspergillus tubingensis TSIP 9 lipid yield 121.4 ± 2.7 mg/g-EFB (empty fruit bunch), the integrated biotechnology can greatly facilitate the conversion of lignocellulosic biomass to biodiesel feedstock is a cost-effective and sustainable biotransformation. Zhang et al. (84) deeply analyzed the effects of corn stover hydrolyzate on lipid accumulation by using xylose metabolism engineering strains of M. circinelloides strains. The results showed that the fatty acid contents of the engineered M. circinelloides strains were increased by 19.8% (in Mc-XI) and 22.3% (in Mc-XK), respectively, compared with the control strain.

Glucose and xylose coexist in lignocellulose hydrolysate. Lipid-producing yeasts consume glucose first and then xylose, and even some lipid-producing yeasts are unable to utilize xylose. Therefore, lignocellulose hydrolysate suffers from long fermentation cycle and low substrate utilization.

Jerusalem artichoke is a kind of perennial plant resistant to barren, cold and drought. The planting of Jerusalem artichoke should not occupy cultivated land and other agricultural lands (99). The storage form of sugar in Jerusalem artichoke is inulin, which is a polyfructose linked by β-2,1 glycosidic bond with a glucose residue at the end (100). Yeast or molds can accumulate large amounts of lipids from inulin hydrolysates (101–103). In the medium containing inulin, fatty acids can be produced and the lipid content and biomass of cells can be changed. By converting the inulinase gene, the gene accumulates higher fatty acids (100). Aurantiochytrium sp. YLH70 can produce lipid in a medium with 695 mL/L hydrolysate of Jerusalem artichoke. The biomass higher biomass (32.71 g/L) and DHA content (46.9% of the total fatty acid) (88).

The combination of low-cost organic compounds contained in agro-commercial waste and the cultivation of lipid-producing microorganisms can effectively achieve the effect of accumulating lipids (104, 105). Lipid-producing microorganisms use some forms of carbon sources and nutrients for growth and fatty acid accumulation. Organic waste usually contains organic particles, which may be an ideal and inexpensive substrate for microbial fatty acid production, but the chemical composition of organic waste affects the lipid production of different species (104, 106). Lipid-producing yeasts can also transform commercial organic wastes into lipid (105, 107). Crude glycerol is a by-product of biodiesel industry, which is usually treated as commercial waste (64). The engineered strain of the filamentous fungus Ashbya gossypii can produce microbial lipids, whose efficiency is improved by three genomic manipulation methods. Using organic commercial waste as a raw material, the strain can accumulate lipid at about 40% of DCW (85). The use of commercial waste to produce lipid is also of great significance to environmental governance.

The synthesis of FAs in microbial cells requires acetyl-CoA acting as the precursor of FAs and a sufficient supply of NADPH to provide reducibility for the synthesis. It is generally believed that when nitrogen is depleted, the activity of AMP-deaminase increases. This can supplement NH4+ for various metabolisms, decrease intracellular AMP level and the activity of isocitrate dehydrogenase (IDH) activated and cause the accumulation of isocitrate in mitochondria (108). Aconitase (AT) in mitochondria is able to catalyze the conversion of over-accumulated isocitrate to citric acid. The citric acid is then transported to the cytoplasm, where the ATP-citrate lyase (ACL) helps its cracking catalysis to acetyl-CoA and oxaloacetic acid (OAA). As a result, abundant acetyl-CoA is produced as the precursor of FAs (109). Acetyl-CoA directly participates in the FA synthesis, while oxaloacetate is first reduced to malate dehydrogenase (MD) and then undergoes oxidative decarboxylation in the presence of malic enzyme (ME) to release NADPH (110). Studies have shown that ME can regulate lipid accumulation in oleaginous microorganisms (111). Accordingly, if the activity of ME is inhibited, the lipid accumulation will decrease. This is because although many reactions in the cellular metabolic network can produce NADPH, the NADPH required by FA synthesis comes almost entirely from ME-catalyzed reactions (111). Catalyzed by acetyl-CoA carboxylase (ACC), acetyl-CoA and CO₂ were transformed into monoyl-CoA. Multiple reactions can be continued in the presence of FAS. Acetyl-CoA combines with ACP to form acetyl-ACP, and malonyl-CoA and acetyl-CoA yield acyl-CoA via a condensation reaction. The three steps of reduction, dehydration and re-reduction are continued, and the FA chain extends by two carbon atoms. NADPH is taken as the reducing cofactor by FAS, and two NADPH molecules are required in each step of the acyl-CoA chain elongation. The chain is repeatedly extended to the desired length of the synthetic organism, and then some FAs are desaturated to form unsaturated FAs (112–114). The related reactions and enzymes are shown in Figure 2.

The synthesis of some special FAs in microorganisms may be related to PKS. PKS is a sophisticated molecular machine responsible for synthesizing polyketides, which are natural products from the secondary metabolism with similarities to FA (115–117).

