ι-Carrageenan catabolism is initiated by key sulfatases in the marine bacterium Pseudoalteromonas haloplanktis LL1.

Marine bacteria contribute substantially to cycle macroalgae polysaccharides in marine environments. Carrageenans are the primary cell wall polysaccharides of red macroalgae. The carrageenan catabolism mechanism and pathways are still largely unclear. Pseudoalteromonas is a representative bacterial genus that can utilize carrageenan. We previously isolated the strain Pseudoalteromonas haloplanktis LL1 that could grow on ι-carrageenan but produce no ι-carrageenase. Here, through a combination of bioinformatic, biochemical, and genetic analyses, we determined that P. haloplanktis LL1 processed a desulfurization-depolymerization sequential pathway for ι-carrageenan utilization, which was initiated by key sulfatases PhSulf1 and PhSulf2. PhSulf2 acted as an endo/exo-G4S (4-O-sulfation-ß-D-galactopyranose) sulfatase, while PhSulf1 was identified as a novel endo-DA2S sulfatase that could function extracellularly. Because of the unique activity of PhSulf1 toward ι-carrageenan rather than oligosaccharides, P. haloplanktis LL1 was considered to have a distinct ι-carrageenan catabolic pathway compared to other known ι-carrageenan-degrading bacteria, which mainly employ multifunctional G4S sulfatases and exo-DA2S (2-O-sulfation-3,6-anhydro-α-D-galactopyranose) sulfatase for sulfate removal. Furthermore, we detected widespread occurrence of PhSulf1-encoding gene homologs in the global ocean, indicating the prevalence of such endo-acting DA2S sulfatases as well as the related ι-carrageenan catabolism pathway. This research provides valuable insights into the enzymatic processes involved in carrageenan catabolism within marine ecological systems.IMPORTANCECarrageenan is a type of linear sulfated polysaccharide that plays a significant role in forming cell walls of marine algae and is found extensively distributed throughout the world's oceans. To the best of our current knowledge, the ι-carrageenan catabolism in marine bacteria either follows the depolymerization-desulfurization sequential process initiated by ι-carrageenase or starts from the desulfurization step catalyzed by exo-acting sulfatases. In this study, we found that the marine bacterium Pseudoalteromonas haloplanktis LL1 processes a distinct pathway for ι-carrageenan catabolism employing a specific endo-acting DA2S-sulfatase PhSulf1 and a multifunctional G4S sulfatase PhSulf2. The unique PhSulf1 homologs appear to be widely present on a global scale, indicating the indispensable contribution of the marine bacteria containing the distinct ι-carrageenan catabolism pathway. Therefore, this study would significantly enrich our understanding of the molecular mechanisms underlying carrageenan utilization, providing valuable insights into the intricate roles of marine bacteria in polysaccharide cycling in marine environments.

Biotechnological application of Aureobasidium spp. as a promising chassis for biosynthesis of ornithine-urea cycle-derived bioproducts.

The ornithine-urea cycle (OUC) in fungal cells has biotechnological importance and many physiological functions and is closely related to the acetyl glutamate cycle (AGC). Fumarate can be released from argininosuccinate under the catalysis of argininosuccinate lyase in OUC which is regulated by the Ca2+ signaling pathway and over 93.9 ± 0.8 g/L fumarate can be yielded by the engineered strain of Aureobasidium pullulans var. aubasidani in the presence of CaCO3. Furthermore, 2.1 ± 0.02 mg of L-ornithine (L-Orn)/mg of the protein also can be synthesized via OUC by the engineered strains of Aureobasidum melanogenum. Fumarate can be transformed into many drugs and amino acids and L-Orn can be converted into siderophores (1.7 g/L), putrescine (33.4 g/L) and L-piperazic acid (L-Piz) (3.0 g/L), by different recombinant strains of A. melanogenum. All the fumarate, L-Orn, siderophore, putrescine and L-Piz have many applications. As the yeast-like fungi and the promising chassis, Aureobasidium spp, have many advantages over any other fungal strains. Further genetic manipulation and bioengineering will enhance the biosynthesis of fumarate and L-Orn and their derivates.

OUC in fungal cells has biotechnological importance and many physiological functions; OUC is closely related to acetyl glutamate cycle (AGC). Fumarate, L-Orn, siderophore, putrescine and L-Piz produced from OUC have many applications.

Novel chromosomes and genomes provide new insights into evolution and adaptation of the whole genome duplicated yeast-like fungus TN3-1 isolated from natural honey.

