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Asset Tag: Industrial biotechnology

September 22, 2019

Heterologous expression guides identification of the biosynthetic gene cluster of chuangxinmycin, an indole alkaloid antibiotic.

The indole alkaloid antibiotic chuangxinmycin, from Actinobacteria Actinoplanes tsinanensis, containing a unique thiopyrano[4,3,2- cd]indole scaffold, is a potent and selective inhibitor of bacterial tryptophanyl-tRNA synthetase. The chuangxinmycin biosynthetic gene cluster was identified by in silico analysis of the genome sequence, then verified by heterologous expression. Systemic gene inactivation and intermediate identification determined the minimum set of genes for unique thiopyrano[4,3,2- cd]indole formation and the concerted action of a radical S-adenosylmethionine protein plus an unknown protein for addition of the 3-methyl group. These findings set a solid foundation for comprehensively investigating the biosynthesis, optimizing yield, and generating new analogues of chuangxinmycin.

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September 22, 2019

Genome-wide comparison reveals a probiotic strain Lactococcus lactis WFLU12 isolated from the gastrointestinal tract of olive flounder (Paralichthys Olivaceus) harboring genes supporting probiotic action.

Our previous study has shown that dietary supplementation with Lactococcus lactis WFLU12 can enhance the growth of olive flounder and its resistance against streptococcal infection. The objective of the present study was to use comparative genomics tools to investigate genomic characteristics of strain WFLU12 and the presence of genes supporting its probiotic action using sequenced genomes of L. lactis strains. Dispensable and singleton genes of strain WFLU12 were found to be more enriched in genes associated with metabolism (e.g., energy production and conversion, and carbohydrate transport and metabolism) than pooled dispensable and singleton genes in other L. lactis strains, reflecting WFLU12 strain-specific ecosystem origin and its ability to metabolize different energy sources. Strain WFLU12 produced antimicrobial compounds that could inhibit several bacterial fish pathogens. It possessed the nisin gene cluster (nisZBTCIPRKFEG) and genes encoding lysozyme and colicin V. However, only three other strains (CV56, IO-1, and SO) harbor a complete nisin gene cluster. We also found that L. lactis WFLU12 possessed many other important functional genes involved in stress responses to the gastrointestinal tract environment, dietary energy extraction, and metabolism to support the probiotic action of this strain found in our previous study. This strongly indicates that not all L. lactis strains can be used as probiotics. This study highlights comparative genomics approaches as very useful and powerful tools to select probiotic candidates and predict their probiotic effects.

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September 22, 2019

Inferring the minimal genome of Mesoplasma florum by comparative genomics and transposon mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

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September 22, 2019

The genome of Ectocarpus subulatus highlights unique mechanisms for stress tolerance in brown algae

Brown algae are multicellular photosynthetic organisms belonging to the stramenopile lineage. They are successful colonizers of marine rocky shores world-wide. The genus Ectocarpus, and especially strain Ec32, has been established as a genetic and genomic model for brown algae. A related species, Ectocarpus subulatus Kuetzing, is characterized by its high tolerance of abiotic stress. Here we present the genome and metabolic network of a haploid male strain of E. subulatus, establishing it as a comparative model to study the genomic bases of stress tolerance in Ectocarpus. Our analyses indicate that E. subulatus has separated from Ectocarpus sp. Ec32 via allopatric speciation. Since this event, its genome has been shaped by the activity of viruses and large retrotransposons, which in the case of chlorophyll-binding proteins, may be related to the expansion of this gene family. We have identified a number of further genes that we suspect to contribute to stress tolerance in E. subulatus, including an expanded family of heat shock proteins, the reduction of genes involved in the production of halogenated defense compounds, and the presence of fewer cell wall polysaccharide-modifying enzymes. However, 96% of genes that differed between the two examined Ectocarpus species, as well as 90% of genes under positive selection, were found to be lineage-specific and encode proteins of unknown function. This underlines the uniqueness of brown algae with respect to their stress tolerance mechanisms as well as the significance of establishing E. subulatus as a comparative model for future functional studies.

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September 22, 2019

Strain-level genetic diversity of Methylophaga nitratireducenticrescens confers plasticity to denitrification capacity in a methylotrophic marine denitrifying biofilm.

