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The fungus, Mucor lusitanicus , is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter ( mt ) and 2-oxoglutarate: malate antiporter ( sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation., Competing Interests: 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., (Copyright © 2021 Wang, Mohamed, Bao, Wu, Shi, Song and Yang.)

Canthaxanthin is a reddish-orange xanthophyll with strong antioxidant activity and higher bioavailability than carotenes, primarily used in food, cosmetics, aquaculture, and pharmaceutical industries. The spiking market for natural canthaxanthin promoted researchers toward genetic engineering of heterologous hosts for canthaxanthin production. Mucor circinelloides is a dimorphic fungus that produces β-carotene as the major carotenoid and is considered as a model organism for carotenogenic studies. In this study, canthaxanthin-producing M. circinelloides strain was developed by integrating the codon-optimized β-carotene ketolase gene ( bkt ) of the Haematococcus pluvialis into the genome of the fungus under the control of strong promoter zrt1 . First, a basic plasmid was constructed to disrupt crgA gene, a negative regulator of carotene biosynthesis resulted in substantial β-carotene production, which served as the building block for canthaxanthin by further enzymatic reaction of the ketolase enzyme. The genetically engineered strain produced a significant amount (576 ± 28 μg/g) of canthaxanthin, which is the highest amount reported in Mucor to date. Moreover, the cell dry weight of the recombinant strain was also determined, producing up to more than 9.0 g/L, after 96 h. The mRNA expression level of bkt in the overexpressing strain was analyzed by RT-qPCR, which increased by 5.3-, 4.1-, and 3-folds at 24, 48, and 72 h, respectively, compared with the control strain. The canthaxanthin-producing M. circinelloides strain obtained in this study provided a basis for further improving the biotechnological production of canthaxanthin and suggested a useful approach for the construction of more valuable carotenoids, such as astaxanthin., Competing Interests: 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., (Copyright © 2021 Naz, Yang, Nosheen, Sun, Nazir, Mohamed, Fazili, Ullah, Li, Yang, Garre and Song.)

Cerrena unicolor is an ecologically and biotechnologically important wood-degrading basidiomycete with high lignocellulose degrading ability. Biological and genetic investigations are limited in the Cerrena genus and, thus, hinder genetic modification and commercial use. The aim of the present study was to provide a global understanding through genomic and experimental research about lignocellulosic biomass utilization by Cerrena unicolor . In this study, we reported the genome sequence of C. unicolor SP02 by using the Illumina and PacBio 20 platforms to obtain trustworthy assembly and annotation. This is the combinational 2nd and 3rd genome sequencing and assembly of C. unicolor species. The generated genome was 42.79 Mb in size with an N50 contig size of 2.48 Mb, a G + C content of 47.43%, and encoding of 12,277 predicted genes. The genes encoding various lignocellulolytic enzymes including laccase, lignin peroxidase, manganese peroxidase, cytochromes P450, cellulase, xylanase, α-amylase, and pectinase involved in the degradation of lignin, cellulose, xylan, starch, pectin, and chitin that showed the C. unicolor SP02 potentially have a wide range of applications in lignocellulosic biomass conversion. Genome-scale metabolic analysis opened up a valuable resource for a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases that provide insights into the genetic basis and molecular mechanisms for lignocellulosic degradation. The C. unicolor SP02 model can be used for the development of efficient microbial cell factories in lignocellulosic industries. The understanding of the genetic material of C. unicolor SP02 coding for the lignocellulolytic enzymes will significantly benefit us in genetic manipulation, site-directed mutagenesis, and industrial biotechnology.