Aureobasidium melanogenum TN3-1 strain and A. melanogenum P16 strain were isolated from the natural honey and the mangrove ecosystem, respectively. The former can produce much higher pullulan from high concentration of glucose than the latter. In order to know what happened to their genomes, the PacBio sequencing and Hi-C technologies were used to create the first high-quality chromosome-level reference genome assembly of A. melanogenum TN3-1 (51.61 Mb) and A. melanogenum P16 (25.82 Mb) with the contig N50 of 2.19 Mb and 2.26 Mb, respectively. Based on the Hi-C results, a total of 93.33% contigs in the TN3-1 strain and 92.31% contigs in the P16 strain were anchored onto 24 and 12 haploid chromosomes, respectively. The genomes of the TN3-1 strain had two subgenomes A and B. Synteny analysis showed that the genomic contents of the two subgenomes were asymmetric with many structural variations. Intriguingly, the TN3-1 strain was revealed as a recent hybrid/fusion between the ancestor of A. melanogenum CBS105.22/CBS110374 and the ancestor of another unidentified strain of A. melanogenum similar to P16 strain. We estimated that the two ancient progenitors diverged around 18.38 Mya and merged around 10.66-9.98 Mya. It was found that in the TN3-1 strain, telomeres of each chromosome contained high level of long interspersed nuclear elements (LINEs), but had low level of the telomerase encoding gene. Meanwhile, there were high level of transposable elements (TEs) inserted in the chromosomes of the TN3-1 strain. In addition, the positively selected genes of the TN3-1 strain were mainly enriched in the metabolic processes related to harsh environmental adaptability. Most of the stress-related genes were found to be related to the adjacent LTRs, and the glucose derepression was caused by the mutation of the Glc7-2 in the Snf-Mig1 system. All of these could contribute to its genetic instability, genome evolution, high stress resistance, and high pullulan production from glucose.

Liamocins biosynthesis, its regulation in Aureobasidium spp., and their bioactivities.

Liamocins synthesized by Aureobasidium spp. are glycolipids composed of a single mannitol or arabitol headgroup linked to either three, four or even six 3,5-dihydroxydecanoic ester tail-groups. The highest titer of liamocin achieved was over 40.0 g/L. The substrates for liamocins synthesis include glucose, sucrose, xylose, mannitol, and others. The Pks1 is responsible for the biosynthesis of the tail-group 3,5-dihydroxydecanoic acid, both mannitol dehydrogenase (MDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH) catalyze the mannitol biosynthesis and the arabitol biosynthesis is controlled by arabitol dehydrogenase (ArDH). The ester bond formation between 3,5-dihydroxydecanoic acid and mannitol or arabitol is catalyzed by the esterase (Est1). Liamocin biosynthesis is regulated by the specific transcriptional activator (Gal1), global transcriptional activator (Msn2), various signaling pathways, acetyl-CoA flux while Pks1 activity is controlled by PPTase activity. The synthesized liamocins have high bioactivity against the pathogenic bacteria Streptococcus spp. and some kinds of cancer cells while Massoia lactone released liamocins which exhibited obvious antifungal and anticancer activities. Therefore, liamocins and Massoia lactone have many applications in various sectors of biotechnology.

Massoia Lactone Displays Strong Antifungal Property Against Many Crop Pathogens and Its Potential Application.

Massoia lactone could be released from liamocins produced by Aureobasidium melanogenum M39. The obtained Massoia lactone was very stable and highly active against many fungal crop pathogens which cause many plant diseases and food unsafety. Massoia lactone treatment not only could effectively inhibit their hyphal growth and spore germination, but also caused pore formation in cell membrane, reduction of ergosterol content, rise in intracellular ROS levels, and leakage of intracellular components, consequently leading to cellular necrosis and cell death. The direct contact of Massoia lactone with Fusarium graminearum spores could stop the development of Fusarium head blight symptom in the diseased wheats. Therefore, Massoia lactone could be a promising candidate for development as an effective and green bio-fungicide because of its high anti-fungal activity and the multiplicity of mode of its action.

Molecular evolution and regulation of DHN melanin-related gene clusters are closely related to adaptation of different melanin-producing fungi.

Many genes responsible for melanin biosynthesis in fungi were physically linked together. The PKS gene clusters in most of the melanin-producing fungi were regulated by the Cmr1. It was found that a close rearrangement of the PKS gene clusters had evolved in most of the melanin-producing fungi and various functions of melanin in them were beneficial to their adaptation to the changing environments. The melanin-producing fungi had undergone at least five large-scale differentiations, making their PKS gene clusters be quickly evolved and the fungi be adapted to different harsh environments. The recent gene losses and expansion were remarkably frequent in the PKS gene clusters, leading to their rapid evolution and adaptation of their hosts to different environments. The PKS gene and the CMR1 gene in them were subject to a strong co-evolution, but the horizontal gene transfer events might have occurred in the genome-duplicated species, Aspergillus and Penicillium.