The biofilm of a methanol-fed, fluidized denitrification system treating a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans NL23 and Methylophaga nitratireducenticrescens JAM1 are the principal bacteria involved in the denitrifying activities. Strain NL23 can carry complete nitrate (NO[Formula: see text]) reduction to N2, whereas strain JAM1 can perform 3 out of the 4 reduction steps. A small proportion of other denitrifiers exists in the biofilm, suggesting the potential plasticity of the biofilm in adapting to environmental changes. Here, we report the acclimation of the denitrifying biofilm from continuous operating mode to batch operating mode, and the isolation and characterization from the acclimated biofilm of a new denitrifying bacterial strain, named GP59.The denitrifying biofilm was batch-cultured under anoxic conditions. The acclimated biofilm was plated on Methylophaga specific medium to isolate denitrifying Methylophaga isolates. Planktonic cultures of strains GP59 and JAM1 were performed, and the growth and the dynamics of NO[Formula: see text], nitrite (NO[Formula: see text]) and N2O were determined. The genomes of strains GP59 and JAM1 were sequenced and compared. The transcriptomes of strains GP59 and JAM1 were derived from anoxic cultures.During batch cultures of the biofilm, we observed the disappearance of H. nitrativorans NL23 without affecting the denitrification performance. From the acclimated biofilm, we isolated strain GP59 that can perform, like H. nitrativorans NL23, the complete denitrification pathway. The GP59 cell concentration in the acclimated biofilm was 2-3 orders of magnitude higher than M. nitratireducenticrescens JAM1 and H. nitrativorans NL23. Genome analyses revealed that strain GP59 belongs to the species M. nitratireducenticrescens. The GP59 genome shares more than 85% of its coding sequences with those of strain JAM1. Based on transcriptomic analyses of anoxic cultures, most of these common genes in strain GP59 were expressed at similar level than their counterparts in strain JAM1. In contrast to strain JAM1, strain GP59 cannot reduce NO[Formula: see text] under oxic culture conditions, and has a 24-h lag time before growth and NO[Formula: see text] reduction start to occur in anoxic cultures, suggesting that both strains regulate differently the expression of their denitrification genes. Strain GP59 has the ability to reduce NO[Formula: see text] as it carries a gene encoding a NirK-type NO[Formula: see text] reductase. Based on the CRISPR sequences, strain GP59 did not emerge from strain JAM1 during the biofilm batch cultures but rather was present in the original biofilm and was enriched during this process.These results reinforce the unique trait of the species M. nitratireducenticrescens among the Methylophaga genus as facultative anaerobic bacterium. These findings also showed the plasticity of denitrifying population of the biofilm in adapting to anoxic marine environments of the bioreactor.

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September 22, 2019

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol.Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation.We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.

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September 22, 2019

Solar-panel and parasol strategies shape the proteorhodopsin distribution pattern in marine Flavobacteriia.

Proteorhodopsin (PR) is a light-driven proton pump that is found in diverse bacteria and archaea species, and is widespread in marine microbial ecosystems. To date, many studies have suggested the advantage of PR for microorganisms in sunlit environments. The ecophysiological significance of PR is still not fully understood however, including the drivers of PR gene gain, retention, and loss in different marine microbial species. To explore this question we sequenced 21 marine Flavobacteriia genomes of polyphyletic origin, which encompassed both PR-possessing as well as PR-lacking strains. Here, we show that the possession or alternatively the lack of PR genes reflects one of two fundamental adaptive strategies in marine bacteria. Specifically, while PR-possessing bacteria utilize light energy (“solar-panel strategy”), PR-lacking bacteria exclusively possess UV-screening pigment synthesis genes to avoid UV damage and would adapt to microaerobic environment (“parasol strategy”), which also helps explain why PR-possessing bacteria have smaller genomes than those of PR-lacking bacteria. Collectively, our results highlight the different strategies of dealing with light, DNA repair, and oxygen availability that relate to the presence or absence of PR phototrophy.

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September 22, 2019

Genomic comparison between members of the Salinibacteraceae family, and description of a new species of Salinibacter (Salinibacter altiplanensis sp. nov.) isolated from high altitude hypersaline environments of the Argentinian Altiplano.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T=CECT 9105T=IBRC-M 11031T). Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019

Whole genome sequence of an edible and potential medicinal fungus, Cordyceps guangdongensis.

Cordyceps guangdongensis is an edible fungus which was approved as a novel food by the Chinese Ministry of Public Health in 2013. It also has a broad prospect of application in pharmaceutical industries, with many medicinal activities. In this study, the whole genome of C. guangdongensis GD15, a single spore isolate from a wild strain, was sequenced and assembled with Illumina and PacBio sequencing technology. The generated genome is 29.05 Mb in size, comprising nine scaffolds with an average GC content of 57.01%. It is predicted to contain a total of 9150 protein-coding genes. Sequence identification and comparative analysis indicated that the assembled scaffolds contained two complete chromosomes and four single-end chromosomes, showing a high level assembly. Gene annotation revealed a diversity of transposons that could contribute to the genome size and evolution. Besides, approximately 15.57% and 12.01% genes involved in metabolic processes were annotated by KEGG and COG respectively. Genes belonging to CAZymes accounted for 3.15% of the total genes. In addition, 435 transcription factors, involved in various biological processes, were identified. Among the identified transcription factors, the fungal transcription regulatory proteins (18.39%) and fungal-specific transcription factors (19.77%) represented the two largest classes of transcription factors. This genomic resource provided a new insight into better understanding the relevance of phenotypic characters and genetic mechanisms in C. guangdongensis. Copyright © 2018 Zhang et al.

