These results reveal that not all fermentable fibers are equally capable of stimulating SCFA production, and they highlight the importance of the composition of an individual’s microbiota in determining whether or not they respond to a specific dietary supplement. In particular, R. bromii or C. chartatabidum may be required for enhanced butyrate production in response to RS. Bifidobacteria, though proficient at degrading RS and inulin, may not contribute to the butyrogenic effect of those fermentable fibers in the short term.

BackgroundThe fermentation of dietary fiber to various organic acids is a beneficial function provided by the microbiota in the human large intestine. In particular, butyric acid contributes to host health by facilitating maintenance of epithelial integrity, regulating inflammation, and influencing gene expression in colonocytes. We sought to increase the concentration of butyrate in 20 healthy young adults through dietary supplementation with resistant starch (unmodified potato starch—resistant starch (RS) type 2).MethodsFecal samples were collected from individuals to characterize butyrate concentration via liquid chromatography and composition of the microbiota via surveys of 16S rRNA-encoding gene sequences from the Illumina MiSeq platform. Random Forest and LEfSe analyses were used to associate responses in butyrate production to features of the microbiota.ResultsRS supplementation increased fecal butyrate concentrations in this cohort from 8 to 12 mmol/kg wet feces, but responses varied widely between individuals. Individuals could be categorized into three groups based upon butyrate concentrations before and during RS: enhanced, high, and low (n = 11, 3, and 6, respectively). Fecal butyrate increased by 67 % in the enhanced group (from 9 to 15 mmol/kg), while it remained ≥11 mmol/kg in the high group and ≤8 mmol/kg in the low group. Microbiota analyses revealed that the relative abundance of RS-degrading organisms—Bifidobacterium adolescentis or Ruminococcus bromii—increased from ~2 to 9 % in the enhanced and high groups, but remained at ~1.5 % in the low group. The lack of increase in RS-degrading bacteria in the low group may explain why there was no increase in fecal butyrate in response to RS. The microbiota of individuals in the high group were characterized by an elevated abundance of the butyrogenic microbe Eubacterium rectale (~6 % in high vs. 3 % in enhanced and low groups) throughout the study.ConclusionsWe document the heterogeneous responses in butyrate concentrations upon RS supplementation and identify characteristic of the microbiota that appear to underlie this variation. This study complements and extends other studies that call for personalized approaches to manage beneficial functions provided by gut microbiomes.

The steady-state growth rate of Saccharomyces cerevisiae was varied by growing the cells in different media. The total amount of ribonucleic acid (RNA) per cell was found to decrease as a nonlinear function of decreasing growh rate. The RNA from cells growing in different media was analyzed by polyacrylamide gel electrophoresis. Although the amounts of both ribosomal RNA and transfer RNA decreased with decreasing growth rate, the ratio of ribosomal to transfer RNA was not constant. As the growth rate was reduced the ribosomal RNA fraction decreased slightly, whereas the transfer RNA fraction increased slightly. Thus the levels of ribosomal and transfer RNA were regulated to similar yet different extents. The levels of the different ribosomal RNA species were more closely coordinated. At all growth rates the ribosomal RNAs (including 5S RNA) were present in equimolar amounts. The rate of protein synthesis in yeast cells also decreased with decreasing growth rate. The low rates of protein synthesis did not appear to be due to limiting numbers of ribosomes or transfer RNA molecules.

Most of the S. spinosa genes involved in spinosyn biosynthesis are found in one 74 kb cluster, though it does not contain all of the genes required for the essential deoxysugars. Characterization of the clustered genes suggests that the spinosyns are synthesized largely by mechanisms similar to those used to assemble complex macrolides in other actinomycetes. However, there are several unusual genes in the spinosyn cluster that could encode enzymes that generate the most striking structural feature of these compounds, a tetracyclic polyketide aglycone nucleus.

A gene coding for potato multicystatin (PMC), the crystalline inhibitor of cysteine proteases which is found in tubers, was isolated and characterized. The deduced polypeptide product of this genomic sequence is 757 amino acids long and has a molecular mass of 86,778 Da. It consists exclusively of eight closely related domains, with 53-89% identity of residues. Each repeated unit is homologous to the cystatin superfamily of cysteine protease inhibitors. To date, no other member of this family has been found to contain so many inhibitor domains in one polypeptide. Eight introns are proposed in the 3.5 kb of genomic DNA coding for PMC, one in each cystatin unit. There is a family of 4 to 6 such large genes in potato, while in pea and maize the homologues are much smaller, and probably code for single-domain cystatins. PMC transcripts are abundant in tubers, but scarce in undamaged leaves or stems of field-grown potatoes. The tuber messages are derived from at least four genes (including the cloned example). The pattern of gene expression, as well as the properties of the protein, suggest that PMC has a role in the plant's defense system.