Liamocin overproduction by the mutants of Aureobasidium melanogenum 9-1 for effectively killing spores of the pathogenic fungi from diseased human skin by Massoia lactone.

Liamocins and Massoia lactone have many applications. In this study, the glucose-derepressed mutant Δcrea5 in which the CREA gene was removed could produce 36.5 g/L of liamocins. Furthermore, overexpression of the MSN2 gene in the mutant Δcrea5 made the transformant M60 produce 41.4 g/L of liamocins and further overexpression of the GAL1 gene in the transformant M60 rendered the transformant G40 to produce 49.5 ± 0.4 g/L of liamocins during the 10-L fermentation while their wild type strain 9-1 made only 26.3 g/L of liamocins. The expressed transcription activators Msn2 and Gal1 were localized in the nuclei, promoting expression of the genes responsible for liamocins biosynthesis and sugar transport. Massoia lactone prepared from the produced liamocins could actively kill the spores of the pathogenic fungi from the diseased human skin by inhibiting spore germination and causing cellular necrosis of the fungal spores.

The Genome-Wide Mutation Shows the Importance of Cell Wall Integrity in Growth of the Psychrophilic Yeast Metschnikowia australis W7-5 at Different Temperatures.

In this study, it was found that a Cre/loxP system could be successfully used as a tool for editing the genome of the psychrophilic yeast Metschnikowia australis W7-5 isolated from Antarctica. The deletion and over-expression of the TPS1 gene for trehalose biosynthesis, the GSY gene for glycogen biosynthesis, and the GPD1 and GPP genes for glycerol biosynthesis had no influence on cell growth of the mutants and transformants compared to cell growth of their wild-type strain M. australis W7-5, indicating that trehalose, glycogen, and glycerol had no function in growth of the psychrophilic yeast at different temperatures. However, removal of the SLT2 gene encoding the mitogen-activated protein kinase in the cell wall integrity (CWI) signaling pathway and the SWI4 and SWI6 genes encoding the transcriptional activators Swi4/6 had the crucial influence on cell growth of the psychrophilic yeast at the low temperature, especially at 25 °C and expression of the genes related to cell wall and lipid biosynthesis. Therefore, the cell wall could play an important role in growth of the psychrophilic yeast at different temperatures and biosynthesis of cell wall was actively regulated by the CWI signaling pathway. This was the first time to show that the genome of the psychrophilic yeast was successfully edited and the molecular evidences were obtained to elucidate mechanisms of low temperature growth of the psychrophilic yeast from Antarctica.

Genetic evidences for the core biosynthesis pathway, regulation, transport and secretion of liamocins in yeast-like fungal cells.

So far, it has been still unknown how liamocins are biosynthesized, regulated, transported and secreted. In this study, a highly reducing polyketide synthase (HR-PKS), a mannitol-1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH) and an esterase (Est1) were found to be closely related to core biosynthesis of extracellular liamocins in Aureobasidium melanogenum 6-1-2. The HR-PKS was responsible for biosynthesis of 3,5-dihydroxydecanoic acid. The MPDH and MtDH were implicated in mannitol biosynthesis and the ArDH was involved in arabitol biosynthesis. The Est1 catalyzed ester bond formation of them. A phosphopantetheine transferase (PPTase) activated the HR-PKS and a transcriptional activator Ga11 activated expression of the PKS1 gene. Therefore, deletion of the PKS1 gene, all the three genes encoding MPDH, MtDH and ArDH, the EST1, the gene responsible for PPTase and the gene for Ga11 made all the disruptants (Δpks13, Δpta13, Δest1, Δp12 and Δg11) totally lose the ability to produce any liamocins. A GLTP gene encoding a glycolipid transporter and a MDR1 gene encoding an ABC transporter took part in transport and secretion of the produced liamocins into medium. Removal of the GLTP gene and the MDR1 gene resulted in a Δgltp1 mutant and a Δmdr16 mutant, respectively, that lost the partial ability to secrete liamocins, but which cells were swollen and intracellular lipid accumulation was greatly enhanced. Hydrolysis of liamocins released 3,5-dihydroxydecanoic acid, mannitol, arabitol and acetic acid. We proposed a core biosynthesis pathway, regulation, transport and secretion of liamocins in A. melanogenum.

