Microbial Utilization of Next-Generation Feedstocks for the Biomanufacturing of Value-Added Chemicals and Food Ingredients.

Global shift to sustainability has driven the exploration of alternative feedstocks beyond sugars for biomanufacturing. Recently, C1 (CO2, CO, methane, formate and methanol) and C2 (acetate and ethanol) substrates are drawing great attention due to their natural abundance and low production cost. The advances in metabolic engineering, synthetic biology and industrial process design have greatly enhanced the efficiency that microbes use these next-generation feedstocks. The metabolic pathways to use C1 and C2 feedstocks have been introduced or enhanced into industrial workhorses, such as Escherichia coli and yeasts, by genetic rewiring and laboratory evolution strategies. Furthermore, microbes are engineered to convert these low-cost feedstocks to various high-value products, ranging from food ingredients to chemicals. This review highlights the recent development in metabolic engineering, the challenges in strain engineering and bioprocess design, and the perspectives of microbial utilization of C1 and C2 feedstocks for the biomanufacturing of value-added products.

Induced Hatching of Quiescent Aedes aegypti (Diptera: Culicidae) Eggs by Labile Glutathione-Stabilizable Compounds From Yeast Extract.

Aedes aegypti (Linnaeus, 1762) is the insect vector that transmits several deadly human diseases. Although the egg stage is an important phase of its life cycle, the biology of mosquito egg remains poorly understood. Here, we report our investigations on the chemical factors that induced hatching of Ae. aegypti eggs. Commercial yeast extracts were able to increase egg hatching rate in a dose-dependent manner, with a hatching rate that ranged from approximately 10% with 1 g/liter to 80% with 20 g/liter of yeast extract. Notably, the addition of glutathione, a reducing agent that showed no significant effect on egg hatching by itself, enhanced and stabilized the activity of yeast extract for at least 70 h. Because dissolved oxygen in different treatments was maintained at high levels in a narrow range (92-95%), we proposed that yeast extract contains hatching inducing compound (HIC) which is able to trigger egg hatching independent of dissolved oxygen level. The HIC in yeast extract could prove to be a potential starting point to design an effective tool to forcefully induce mosquito eggs to hatch under unfavorable conditions, functioning as a novel method for vector control.

Understanding and exploiting the fatty acid desaturation system in Rhodotorula toruloides.

BACKGROUND: Rhodotorula toruloides is a robust producer of triacylglycerol owing to its fast growth rate and strong metabolic flux under conditions of high cell density fermentation. However, the molecular basis of fatty acid biosynthesis, desaturation and regulation remains elusive. RESULTS: We present the molecular characterization of four fatty acid desaturase (FAD) genes in R. toruloides. Biosynthesis of oleic acid (OA) and palmitoleic acid (POA) was conferred by a single-copy ∆9 Fad (Ole1) as targeted deletion of which abolished the biosynthesis of all unsaturated fatty acids. Conversion of OA to linoleic acid (LA) and α-linolenic acid (ALA) was predominantly catalyzed by the bifunctional ∆12/∆15 Fad2. FAD4 was found to encode a trifunctional ∆9/∆12/∆15 FAD, playing important roles in lipid and biomass production as well as stress resistance. Furthermore, an abundantly transcribed OLE1-related gene, OLE2 encoding a 149-aa protein, was shown to regulate Ole1 regioselectivity. Like other fungi, the transcription of FAD genes was controlled by nitrogen levels and fatty acids in the medium. A conserved DNA motif, (T/C)(G/A)TTGCAGA(T/C)CCCAG, was demonstrated to mediate the transcription of OLE1 by POA/OA. The applications of these FAD genes were illustrated by engineering high-level production of OA and γ-linolenic acid (GLA). CONCLUSION: Our work has gained novel insights on the transcriptional regulation of FAD genes, evolution of FAD enzymes and their roles in UFA biosynthesis, membrane stress resistance and, cell mass and total fatty acid production. Our findings should illuminate fatty acid metabolic engineering in R. toruloides and beyond.

Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov., endophytically associated phyllosphere bacteria isolated from economically important crop plants.

In this study, two endophytic bacterial strains designated JS21-1T and S6-262T isolated from leaves of Elaeis guineensis and stem tissues of Jatropha curcas respectively, were subjected for polyphasic taxonomic approach. On R2A medium, colonies of strains JS21-1T and S6-262T are orange and yellow, respectively. Phylogenetic analyses using 16S rRNA gene sequencing and whole-genome sequences placed the strains in distinct clades but within the genus Sphingomonas. The DNA G + C content of JS21-1T and S6-262T were 67.31 and 66.95%, respectively. Furthermore, the average nucleotide identity and digital DNA-DNA hybridization values of strains JS21-1T and S6-262T with phylogenetically related Sphingomonas species were lower than 95% and 70% respectively. The chemotaxonomic studies indicated that the major cellular fatty acids of the strain JS21-1T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), C16:0, and C14:0 2OH; strain S6-262T possessed summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The major quinone was Q10, and the unique polyamine observed was homospermidine. The polar lipid profile comprised of mixture of sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and certain uncharacterised phospholipids and lipids. Based on this polyphasic evidence, strains JS21-1T and S6-262T represent two novel species of the genus Sphingomonas, for which the names Sphingomonas palmae sp. nov. and Sphingomonas gellani sp. nov. are proposed, respectively. The type strain of Sphingomonas palmae sp. nov. is JS21-1T (= DSM 27348T = KACC 17591T) and the type strain of Sphingomonas gellani sp. nov. is S6-262T (= DSM 27346T =  KACC 17594T).

Chitinasiproducens palmae gen. nov., sp. nov., a new member of the family Burkholderiaceae isolated from leaf tissues of oil palm (Elaeis guineensis Jacq.).

A Gram-stain-negative, aerobic, rod-shaped, leaf-associated bacterium, designated JS23T, was isolated from surface-sterilized leaf tissue of an oil palm grown in Singapore and was investigated by polyphasic taxonomy. Phylogenetic analyses based on 16S rRNA gene sequences and 180 conserved genes in the genome of several members of Burkholderiaceae revealed that strain JS23T formed a distinct evolutionary lineage independent of other taxa within the family Burkholderiaceae. The predominant ubiquinone was Q-8. The primary polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminophospholipid. The major fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c /C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c /C18 : 1 ω6c). The size of the genome is 5.36 Mbp with a DNA G+C content of 66.2 mol%. Genomic relatedness measurements such as average nucleotide identity, genome-to-genome distance and digital DNA-DNA hybridization clearly distinguished strain JS23T from the closely related genera Burkholderia, Caballeronia, Mycetohabitans, Mycoavidus, Pandoraea, Paraburkholderia, Robbsia and Trinickia. Furthermore, average amino acid identity values and the percentages of conserved proteins, 56.0-68.4 and 28.2-45.5, respectively, were well below threshold values for genus delineation and supported the assignment of JS23T to a novel genus. On the basis of the phylogenetic, biochemical, chemotaxonomic and phylogenomic evidence, strain JS23T is proposed to represent a novel species of a new genus within the family Burkholderiaceae, for which the name Chitinasiproducens palmae gen. nov., sp. nov., is proposed with the type strain of JS23T (= DSM 27307T=KACC 17592T).

Phytobacter palmae sp. nov., a novel endophytic, N2 fixing, plant growth promoting Gammaproteobacterium isolated from oil palm (Elaeis guineensis Jacq.).