Previously, we presented a novel approach for increasing the consumption of xylose and the lipid yield by overexpressing the genes coding for xylose isomerase (XI) and xylulokinase (XK) in Mucor circinelloides. In the present study, an in-depth analysis of lipid accumulation by xylose metabolism engineered M. circinelloides strains (namely Mc-XI and Mc-XK) using corn straw hydrolysate was to be explored. The results showed that the fatty acid contents of the engineered M. circinelloides strains were, respectively, increased by 19.8% (in Mc-XI) and 22.3% (in Mc-XK) when compared with the control strain, even though a slightly decreased biomass in these engineered strains was detected. Moreover, the xylose uptake rates of engineered strains in the corn straw hydrolysate were improved significantly by 71.5% (in Mc-XI) and 68.8% (in Mc-XK), respectively, when compared with the control strain. Maybe the increased utilization of xylose led to an increase in lipid synthesis. When the recombinant M. circinelloides strains were cultured in corn straw hydrolysate medium with the carbon-to-nitrogen ratio (C/N ratio) of 50 and initial pH of 6.0, at 30 °C and 500 rpm for 144 h, a total biomass of 12.6-12.9 g/L with a lipid content of 17.2-17.7% (corresponding to a lipid yield of 2.17-2.28 g/L) was achieved. Our study provides a foundation for the further application of the engineered M. circinelloides strains to produce lipid from lignocelluloses.

Background: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet., Objectives: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation., Results: Two genes for α-subunit, one for β-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain., Conclusion: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-β, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Mucor circinelloides is a valuable oleaginous filamentous fungus rich in γ-linolenic acid (GLA, 18:3; n-6), which is beneficial for human health. Our previous comparative proteomic analysis between high lipid-producing M. circinelloides WJ11 and low lipid-producing M. circinelloides CBS 277.49 indicated that glucose 6-phosphate dehydrogenase (G6PDH) and β-isopropylmalate dehydrogenase (IPMDH) were closely involved in lipid accumulation. Transcription analysis suggested that in the strain WJ11, g 6 pdh 1 and g 6 pdh 2, which encode G6PDH, and leuB , which encodes IPMDH, could be the key genes regulating lipid accumulation. To further analyze the effects of these three genes (i.e., g 6 pdh 1, g 6 pdh 2, and leuB ) on lipid accumulation, we respectively overexpressed these genes from M. circinelloides WJ11 in defective CBS 277.49 strains in this study. The results showed that overexpression of g 6 pdh 1 and g 6 pdh 2 genes from strain WJ11 increased the fatty acid content of cell dry weight by 23-38 and 41-47%, respectively, compared with the control strain. Furthermore, overexpression of the leuB gene from strain WJ11 increased the fatty acid content of cell dry weight by up to 67-73%. These results suggest that g 6 pdh 1, g 6 pdh 2, and especially leuB genes play important roles in regulating fatty acid synthesis in M. circinelloides .

Objectives: To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database., Results: Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 °C, and stability between 40 and 50 °C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates., Conclusion: The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.

Background: Mitochondrial and cytoplasmic malate transporter proteins are responsible for transmembrane transport of malate, thereby linking malate metabolism in various subcellular regions of the cell. These transporters play an important role in fatty acid biosynthesis of oleaginous microorganisms. Our previous studies have found that lipid content of the recombinant Mucor circinelloides (M. circinelloides) strain with mitochondrial malate transporter (mt) gene overexpression was increased by 70%, while that of strain with mt gene knockout was decreased by 27%. However, the mechanism of malate transporter promoting the transport of mitochondrial malate and citrate related to lipid accumulation is not clear. Therefore, 13 C-labeled glucose metabolic flux analysis was carried out to identify the metabolic network topology and estimate intracellular fluxes of genetically engineered M. circinelloides strains for the purpose of better understanding the roles of malate transporters in citrate transport systems and lipid accumulation., Results: The metabolic flux distribution analysis suggested that tricarboxylic acid (TCA) cycle flux ratio of mt-overexpression strains was decreased compared to that of the control strain, but in contrast, glyoxylic acid (GOX) cycle flux ratio was increased. Accordingly, the mt-knockout strain showed an opposite phenomenon with a higher TCA cycle flux ratio and a lower GOX cycle flux ratio than the control strain. GOX cycle might be more effective than TCA cycle in producing malate and oxaloacetate replenishment. Moreover, a relatively higher flux ratio of the pentose phosphate (PP) pathway was obtained in mt-overexpression strains, but no significant difference in the malic enzyme flux between recombinant strains and the control strain. Our results confirmed that PP pathway might play an important role for supplying NADPH and malic enzyme is not a limiting factor for fatty acid synthesis in oleaginous fungus M. circinelloides strains., Conclusion: Intracellular metabolic flux information suggested that mt-overexpression strains had higher flux in PP pathway and GOX cycle, lower flux in TCA cycle, and no difference in malic enzyme cycle. Together, the role of malate transporter was assumed to further participate in transporting cycle of acetyl-CoA and drive PP pathway to supply NADPH required for lipid accumulation in recombinant M. circinelloides strains.

Natural and de-novo biosynthesized phyto-compounds have gained much significance because of their non-controversial nutritional, health and safety benefits as compared with chemically synthesized commercially rivalry antioxidants. However, none of natural de-novo biosynthesized phyto-compounds has been commercially available and used in customary food business and processing. In this study, efficacy of sesame seed extracts (SSEs) in stabilizing sunflower oil during storage has been studied. Fine powder of sesame seed was extracted in different solvents. The results showed that significant differences in extractability of different solvents and maximum extraction yield (29.48%) were achieved with methanol. The antioxidant components and capability of different extracts were further investigated and evaluated via total phenolic contents, DPPH radical scavenging activity and β-carotene/linoleic acid calorimetric assays respectively. Being highest in yield and antioxidant potential, methanolic extract was used; three different concentrations of SSE (500, 750, and 1000 μL) were added in 100 mL of sunflower oil to further evaluate its oxidative stability. Sensory and oxidative analysis of baked product from these groups was also evaluated., Competing Interests: Compliance with ethical standardsThe authors declare no conflict of interest.

In this study, optimization of growth, lipid and DHA production of Aurantiochytrium SW1 was carried out using response surface methodology (RSM) in optimizing initial fructose concentration, agitation speed and monosodium glutamate (MSG) concentration. Central composite design was applied as the experimental design and analysis of variance (ANOVA) was used to analyze the data. ANOVA analysis revealed that the process which adequately represented by quadratic model was significant (p < 0.0001) for all the response. All the three factors were significant (p < 0.005) in influencing the biomass and lipid data while only two factors (agitation speed and MSG) gave significant effect on DHA production (p < 0.005). The estimated optimal conditions for enhanced growth, lipid and DHA production were 70 g/L fructose, 250 rpm agitation speed and 10 g/L MSG. Consequently, the quadratic model was validated by applying the estimated optimum conditions, which confirmed the model validity where 19.0 g/L biomass, 9.13 g/L lipid and 4.75 g/L of DHA were produced. The growth, lipid and DHA were 28, 36 and 35% respectively higher than that produced in the original medium prior to optimization.

Objective: We aimed at characterizing a malic enzyme isoform V from Mucor circinelloides., Methods: me1 gene encoding malic enzyme isoform V was amplified and cloned into expression vector pET28a. High-purity recombinant protein BLME1 was obtained by affinity chromatography using. Ni-NTA column and characterized subsequently., Results: The optimum conditions were pH at 8.0 and temperature at 33 degrees C. Under optimum conditions, BLME1 activity achieved 92.8 U/mg. The K(m) for L-malate and NADP+ were 0.74960 ± 0.06120 mmol/L and 0.22070 ± 0.01810 mmol/L, the V(max) for L-malate and NADP+ were 72.820 ± 1.077 U/mg and 86.110 ± 1.665 U/mg, respectively. In addition, ions played important roles in BLME1 activity; several ions such as Mn2+, Mg2+, Co2+, Ni2+ could activate BLME1, whereas Ca2+, Cu2+ could be used as inhibitors. Additionally, the metabolic intermediates such as oxaloacetic acid and α-ketoglutaric acid inhibited the activity of BLME1, whereas succinic acid activated it., Conclusion: A malic enzyme isoform V from Mucor circinelloides was characterized, providing the references for further studies on this enzyme.

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Moonlighting proteins, characterized by their multiple autonomous functions, have been detected in bacteria. Surprisingly, many of these proteins are conserved and involved in metabolic pathway or the cell stress response. They localise to the bacterial surface to take on additional activities, which have been hypothesised to contribute to bacterial virulence or bacterial benefit. In this review, we compare the functions of moonlighting proteins in bacteria, describe the structural basis of moonlighting functions, and summarise the regulation of secretion and localisation of moonlighting proteins.