Melanin biosynthesis in the desert-derived Aureobasidium melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1.

The melanin produced by Aureobasidium melanogenum XJ5-1 obtained from the Taklimakan Desert can play an important role in adaptation of the yeast strain to various stress treatments. It is very important to know how the desert-derived yeast sense, respond and adapt to the harsh environments. However, it is still unclear how melanin is genetically controlled by signaling pathways and transcriptional factors. In this study, it was found that the mitogen-activated protein kinase (MAPK) Slt2 in the cell wall integrity (CWI) signal pathway could regulate activity of the transcriptional activator Swi4; in turn, the Swi4 could control the expression of the CMR1 gene. The melanin-specific transcriptional activator Cmr1 encoded by the CMR1 gene was specifically bound to the promoter with the sequence TTCTCTCCA of the PKS1 gene and strongly stimulated expression of the PKS1 gene and any other genes responsible for melanin biosynthesis, so that a large amount of melanin could be produced by A. melanogenum XJ5-1. Therefore, melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 was controlled mainly by the CWI signal pathway among the cell wall-related signal pathways via a transcriptional activator Cmr and regulation of the melanin biosynthesis in A. melanogenum XJ5-1 was completely different from that of the melanin biosynthesis in any other fungi. This is the first time to show that melanin biosynthesis in the desert-derived A. melanogenum XJ5-1 is controlled mainly by the CWI signal pathway via a transcriptional activator Cmr1. This would provide the fundamentals for further research on the desert-derived yeast to sense, respond and adapt to the harsh environments.

Fungi in mangrove ecosystems and their potential applications.

Mangrove fungi, their ecological role in mangrove ecosystems, their bioproducts, and potential applications are reviewed in this article. Mangrove ecosystems can play an important role in beach protection, accretion promotion, and sheltering coastlines and creeks as barriers against devastating tropical storms and waves, seawater, and air pollution. The ecosystems are characterized by high average and constant temperatures, high salinity, strong winds, and anaerobic muddy soil. The mangrove ecosystems also provide the unique habitats for the colonization of fungi which can produce different kinds of enzymes for industrial uses, recycling of plants and animals in the ecosystems, and the degradation of pollutants. Many mangrove ecosystem-associated fungi also can produce exopolysaccharides, Ca2+-gluconic acid, polymalate, liamocin, polyunsaturated fatty acids, biofuels, xylitol, enzymes, and bioactive substances, which have many potential applications in the bioenergy, food, agricultural, and pharmaceutical industries. Therefore, mangrove ecosystems are rich bioresources for bioindustries and ecology. It is necessary to identify more mangrove fungi and genetically edit them to produce a distinct array of novel chemical entities, enzymes, and bioactive substances.

Genome sequencing of Aureobasidium pullulans P25 and overexpression of a glucose oxidase gene for hyper-production of Ca2+-gluconic acid.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

Genetics of trehalose biosynthesis in desert-derived Aureobasidium melanogenum and role of trehalose in the adaptation of the yeast to extreme environments.

Melanin plays an important role in the stress adaptation of Aureobasidium melanogenum XJ5-1 isolated from the Taklimakan desert. A trehalose-6-phosphate synthase gene (TPS1 gene) was cloned from K5, characterized, and then deleted to determine the role of trehalose in the stress adaptation of the albino mutant K5. No stress response element and heat shock element were found in the promoter of the TPS1 gene. Deletion of the TPS1 gene in the albino mutant rendered a strain DT43 unable to synthesize any trehalose, but DT43 still could grow in glucose, suggesting that its hexokinase was insensitive to inhibition by trehalose-6-phosphate. Overexpression of the TPS1 gene enhanced trehalose biosynthesis in strain ET6. DT43 could not grow at 33 °C, whereas K5, ET6, and XJ5-1 could grow well at this temperature. Compared with K5 and ET6, DT43 was highly sensitive to heat shock treatment, high oxidation, and high desiccation, but all the three strains demonstrated the same sensitivity to UV light and high NaCl concentration. Therefore, trehalose played an important role in the adaptation of K5 to heat shock treatment, high oxidation, and high desiccation.

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications.

PURPOSE: Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article. RESULTS: Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids. CONCLUSIONS: It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.

Cell wall integrity is required for pullulan biosynthesis and glycogen accumulation in Aureobasidium melanogenum P16.

BACKGROUND: Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. METHODS: The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. RESULTS: In this study, the GT6 and GT7 genes encoding different α1,2 mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. GENERAL SIGNIFICANCE: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.