A novel strain S29T with high nitrogen fixing ability was isolated from surface-sterilized leaf tissues of oil palm (Elaeis guineensis) growing in Science Park II, Singapore. On the basis of 16S rRNA gene analysis and multilocus sequence typing with the rpoB, gyrB, infB and atpD genes, strain S29T was a member of the genus Phytobacter, with Phytobacter ursingii ATCC 27989T and Phytobacter diazotrophicus LS 8T as its closest relatives. Unique biochemical features that differentiated strain S29T from its closest relatives were the ability to utilize melibiose, α-cyclodextrin, glycogen, adonital, d-arabitol, m-inositol and xylitol. The major fatty acids were C14 : 0, C16 : 0, C17 : 0, C16 : 1 ω5c and summed feature 2 containing C16 : 1 ω7c and/or C16 : 1 ω6c. The polar fatty acid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid and aminolipids. The draft genome of strain S29T comprised 5, 284, 330 bp with a G + C content of 52.6 %. The average nucleotide identity and digital DNA-DNA hybridization values between strain S29T and the phylogenetically related Enterobacterales species were lower than 95 % and 70 %, respectively. Thus, the polyphasic evidences generated through the phenotypic, chemotaxonomic and genomic methods confirmed that strain S29T represents a novel species of the genus Phytobacter, for which we propose the name Phytobacter palmae sp. nov. with the type strain of S29T (=DSM 27342T=KACC 17598T).

Identification of novel genes in the carotenogenic and oleaginous yeast Rhodotorula toruloides through genome-wide insertional mutagenesis.

BACKGROUND: Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. RESULTS: We have adapted Agrobacterium tumefaciens-mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides. Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides, which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1. Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. CONCLUSIONS: T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

Site-specific phosphorylation of TRANSPARENT TESTA GLABRA1 mediates carbon partitioning in Arabidopsis seeds.

Seed development is dependent on nutrients, such as a source of carbon, supplied by the parent plant. It remains largely unknown how these nutrients are distributed to zygotic and maternal tissues to coordinate storage of reserve compounds and development of protective tissues like seed coat. Here we show that phosphorylation of TRANSPARENT TESTA GLABRA1 (TTG1) is regulated by SHAGGY-like kinases 11/12 (SK11/12) and that this mediates carbon flow to fatty acid synthesis and seed coat traits in Arabidopsis seeds. SK11/12 phosphorylate TTG1 at serine 215, thus preventing TTG1 interaction with TRANSPARENT TESTA2. This compromises recruitment of TTG1 to the GLABRA2 locus and downregulates GLABRA2 expression, which enhances biosynthesis of fatty acids in the embryo, but reduces production of mucilage and flavonoid pigments in the seed coat. Therefore, site-specific phosphorylation of TTG1 by SK11/SK12 regulates carbon partitioning between zygotic and maternal sinks in seeds.

Mapping QTL for Omega-3 Content in Hybrid Saline Tilapia.

Tilapia is one of most important foodfish species. The low omega-3 to omega-6 fatty acid ratio in freshwater tilapia meat is disadvantageous for human health. Increasing omega-3 content is an important task in breeding to increase the nutritional value of tilapia. However, conventional breeding to increase omega-3 content is difficult and slow. To accelerate the increase of omega-3 through marker-assisted selection (MAS), we conducted QTL mapping for fatty acid contents and profiles in a F2 family of saline tilapia generated by crossing red tilapia and Mozambique tilapia. The total omega-3 content in F2 hybrid tilapia was 2.5 ± 1.0 mg/g, higher than that (2.00 mg/g) in freshwater tilapia. Genotyping by sequencing (GBS) technology was used to discover and genotype SNP markers, and microsatellites were also genotyped. We constructed a linkage map with 784 markers (151 microsatellites and 633 SNPs). The linkage map was 2076.7 cM long and consisted of 22 linkage groups. Significant and suggestive QTL for total lipid content were mapped on six linkage groups (LG3, -4, -6, -8, -13, and -15) and explained 5.8-8.3% of the phenotypic variance. QTL for omega-3 fatty acids were located on four LGs (LG11, -18, -19, and -20) and explained 5.0 to 7.5% of the phenotypic variance. Our data suggest that the total lipid and omega-3 fatty acid content were determined by multiple genes in tilapia. The markers flanking the QTL for omega-3 fatty acids can be used in MAS to accelerate the genetic improvements of these traits in salt-tolerant tilapia.