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in Saccharopolyspora spinosa are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.Spinosyns, the active ingredients in Dow AgroSciences' new Naturalyte line of insect control products, are produced by fermentation of the actinomycete Saccharopolyspora spinosa. Spinosyns are macrolides ( Fig. 1) consisting of a 21-carbon tetracyclic lactone to which are attached two deoxysugars: tri-O-methylated rhamnose and forosamine (6). The most active components of the spinosyn family of compounds are spinosyns A and D, which differ from each other by a single methyl substituent at position 6 of the polyketide. Other factors in this family have different levels of methylation and are significantly less active. Both the rhamnose and forosamine moieties are essential for the insecticidal activity of spinosyns (2). Spinosad is highly effective against target insects and has an excellent environmental and mammalian toxicological profile (2,13,14).Spinosyn biosynthesis occurs via the nonglycosylated intermediate, the aglycone (AGL). Rhamnose is the first sugar attached and is tri-O-methylated to yield the intermediate pseudoaglycone.Only after the rhamnose is attached can the forosamine sugar be incorporated (M. C. Broughton, M. L. B. Huber, L. C. Creemer, H. A. Kirst, and J. R. Turner, Abstr. 91st Annu. Meet. Am. Soc. Microbiol. 1991, abstr. K-58, p. 224, 1991. Both trimethyl rhamnose and forosamine are believed to be synthesized from glucose-1-phosphate via the common intermediate 4-keto-6-deoxy-D-glucose (Fig. 2). The biosynthetic pathway for rhamnose (Fig. 2) has been elucidated in enteric bacteria, where the deoxysugar is an element of surface antigens (8, 18). The first step, activation of glucose by addition of a nucleotidyl diphosphate (NDP), is catalyzed by an NDP-glucose synthase (the gtt gene product). The second step, dehydration to NDP-4-keto-6-deoxyglucose, is catalyzed by glucose dehydratase (the gdh gene product). 4-Keto-6-deoxy-D-glucose is the common intermediate to many deoxysugar biosynthetic pathways, and the enzymes encoded by the gtt and gdh genes may supply the precursors for all of them. Rhamnose synthesis requires two additional enzymes, a 3Ј5Ј epimerase (encoded by epi) and a 4Ј ketoreductase (encoded by kre), that are unique to the pathway. They convert the NDP-4-keto-6-deoxyglucose to NDP-L-rhamnose, the activated sugar that is the substrate of th...

From the protein and RNA content of Saccharomyces cerevisiae growing in different media we calculate that ribosome efficiency is changed: incorporation of amino acids into protein decreases from 8.8 amino acids/s per ribosome in fast-growing cells (0.54 doubling/h) to 5.2 amino acids/s per ribosome in slow-growing cells (0.30 doubling/h). We could not detect significant protein turnover in either fast-or slow-growing cultures, so the lower ribosome efficiency does not seem to be an artifact caused by changes in unstable protein production at different growth rates. Nor is the lower ribosome efficiency due to slower migration of ribosomes along mRNA: the times required to complete polypeptides of known molecular weights are the same in slow-growing cells as those previously determined for fast-growing cells [Waldron, Jund & Lacroute (1974) FEBS Lett. 46, 11-16]. We therefore deduce that ribosome efficiency changes in yeast because the fraction of ribosomes engaged in protein synthesis falls (from 84% in fast-growing cells to 50% in slow-growing cells.

Spinosyns, a novel class of insect active macrolides produced by Saccharopolyspora spinosa, are used for insect control in a number of commercial crops. Recently, a new class of spinosyns was discovered from S. pogona NRRL 30141. The butenyl-spinosyns, also called pogonins, are very similar to spinosyns, differing in the length of the side chain at C-21 and in the variety of novel minor factors. The butenyl-spinosyn biosynthetic genes (bus) were cloned on four cosmids covering a contiguous 110-kb region of the NRRL 30141 chromosome. Their function in butenyl-spinosyn biosynthesis was confirmed by a loss-of-function deletion, and subsequent complementation by cloned genes. The coding sequences of the butenyl-spinosyn biosynthetic genes and the spinosyn biosynthetic genes from S. spinosa were highly conserved. In particular, the PKS-coding genes from S. spinosa and S. pogona have 91-94% nucleic acid identity, with one notable exception. The butenyl-spinosyn gene sequence codes for one additional PKS module, which is responsible for the additional two carbons in the C-21 tail. The DNA sequence of spinosyn genes in this region suggested that the S. spinosa spnA gene could have been the result of an in-frame deletion of the S. pogona busA gene. Therefore, the butenyl-spinosyn genes represent the putative parental gene structure that was naturally engineered by deletion to create the spinosyn genes.