The microalga Botryococcus braunii is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the Botryococcus braunii race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of Botryococcus braunii and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.

BackgroundThe exopolysaccharide xanthan is a natural product which is extensively used in industry. It is a thickening agent in many fields, from oil recovery to the food sector. Xanthan is produced by the Gram negative bacterium Xanthomonas campestris pv. campestris (Xcc). We analyzed the lipopolysaccharide (LPS) of three mutant strains of the Xcc wild type B100 to distinguish if the xanthan production can be increased when LPS biosynthesis is affected.ResultsThe Xcc B100 O-antigen (OA) is composed of a linear main chain of rhamnose residues with N-acetylfucosamine (FucNAc) side branches at every second rhamnose. It is the major LPS constituent. The O-antigen was missing completely in the mutant strain H21012 (deficient in wxcB), since neither rhamnose nor FucNAc could be detected as part of the LPS by MALDI-TOF-MS, and only a slight amount of rhamnose and no FucNAc was found by GC analysis. The LPS of two other mutants was analyzed, Xcc H28110 (deficient in wxcK) and H20110 (wxcN). In both of them no FucNAc could be detected in the LPS fraction, while the rhamnose moieties were more abundant than in wild type LPS. The measurements were carried out by GC and confirmed by MALDI-TOF-MS analyses that indicated an altered OA in which the branches are missing, while the rhamnan main chain seemed longer than in the wild type. Quantification of xanthan confirmed our hypothesis that a missing OA can lead to an increased production of the extracellular polysaccharide. About 6.3 g xanthan per g biomass were produced by the Xcc mutant H21012 (wxcB), as compared to the wild type production of approximately 5 g xanthan per g biomass. In the two mutant strains with modified OA however, Xcc H28110 (wxcK) and Xcc H20110 (wxcN), the xanthan production of 5.5 g and 5.3 g, respectively, was not significantly increased.ConclusionsMutations affecting LPS biosynthesis can be beneficial for the production of the extracellular polysaccharide xanthan. However, only complete inhibition of the OA resulted in increased xanthan production. The inhibition of the FucNAc side branches did not lead to increased production, but provoked a novel LPS phenotype. The data suggests an elongation of the linear rhamnan main chain of the LPS OA in both the Xcc H28110 (wxcK) and Xcc H20110 (wxcN) mutant strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0710-y) contains supplementary material, which is available to authorized users.

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Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling.

The ɣ-proteobacterium Xanthomonas campestris pv. campestris (Xcc) is the producer of the biopolymer xanthan, a polysaccharide which is used as a thickener in numerous industrial applications. In this study, we present a global transcriptome profiling of two Xcc strain B100 cultures obtained from fermentation during the growth phase and the subsequent stationary phase associated with xanthan biosynthesis. During the xanthan production phase, highly abundant transcripts belonged to genes encoding for small RNAs, glycogen biosynthesis, and xanthan export. A total of 1850 (40%) genes were differentially transcribed during the stationary phase where 924 were transcriptionally up-regulated and 926 genes were down-regulated. An overview of differentially transcribed genes includes a significant down-regulation of genes involved in transcription, translation, and amino acid biosynthesis pathways. A group of up-regulated genes was involved in cellular response against oxidative stress, such as those coding for superoxide dismutase and catalase. Genes encoding enzymes involved in nucleotide sugar precursor synthesis of xanthan biosynthesis, such as xanA, galU, and ugd, exhibited a transcription pattern that did not change during the growth and stationary phase. Regarding the transcription pattern of the gum gene cluster that govern xanthan biosynthesis, a significant up-regulation of the genes gumB, gumC, and gumD was observed, while the transcript pools of the genes gumG, gumH, gumI, and gumJ were reduced and those of genes gumE, gumF, gumK, gumL, and gumM remained un-changed during the stationary phase compared to the growth phase. The obtained data represents the first analysis of gene expression patterns under xanthan production conditions and provides the bases for future studies aiming at enhancing xanthan yield.