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Associated bacteria of Botryococcus braunii (Chlorophyta).

ABSTRACT: Botryococcus braunii (Chlorophyta) is a green microalga known for producing hydrocarbons and exopolysaccharides (EPS). Improving the biomass productivity of B. braunii and hence, the productivity of the hydrocarbons and of the EPS, will make B. braunii more attractive for industries. Microalgae usually cohabit with bacteria which leads to the formation of species-specific communities with environmental and biological advantages. Bacteria have been found and identified with a few B. braunii strains, but little is known about the bacterial community across the different strains. A better knowledge of the bacterial community of B. braunii will help to optimize the biomass productivity, hydrocarbons, and EPS accumulation. To better understand the bacterial community diversity of B. braunii, we screened 12 strains from culture collections. Using 16S rRNA gene analysis by MiSeq we described the bacterial diversity across 12 B. braunii strains and identified possible shared communities. We found three bacterial families common to all strains: Rhizobiaceae, Bradyrhizobiaceae, and Comamonadaceae. Additionally, the results also suggest that each strain has its own specific bacteria that may be the result of long-term isolated culture.

SUBMITTER: Gouveia JD

PROVIDER: S-EPMC6441321 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe colonial microalga Botryococcus braunii has been brought to people's attention for its conspicuous ability to accumulate a variety of lipids including hydrocarbons. B. braunii strains are classified into 3 races based on the types of hydrocarbons. A and B races are known to accumulate high level of lipids. However, their extreme slow growth rate has impeded its application for renewable biofuel production.ResultsIn this study, we report the transcriptomic response of a moderately growing subisolate from the culture of Botryococcus sp. CCALA-779 upon nitrogen deprivation (ND). We show that the subisolate has an average growth rate of 0.52 g l(-1) day(-1) under photoautotrophic growth conditions and lipid content is enhanced to 75 % of CDW upon ND. Both rDNA sequence and hydrocarbon composition analyses indicate that the subisolate belongs to A race B. braunii. Hence, it is designated as B. braunii 779. We show that B. braunii 779 transcriptome shares homology to majority of the A race but not B race B. braunii ESTs, suggesting that transcriptomes of A race differ from that of B race. We found that many homologous ESTs between A races 779 and Bot-88 are unknown sequences, implying that A race contains many unknown genes. Pathway-based transcriptomic analysis indicates that energy metabolisms are among the top expressed functions in log-phase cells, indicating that the slow growth rate is a result that energy flow is directed to lipid biosynthesis but not population growth. Upon ND, reconfiguration of metabolisms for reducing power is apparent, suggesting that B. braunii 779 is rapidly adapting under ND condition by transcriptomic reprogramming.ConclusionsTaken together, our result shows that the subisolate B. braunii 779, similar to the Gottingen strain, is useful for biofuel production. Difference between transcriptomes of A and B races implies that different races of B. braunii strains belong to different sub-species. Furthermore, there are many novel genes that are unique to A race, suggesting that sequences of many enzymes involved in hydrocarbon biosynthesis are not currently known. We propose that B. braunii transcriptomes provide a rich source for discovery of novel genes involved in hydrocarbon biosynthesis.

Project description:Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

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Associated bacteria of Botryococcus braunii (Chlorophyta).

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