Sphingomonas jatrophae sp. nov. and Sphingomonas carotinifaciens sp. nov., two yellow-pigmented endophytes isolated from stem tissues of Jatropha curcas L.

Two yellow-pigmented isolates, S5-249T and L9-754T, originating from surface-sterilized plant tissues of Jatropha curcas L. (Jatropha) cultivars were characterized using a polyphasic taxonomic approach. Strains S5-249T and L9-754T had 16S rRNA genes sharing 94.2 % sequence similarity with each other and 91.6-97.2 % sequence similarity with those of other species in the genus Sphingomonas, suggesting that they represent two potentially novel species. The 16S rRNA gene sequences of strains S5-249T and L9-754T shared the highest similarity to that of Sphingomonas sanguinis NBRC 13937T (96.1 and 97.2 %, respectively). The genomic DNA G+C contents of strains S5-249T and L9-754T were 66.9 and 68.5 mol%, respectively. The respiratory quinone was determined to be Q-10, and the major polyamine was homospermidine. Strains S5-249T and L9-754T contained summed feature 7 (comprising C18 : 1ω7c, C18 : 1ω9t and/or C18 : 1ω12t), C16 : 1, C14 : 0 2-OH and summed feature 4 (C16 : 1ω7t, iso-C15 : 0 2-OH and C16 : 1ω7c) as the major cellular fatty acids. The predominant polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and sphingoglycolipid. The average nucleotide identity (ANI) values between S. sanguinis NBRC 13937T and the two type strains (S5-249T and L9-754T) were 72.31 and 77.73 %, respectively. Digital DNA-DNA hybridization (dDDH) studies between the novel strains (S5-249T and L9-754T) and other species of the genus Sphingomonas were well below the thresholds used to discriminate between bacterial species. The results of dDDH and physiological tests allowed genotypic and phenotypic differentiation of the strains from each other as well as from the species of the genus Sphingomonas with validly published names. These data strongly support the classification of the strains as representatives of novel species, for which we propose the names Sphingomonas jatrophae sp. nov. (type strain S5-249T=DSM 27345T=KACC 17593T) and Sphingomonas carotinifaciens sp. nov. (type strain L9-754T=DSM 27347T=KACC 17595T).

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

BACKGROUND: Red yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts. RESULTS: The upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4-11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase ß subunit gene (FAS1) exhibited intermediate strength, displaying 50-160 and 20-90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6-3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5-3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation. CONCLUSION: Intronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast, R. toruloides.

Draft Genome Sequences of Rhodosporidium toruloides Strains ATCC 10788 and ATCC 10657 with Compatible Mating Types.

Rhodosporidium toruloides ATCC 10788 (haploid, A1 mating type) and ATCC 10657 (haploid, A2 mating type) were derived from the same diploid parent strain Rhodotorula glutinis ATCC 90781 and are important strains for metabolic engineering. Draft genome sequences of both strains are reported here. The current assembly of strain ATCC 10788 comprises 61 scaffolds with a total size of 20.75 Mbp and a GC content of 62.01%, while that of strain ATCC 10657 comprises 137 scaffolds with a total size of 21.49 Mbp and a GC content of 61.81%. Genome annotation predicts 7,730 and 7,800 protein encoding genes for strain ATCC 10788 and strain ATCC 10657, respectively.

Chania multitudinisentens gen. nov., sp. nov., an N-acyl-homoserine-lactone-producing bacterium in the family Enterobacteriaceae isolated from landfill site soil.