The DHA synthesis in Thraustochytrium, Schizochytrium limacinum, Aurantiochytrium sp. is considered to involve the PKS pathway. The successive condensation reactions of precursors catalyzed by PKS can form a variety of polyketides, and then numerous complex compounds are generated through modification reactions such as methylation, redox, glycosylation and hydroxylation (116, 118). In terms of structure and properties, PKS can be divided into three types: modular type (type I), repetition type (type II) and chalcone type (type III). The PKS found in fungi is mostly type I, which is large multifunctional proteins encoded by a single gene. It has multiple similar modules, and some domains are reused in the compound synthesis (117). The type I has a multidomain architecture whose active sites were distributed on large modules, while the type II is composed of monofunctional enzymes, with catalytic sites separated on different proteins (119). Type III polyketide synthases (PKSs) produce secondary metabolites with diverse biological activities, including antimicrobials (120, 121). In contrast to types I and II, type III PKSs are dimers of ketone synthases that undergo a series of reactions such as initiation of primer substrates, decarboxylation condensation of extended substrates, ring closure of growing polyketide chains and aromatization, and produce a variety of biologically active aromatic compounds (122). The PKS pathway of some species is shown in Figure 3 (119).

At present, TAGs are viewed as an important form of carbon source and energy storage unit in microorganisms. The TAG synthesis pathway is triggered at the point when carbon is abundant but nitrogen is depleted in the medium. The FA biosynthesis in cytosol involves several reactions which convert the precursor, acetyl-CoA, into long-chain FAs (123). The synthesized acyl-CoA has a typical chain length of 18 or 16 carbon atoms. These C18:0 and C16:0 molecules are then delivered to the endoplasmic reticulum (ER) to be further elongated and desaturated (124–126). The TAG synthesis requires a variety of enzymes, and phosphatidic acid (PA) and diglyceride are two key intermediates in anabolic metabolism.

Generally, the TAG synthesis involves the Kennedy pathway, with glycerol-3-phosphate (G3P) and acyl-CoA serving as the direct substrates in the process (126, 127). The first step of TAG assembly is the conversion of G3P into lysophosphatidic acid (LPA) with G3P acyltransferase (SCT1) as the catalyst (128). Subsequently, the LPA acylation occurs to generate PA in the presence of LPA acyltransferase (SLC1). Further, under the action of phosphatidic acid phosphatase (PAP), PA is dephosphorylated to produce diacylglycerol (DAG) (129). Finally, TAG is formed after the DAG acylation at the sn-3 position by an acyl-CoA-independent or acyl-CoA-dependent reaction (130, 131). Regarding the acyl-CoA-independent reaction, glycerophospholipid is the acyl group donor and phospholipid DAG acyl-transferase (LRO1) catalyzes the process. With respect to the acyl-CoA-dependent reaction, acyl-CoA acts as the final donor of acyl group and DAG acyltransferases, i.e., DGA1 or DGA2, are responsible for the catalysis. Furthermore, acting as the acyl-transferase of an acyl-CoA-dependent reaction, the steryl ester synthetase, which is encoded by ARE1, is proved able to promote the DAG acylation (123). The related enzymes and specific reactions are displayed in Figure 2.

The TAGs accumulated in cells store energy for them. Once carbon becomes insufficient, TAGs would be degraded with acetyl-CoA release so that the cellular metabolism can be maintained. Initially, free fatty acids (FFAs) can be produced from TAGs in the presence of intracellular lipases (TGL3 and TGL4) (132). These FFAs are activated by FAA1 to generate acyl-CoAs, which are then transported by specific transporters (Pxa1 and Pxa2) into the peroxisome (133). Alternatively, the transportation of the produced FFAs into the peroxisome takes place first, followed by the activation to acyl-CoAs therein by acyl/aryl-CoA ligase (AAL) (134). Afterward, acyl-CoAs are degraded in the peroxisome via the β-oxidation pathway to generate acetyl-CoA.

Previous studies have shown that the expression of acetyl-CoA carboxylase (encoded by accA, accB, accC and accD) and thioesterase I (encoded by tesA) in Escherichia coli can speed up the FA synthesis by six times. This suggests that the catalytic reaction of acetyl-CoA carbohydrase is a rate-limiting step for FA synthesis (150–153). Overexpression of both heterologous Δ-15 desaturase (Δ-15D) sourced from flax and endogenous genes (SCD, ACC1, DGA1 and Δ-12D) along with the deletion of endogenous MFE1 and PEX10 can yield a superior lipid producer (140). It is able to produce lipid with content of 77.8% and titer of 50 g/L using glucose as the substrate in a 5 L stirred-tank bioreactor (140).