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September 22, 2019

PhdA catalyzes the first step of phenazine-1-carboxylic acid degradation in Mycobacterium fortuitum.

Phenazines are a class of bacterially produced redox-active metabolites that are found in natural, industrial, and clinical environments. In Pseudomonas spp., phenazine-1-carboxylic acid (PCA)-the precursor of all phenazine metabolites-facilitates nutrient acquisition, biofilm formation, and competition with other organisms. While the removal of phenazines negatively impacts these activities, little is known about the genes or enzymes responsible for phenazine degradation by other organisms. Here, we report that the first step of PCA degradation by Mycobacterium fortuitum is catalyzed by a phenazine-degrading decarboxylase (PhdA). PhdA is related to members of the UbiD protein family that rely on a prenylated flavin mononucleotide cofactor for activity. The gene for PhdB, the enzyme responsible for cofactor synthesis, is present in a putative operon with the gene encoding PhdA in a region of the M. fortuitum genome that is essential for PCA degradation. PhdA and PhdB are present in all known PCA-degrading organisms from the ActinobacteriaM. fortuitum can also catabolize other Pseudomonas-derived phenazines such as phenazine-1-carboxamide, 1-hydroxyphenazine, and pyocyanin. On the basis of our previous work and the current characterization of PhdA, we propose that degradation converges on a common intermediate: dihydroxyphenazine. An understanding of the genes responsible for degradation will enable targeted studies of phenazine degraders in diverse environments.IMPORTANCE Bacteria from phylogenetically diverse groups secrete redox-active metabolites that provide a fitness advantage for their producers. For example, phenazines from Pseudomonas spp. benefit the producers by facilitating anoxic survival and biofilm formation and additionally inhibit competitors by serving as antimicrobials. Phenazine-producing pseudomonads act as biocontrol agents by leveraging these antibiotic properties to inhibit plant pests. Despite this importance, the fate of phenazines in the environment is poorly understood. Here, we characterize an enzyme from Mycobacterium fortuitum that catalyzes the first step of phenazine-1-carboxylic acid degradation. Knowledge of the genetic basis of phenazine degradation will facilitate the identification of environments where this activity influences the microbial community structure. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

The consistent differential expression of genetic pathways following exposure of an industrial Pseudomonas aeruginosa strain to preservatives and a laundry detergent formulation.

Pseudomonas aeruginosa is a common contaminant associated with product recalls in the home and personal care industry. Preservation systems are used to prevent spoilage and protect consumers, but greater knowledge is needed of preservative resistance mechanisms used by P. aeruginosa contaminants. We aimed to identify genetic pathways associated with preservative exposure by using an industrial P. aeruginosa strain and implementing RNA-Seq to understand gene expression changes in response to industry relevant conditions. The consistent differential expression of five genetic pathways during exposure to multiple industrial growth conditions associated with benzisothiazolone (BIT) and phenoxyethanol (POE) preservatives, and a laundry detergent (LD) formulation, was observed. A MexPQ-OpmE Resistance Nodulation Division efflux pump system was commonly upregulated in response to POE, a combination of BIT and POE, and LD together with BIT. In response to all industry conditions, a putative sialic acid transporter and isoprenoid biosynthesis gnyRDBHAL operon demonstrated consistent upregulation. Two operons phnBA and pqsEDCBA involved in Pseudomonas quinolone signaling production and quorum-sensing were also consistently downregulated during exposure to all the industry conditions. The ability to identify consistently differentially expressed genetic pathways in P. aeruginosa can inform the development of future targeted preservation systems that maintain product safety and minimise resistance development.

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September 22, 2019

High-oleate yeast oil without polyunsaturated fatty acids.

Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements.We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ?9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the ?12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with >?90% oleate content.High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

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September 22, 2019

Phenotypic diversification by enhanced genome restructuring after induction of multiple DNA double-strand breaks.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

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September 22, 2019

In vitro DNA SCRaMbLE.

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The “in vitro SCRaMbLE system” uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a ß-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

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September 22, 2019

Precise control of SCRaMbLE in synthetic haploid and diploid yeast.

Compatibility between host cells and heterologous pathways is a challenge for constructing organisms with high productivity or gain of function. Designer yeast cells incorporating the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system provide a platform for generating genotype diversity. Here we construct a genetic AND gate to enable precise control of the SCRaMbLE method to generate synthetic haploid and diploid yeast with desired phenotypes. The yield of carotenoids is increased to 1.5-fold by SCRaMbLEing haploid strains and we determine that the deletion of YEL013W is responsible for the increase. Based on the SCRaMbLEing in diploid strains, we develop a strategy called Multiplex SCRaMbLE Iterative Cycling (MuSIC) to increase the production of carotenoids up to 38.8-fold through 5 iterative cycles of SCRaMbLE. This strategy is potentially a powerful tool for increasing the production of bio-based chemicals and for mining deep knowledge.

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