Phylogenetic and taxonomic characterization was performed for bacterium RB-25T, which was isolated from a soil sample collected in a former municipal landfill site in Puchong, Malaysia. Growth occurred at 20-37 °C at pH 5-8 but not in the presence of 9 % (w/v) NaCl or higher. The principal fatty acids were C16:0, C18:1ω7c and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). Ubiquinone-8 was the only isoprenoid quinone detected. Polar lipid analysis revealed the presence of phospholipid, phosphoaminolipid, phosphatidylethanolamine, phosphatidylglycerol and one unidentified aminolipid. DNA G+C content was 50.9 mol% phylogenetic analysis based on 16S rRNA gene sequence showed that strain RB-25T formed a distinct lineage within the family Enterobacteriaceae of the class Gammaproteobacteria. It exhibited a low level of 16S rRNA gene sequence similarity with its phylogenetic neighbours Pantoea rwandensis LMG 26275T (96.6 %), Rahnella aquatilis CIP 78.65T (96.5 %), Pectobacterium betavasculorum ATCC 43762T (96.4 %), Pantoea rodasii LMG 26273T (96.3 %), Gibbsiella dentisursi NUM 1720T (96.3 %) and Serratia glossinae C1T (96.2 %). Multilocus sequence analyses based on fusA, pyrG, rplB, rpoB and sucA sequences showed a clear distinction of strain RB-25T from the most closely related genera. Isolate RB-25T could also be distinguished from members of these genera by a combination of the DNA G+C content, respiratory quinone system, fatty acid profile, polar lipid composition and other phenotypic features. Strain RB-25T represents a novel species of a new genus, for which the name Chaniamultitudinisentens gen. nov., sp. nov. is proposed. The type strain is RB-25T (=DSM 28811T=LMG 28304T).

Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas.

BACKGROUND: Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant-microbe interactions. RESULTS: An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to α, ß, γ-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female-male flower ratio. CONCLUSION: The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria-plant interaction may significantly contribute to Jatropha's tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant's nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

BACKGROUND: Rhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts. RESULTS: We have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a D-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named P DAO1-in1m1 , showed very similar luciferase activity to the wild-type promoter upon induction with D-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source. CONCLUSION: The intron 1-containing DAO1 promoters coupled with a DAO1 null mutant makes an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium and Rhodotorula genera. The system will be a valuable tool for metabolic engineering and enzyme expression in these yeast hosts.

Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

Draft Genome Sequence of Methylobacterium sp. Strain L2-4, a Leaf-Associated Endophytic N-Fixing Bacterium Isolated from Jatropha curcas L.

Methylobacterium sp. strain L2-4 is an efficient nitrogen-fixing leaf colonizer of biofuel crop Jatropha curcas. This strain is able to greatly improve the growth and seed yield of Jatropha curcas and is the second reported genome sequence of plant growth-promoting bacteria isolated from Jatropha curcas.

Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is a ß-carotenoid accumulating, oleaginous yeast that has great biotechnological potential. The lack of reliable and efficient genetic manipulation tools have been a major hurdle blocking its adoption as a biotechnology platform. RESULTS: We report for the first time the development of a highly efficient targeted gene deletion method in R. toruloides ATCC 10657 via Agrobacterium tumefaciens-mediated transformation. To further improve targeting frequency, the KU70 and KU80 homologs in R. toruloides were isolated and characterized in detail. A KU70-deficient mutant (∆ku70e) generated with the hygromycin selection cassette removed by the Cre-loxP recombination system showed a dramatically improved targeted gene deletion frequency, with over 90% of the transformants being true knockouts when homology sequence length of at least 1 kb was used. Successful gene targeting could be made with homologous flanking sequences as short as 100 bp in the ∆ku70e strain. KU70 deficiency did not perturb cell growth although an elevated sensitivity to DNA mutagenic agents was observed. Compared to the other well-known oleaginous yeast, Yarrowia lipolytica, R. toruloides KU70/KU80 genes contain much higher density of introns and are the most GC-rich KU70/KU80 genes reported. CONCLUSIONS: The KU70-deficient mutant generated herein was effective in improving gene deletion frequency and allowed shorter homology sequences to be used for gene targeting. It retained the key oleaginous and fast growing features of R. toruloides. The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina.

Improved dominant selection markers and co-culturing conditions for efficient Agrobacterium tumefaciens-mediated transformation of Ustilago scitaminea.

Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen.