Mucor circinelloides is one of few oleaginous fungi that produces a useful oil rich in γ-linolenic acid, but it usually only produces <25% total lipid. Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell dry weight. In this study, we have systematically analyzed the global changes in protein levels between the high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation phase through comparative proteome analysis. Proteome analysis demonstrated that the branched-chain amino acid and lysine metabolism, glycolytic pathway, and pentose phosphate pathway in WJ11 were up-regulated, while the activities of tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were retarded compared with CBS 277.49. The coordinated regulation at proteome level indicate that more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in WJ11 compared with CBS 277.49.Keywords: amino acid metabolism; carbon metabolism; comparative proteome; lipid accumulation; Mucor circinelloides;

To generate lycopene-overproducing strains of the fungus Mucor circinelloides with interest for industrial production and to gain insight into the catalytic mechanism of lycopene cyclase and regulatory process during lycopene overaccumulation.Three lycopene-overproducing mutants were generated by classic mutagenesis techniques from a β-carotene-overproducing strain. They carried distinct mutations in the carRP gene encoding lycopene cyclase that produced loss of enzymatic activity to different extents. In one mutant (MU616), the lycopene cyclase was completely destroyed, and a 43.8% (1.1 mg/g dry mass) increase in lycopene production was observed in comparison to that by the previously existing lycopene overproducer. In addition, feedback regulation of the end product was suggested in lycopene-overproducing strains.A lycopene-overaccumulating strain of the fungus M. circinelloides was generated that could be an alternative for the industrial production of lycopene. Vital catalytic residues for lycopene cyclase activity and the potential mechanism of lycopene formation and accumulation were identified.

Conjugated linoleic acid (CLA) has been shown to exert various potential physiological properties including anti-carcinogenic, anti-obesity, anti-cardiovascular and anti-diabetic activities, and consequently has been considered as a promising food supplement. Bacterial biosynthesis of CLA is an attractive approach for commercial production due to its high isomer-selectivity and convenient purification process. Many bacterial species have been reported to convert free linoleic acid (LA) to CLA, hitherto only the precise CLA-producing mechanisms in Propionibacterium acnes and Lactobacillus plantarum have been illustrated completely, prompting the development of recombinant technology used in CLA production. The purpose of the article is to review the bacterial CLA producers as well as the recent progress on describing the mechanism of microbial CLA-production. Furthermore, the advances and potential in the heterologous expression of CLA genetic determinants will be presented.

Functional genomics of filamentous fungi has gradually uncovered gene information for constructing ‘cell factories’ and controlling pathogens. Available gene manipulation methods of filamentous fungi include random integration methods, gene targeting technology, gene editing with artificial nucleases and RNA technology. This review describes random gene integration constructed by restriction enzyme-mediated integration (REMI); Agrobacterium-mediated transformation (AMT); transposon-arrayed gene knockout (TAGKO); gene targeting technology, mainly about homologous recombination; and modern gene editing strategies containing transcription activator-like effector nucleases (TALENs) and a clustered regularly interspaced short palindromic repeat/associated protein system (CRISPR/Cas) developed in filamentous fungi and RNA technology including RNA interference (RNAi) and ribozymes. This review describes historical and modern gene manipulation methods in filamentous fungi and presents the molecular tools available to researchers investigating filamentous fungi. The biggest difference of this review from the previous ones is the addition of successful application and details of the promising gene editing tool CRISPR/Cas9 system in filamentous fungi.

Eggs enriched with long-chain polyunsaturated fatty acids (PUFAs) are considered an important nutrition source. Mortierella alpina is a food-grade oleaginous fungus with a high level of PUFAs, especially arachidonic acid (AA) and eicosapentaenoic acid (EPA). In this study, hens were randomly assigned to three diet groups: control, 5% and 10% M. alpina. Our data indicated that EPA, being converted to docosahexaenoic acid (DHA), and AA were accumulated in the yolk after 10 days of feeding. The amount of DHA tripled and AA doubled compared with the control eggs. The ratio of ω-6 to ω-3 PUFAs in those eggs decreased from approximately 13:1 to 8:1. These results suggest that M. alpina may represent a valuable source for producing functional eggs enriched with DHA and AA.

Lactobacillus plantarum ZS2058 is an efficient producer of conjugated linoleic acid (CLA) in vitro. To investigate whether L. plantarum ZS2058 produces CLA in vivo and exerts beneficial effects through CLA, an acute colitis model was induced with dextran sodium sulfate (DSS) in C57BL6/J mice. The mice were treated with L. plantarum ZS2058, L. plantarum ST-III, CLA, or vehicle 7 days before modeling until the end of modeling which lasts for seven days. Compared to L. plantarum ST-III, L. plantarum ZS2058 significantly inhibited the increase of the disease activity index (DAI), colon shortening and myeloperoxidase activity in colitic mice. L. plantarum ZS2058 treatment improved the histological damage, protected the colonic mucous layer integrity and significantly attenuated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), while up-regulated the expression of colonic anti-inflammatory cytokine IL-10 and nuclear receptor PPARγ. Furthermore, colonic CLA concentrations were significantly increased in response to L. plantarum ZS2058 treatment, which demonstrates that L. plantarum ZS2058 prevents colitis via producing CLA locally.

Dietary fatty acids (FA) are increasingly recognized as major biologic regulators and have properties that relate to health outcomes and disease. Conjugated linoleic acid (CLA) is a generic term denoting a group of isomers of linoleic acid (C18:2, n-6) with a conjugated double bond. CLA has attracted increased research interest because of its health-promoting benefits and biological functions. In a variety of studies, CLA has been shown to impact immune function and has protective effects against cancer, obesity, diabetes, and atherosclerosis in animal studies and in different human cell lines. Studies investigating the mechanisms involved in the biological functions of CLA are emerging with results from both in vivo and in vitro studies. Most of the biological effects have been attributed to the c9,t11-CLA and t10,c12-CLA isomers. The purpose of this review is to discuss the effects of CLA on health and disease and the possible mechanisms for CLA activities.

To increase the commercial value of oleaginous fungus Mortierella alpina by incorporation of trans-10,cis-12 conjugated linoleic acid (CLA) into the polyunsaturated fatty acids (PUFAs) of M. alpina via Propionibacterium acnes isomerase (PAI) conversion.The PAI gene and the codon optimization version were heterologously expressed in M. alpina via Agrobacterium tumefaciens-mediated transformation (ATMT). Coding usage modification significantly improved the translation of PAI transcripts and trans-10,cis-12 CLA was produced up to 1.2 mg l−1, which corresponds to approx. 0.05 % of the total fatty acid (TFA). Since PAI prefers free linoleic acid as a substrate rather than any other forms, 5 μM long-chain acyl CoA synthetase inhibitor was added and the trans-10,cis-12 CLA content increased approx. 24-fold to 29 mg l−1, reaching up to 1.2 % (w/w) of the TFA in M. alpina.Heterologous expression of PAI in M. alpina by ATMT methods is a practicable way in biosynthesis of CLA and this system may be a feasible platform for industrial production of CLA.

Malic enzyme (ME) catalyses the oxidative decarboxylation of l-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n−6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina.

The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms.

Lactose intolerance is a serious global health problem. A lactose hydrolysis enzyme, thermostable \(\upbeta \)-galactosidase, BgaB (from Geobacillus stearothermophilus) has attracted the attention of industrial biologists because of its potential application in processing lactose-containing products. However, this enzyme experiences galactose product inhibition. Through homology modeling and molecular dynamics (MD) simulation, we have identified the galactose binding sites in the thermostable \(\upbeta \)-galactosidase BgaB (BgaB). The binding sites are formed from Glu303, Asn310, Trp311, His354, Arg109, Phe341, Try272, Asn147, Glu148, and H354; these residues are all important for enzyme catalysis. A ligand–receptor binding model has been proposed to guide site-directed BgaB mutagenesis experiments. Based upon the model and the MD simulations, we recommend mutating Arg109, Phe341, Trp311, Asn147, Asn310, Try272, and His354 to reduce galactose product inhibition. In vitro site-directed mutagenesis experiments confirmed our predictions. The success rate for mutagenesis was 66.7 %. The best BgaB mutant, F341T, can hydrolyze lactose completely, and is the most promising enzyme for use by the dairy industry. Thus, our study is a successful example of optimizing enzyme catalytic chemical reaction by computer-guided modifying the catalytic site of a wild-type enzyme.

The glycoside hydrolase family 42 (GH42) of thermophilic microorganisms consists of thermostable β-galactosidases that display significant variations in their temperature optima and stabilities. In this study, we compared the substrate binding modes of 2 GH42 β-galactosidases, BgaB from Geobacillus stearothermophilus and A4-β-Gal from Thermus thermophilus A4. The A4-β-Gal has a catalytic triad (Glu312-Arg32-Glu35) with an extended hydrogen bond network that has not been observed in BgaB. In this study, we performed site-saturation mutagenesis of Ile42 in BgaB (equivalent to Glu312 in A4-β-Gal) to study the effects of different residues on thermostability, catalytic function, and the extended hydrogen bond network. Our experimental results suggest that substitution of Ile42 with polar AA enhanced the thermostability but decreased the catalytic efficiency of BgaB. Polar AA substitution for Ile42 simultaneously affected thermostability, catalytic efficiency, and the hydrogen bond network, suggesting that Ile42 is responsible for functional discrimination between members of the GH42 family. These observations could lead to a novel strategy for investigating the functional evolution of the GH42 β-galactosidases.

•We sequenced the whole genome of a high CLA producer, Lactobacillus plantarum ZS2058, which could provide more clues to explain the probiotic effects of this strain.•The genome was deep analyzed especially for the CLA production related genes and gene cluster.•The exopolysaccharides (EPS) operon was identified, which indicated the strain might have more health-associated benefits.Lactobacillus plantarum ZS2058 was isolated from sauerkraut and identified to synthesize the beneficial metabolite conjugated linoleic acid. The genome contains a 319,7363-bp chromosome and three plasmids. The sequence will facilitate identification and characterization of the genetic determinants for its putative biological benefits.