Han et al. (147) overexpressed in Schizochytrium sp. ATCC 20888 using the strong constitutive promoter ccg1, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC). The lipid content of overexpressed strains obtained by fermentation culture can reach a maximum of 73.0%, an increase of 38.3%. However, the ACC1 gene from mold M. rouxii is expressed in the Hansenula polymorpha, and the fat content is only 40% higher than the original (142), possibly because the fungus has a more powerful metabolic regulation system.

Diacylglycerol acyltransferase (DGAT) catalyzes the conversion of DAG and acetyl-CoA to TAG, which is the last step in the TAG synthesis (32, 154, 155). When grown in batch conditions and minimal medium, the resulting strain consumes 12 g/L cellulose and accumulates 14% (DCW) lipids (156). Blazeck et al. (141) synergistically regulated multiple key genes related to the degradation and biosynthesis of lipids in Y. lipolytica with a combinatorial strategy, including MFE1, AMPD, PEX10, MAE, DGA1, ACL1, ACL2 and DGA2, with 57 distinct genotypes generated. The double deletion of MFE1 and PEX10 and the overexpression of DGA1 were most effective for modification. After the optimization of bioreaction conditions, the engineered strain had a lipid content of 71% (DCW) and a lipid titer of 25 g/L. Markedly, a 60-fold improvement was realized over the original strain.

IDH and ME probably play a crucial role in the accumulation of lipids. When glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (PGD), IDH and ME are overexpressed in M. alpina, total FAs can be increased by 1.7 times; while ME2 is more effective in desaturation, and the content of arachidonic acid (AA) is increased by 1.5 times compared to the control (157). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is an enzyme highly conserved in the glycolytic pathway. The lipid-producing filamentous fungus M. alpina was used to characterize two copies of the gene encoding GAPDH, and the overexpression strain MA-GAPDH1 increases the lipid content by about 13% (143). First, the total lipid accumulation was increased by overexpressing a malic enzyme from Crypthecodinium cohnii to elevate NADPH supply. Then, the inhibition effect on acetyl-CoA carboxylase was relieved by overexpressing a codon-optimized ELO3 gene from M. alpina. After the above two-step engineering, contents of DHA was increased by 1.39-fold, reaching a level of 26.70% of dry cell weight, respectively (148).

The PKS cluster genes are supposed to synthesize PUFAs in S. limacinum. Ling et al. (146) improved lipid production domain expression by homologous recombination knocking out two enolate reductase (ER) genes located on the PKS cluster. The addition of triclosan as a modulator of the ER domain resulted in a 51.74% increase in PUFA production and a 47.63% increase in lipid production.

The main competitive pathways blocking the lipid accumulation in microbial cells include β-oxidation of FAs, synthesis of phospholipids and conversion of phospho-enol-pyruvic acid (PEP) to oxalacetic acid. The β-oxidation occurs in peroxisomes, and peroxisome biogenesis is generally downregulated by the deletion of PEX3, PEX10 and PEX11 so that the degradation of TAGs can be prevented in commercial strains (158, 159). As an important intermediate metabolite, malate, its subcellular location and concentration have significant effects on fungal lipid metabolism. Yang et al. (149) deleted the two plasma membrane malate transporters “2-oxoglutarate:malate antiporter” (named SoDIT-a and SoDIT-b) of M. circinelloides WJ11 and analyzed their effects on growth ability, lipid accumulation and metabolism. Their results showed that the lipid content of the mutant was increased by ~10–40% compared to the control strain, indicating that defects in plasma membrane malate transport lead to an increase in malate for lipid synthesis.

Additionally, the genes involved in the β-oxidation pathway are often the deletion targets to increase lipid accumulation (160). After the characterization and deletion of the YlGSY1 gene encoding glycogen synthase, Bhutada et al. (144) increased the TAG accumulation of the engineered strain by 60% as compared with the wild-type strains. This proves that glycogen synthesis is a competing pathway, and its elimination is beneficial for the production of neutral lipids.

In addition to the above-mentioned polyunsaturated fatty acids, some of these strains can also produce high levels of squalene and carotenoids, two other compounds of commercial value with rapidly growing market potential (182). Squalene has antioxidant and anticancer activities with broad applications in food and cosmetics industries. Besides, squalene has been used as vaccine adjuvant in vaccine formulations (43, 183). Since, the demand for squalene has increased during the last decade, microbial production of squalene has been investigated as a promising alternative source for traditional extraction methods from shark liver or plant lipids (184). Microbial strains are capable of producing non-polluting, low-cost, high-quality and sustainable sources of squalene, which is the main direction of the lipid-based biofuel industry.

Aurantiochytrium strains have the potential to produce large amounts of squalene, and Aurantiochytrium is known for its potential to produce large amounts of polyunsaturated DHA on a large scale (185). Furthermore, Thraustochytrid reported the co-production of squalene and DHA from inexpensive feedstocks such as organic solvent pretreatment spruce hydrolysis (186).