CreA is directly involved in pullulan biosynthesis and regulation of Aureobasidium melanogenum P16.

Aureobasidium melanogenum P16 is a high pullulan-producing yeast. However, glucose repression on its pullulan biosynthesis must be relieved. After the gene encoding a glucose repressor was cloned, characterized and analyzed, it was found that the repressor belonged to one member of the CreA in filamentous fungi, not to one member of the Mig1 in yeasts. After the CREA gene was fully removed from the yeast strain P16, the glucose repression in the disruptant DG41 was relieved. At the same time, the pullulan production by the disruptant DG41 was enhanced compared to that by its wild-type strain P16, and the transcriptional levels of the gene encoding a glucosyltransferase, three genes encoding glucose transporters, the gene encoding a 6-P-glucose kinase and the genes encoding α-amylase, glucoamylase and pullulanase in the disruptant DG41 were also promoted. However, the transcriptional levels of the genes encoding the CreA and another two glucose transporters were greatly reduced. During the 10-liter fermentation, the disruptant DG41 produced 64.93 ± 1.33 g/l pullulan from 120 g/l of glucose, while its wild-type strain P16 produced only 52.0 ± 1.95 g/l pullulan within 132 h. After the CREA gene was complemented in the disruptant D373, the pullulan production by the transformant BC4 was greatly reduced compared to that by its wild-type strain P16, and the transcriptional levels of the many genes in the transformant BC4 were also decreased. All the results confirmed that the CreA played an important role in the regulation of pullulan biosynthesis in A. melanogenum P16, and that glucose derepression on pullulan biosynthesis could improve pullulan production from glucose. This study opened the possibility for improving the industrial production of this exopolysaccharide by genetic engineering.

Melanin production by a yeast strain XJ5-1 of Aureobasidium melanogenum isolated from the Taklimakan desert and its role in the yeast survival in stress environments.

The yeast strain XJ5-1 isolated from the Taklimakan desert soil was identified to be a strain of Aureobasdium melanogenum and could produce a large amount of melanin when it was grown in the PDA medium, but its melanin biosynthesis and expression of the PKS gene responsible for the melanin biosynthesis was significantly repressed in the presence of (NH4)2SO4. However, A. melanogenum P5 strain isolated from a mangrove ecosystem grown in both the presence and the absence of (NH4)2SO4 did not produce any melanin. The cell size of A. melanogenum XJ5-1 strain was much higher than that of A. melanogenum P5 strain. The melanized cells of the yeast strain XJ5-1 had higher tolerance to UV radiation, oxidation (200.0 mM H2O2), heat treatment (40 °C), salt shock (200.0 g/L NaCl), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than the non-melanized cells of the same yeast strain XJ5-1. At the same time, the melanized cells of the yeast strain XJ5-1 also had higher tolerance to UV radiation, oxidation (200.0 mM H2O2), desiccation and strong acid hydrolysis (6.0 M HCl) at high temperature (80 °C) than A. melanogenum P5 strain, but had similar resistance to heat treatment (40 °C) and salt shock (200.0 g/L NaCl) compared to those of A. melanogenum P5 strain. All the results revealed that many characteristics of A. melanogenum XJ5-1 isolated from the Taklimakan desert soil was different from those of A. melanogenum P5 strain isolated from the mangrove ecosystem.

Poly(ß-L-malic acid) (PMLA) from Aureobasidium spp. and its current proceedings.

Poly(ß-L-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(ß-L-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Enhanced citric acid production by a yeast Yarrowia lipolytica over-expressing a pyruvate carboxylase gene.

In this study, after the expression of a pyruvate carboxylase gene (PYC) cloned from Meyerozyma guilliermondii in a marine-derived yeast Yarrowia lipolytica SWJ-1b, a transformant PG86 obtained had much higher PYC activity than Y. lipolytica SWJ-1b. At the same time, the PYC gene expression and citric acid (CA) production by the transformant PG86 were also greatly enhanced. When glucose concentration in the medium was 60.0 g L(-1), CA concentration formed by the transformant PG86 was 34.02 g L(-1), leading to a CA yield of 0.57 g g(-1) of glucose. During a 10-L fed-batch fermentation, the final concentration of CA was 101.0 ± 1.3 g L(-1), the yield was 0.89 g g(-1) of glucose, the productivity was 0.42 g L(-1) h(-1) and only 5.93 g L(-1) reducing sugar was left in the fermented medium within 240 h of the fed-batch fermentation. HPLC analysis showed that most of the fermentation products were CA.