Temporal and fluoride control of secondary metabolism regulates cellular organofluorine biosynthesis

Abstract

Elucidating mechanisms of natural organofluorine biosynthesis is essential for a basic understanding of fluorine biochemistry in living systems as well as for expanding biological methods for fluorine incorporation into small molecules of interest. To meet this goal we have combined massively parallel sequencing technologies, genetic knockout, and in vitro biochemical approaches to investigate the fluoride response of the only known genetic host of an organofluorine-producing pathway, Streptomyces cattleya. Interestingly, we have discovered that the major mode of S. cattleya's resistance to the fluorinated toxin it produces, fluoroacetate, may be due to temporal control of production rather than the ability of the host's metabolic machinery to discriminate between fluorinated and non-fluorinated molecules. Indeed, neither the acetate kinase/phosphotransacetylase acetate assimilation pathway nor the TCA cycle enzymes (citrate synthase and aconitase) exclude fluorinated substrates based on in vitro biochemical characterization. Furthermore, disruption of the fluoroacetate resistance gene encoding a fluoroacetyl-CoA thioesterase (FlK) does not appear to lead to an observable growth defect related to organofluorine production. By showing that a switch in central metabolism can mediate and control molecular fluorine incorporation, our findings reveal a new potential strategy toward diversifying simple fluorinated building blocks into more complex products.

Figure 1

Analysis of the fluoride response in S. cattleya. (a) Growth and fluoride uptake in S. cattleya cultures in GYM media at pH 5. Growth of cultures measured by OD_{600 nm} (filled circles) and fluoride concentration (open circles) with (red) and without (black) 2 mM sodium fluoride added 24 h after inoculation. (b) Monitoring organofluorine production by ¹⁹F NMR in the culture supernatant. (c) Transcriptional landscape of S. cattleya 48 h after the addition (red) or no addition (black) of fluoride. Values are quantile normalized reads per kb for predicted protein coding sequences. The fluorinase (flA) is indicated by a star. (d) RNA sequencing reads mapped onto the fluorinase gene cluster 48 h after the addition of fluoride (red) compared samples with no added fluoride (black).

Figure 2

Time– and fluoride–dependent changes in transcript levels of predicted pathway genes and their paralogs. Transcript levels were quantified by RNA sequencing 0.5, 2, 6, and 48 h after the addition of 2 mM sodium fluoride and compared to no addition. Values at 48 h are the mean of two replicates and error bars indicate one standard deviation. Transcription levels of reads per kb were normalized to the maximum value for each predicted open reading frame in parentheses. (a) FlA (36,036 reads/kb) and FlB (1,091 reads/kb) (b) the predicted fluoroacetaldehyde dehydrogenase gene (FAldH, 2,677 reads/kb) and the fluorothreonine transaldolase gene (FT transaldolase, 975 reads/kb), (c) methylthioadenosine phosphorylase paralogs (SCAT_3258, 432 reads/kb; SCAT_2201, 411 reads/kb), (d) MRI1 (192 reads/kb) and MRI2 (278 reads/kb), and (e) F1P aldolase paralogs (SCAT_1042, 241 reads/kb; SCAT_p1616, 78 reads/kb).

Figure 3

Organofluorine production in S. cattleya and MRI knockout strains. ¹⁹F NMR analysis of culture supernatants from wildtype, Δmri1, Δmri2, and Δmri1Δmri2 strains of S. cattleya 6 d after the addition of sodium fluoride (2 mM).

Figure 4

Transcription patterns for TCA cycle genes with respect to fluoride and time. Transcript levels were quantified by RNA sequencing 0.5, 2, 6, and 48 h after the addition of 2 mM sodium fluoride and compared to no addition. Values at 48 h are the mean of two replicates and error bars indicate one standard deviation. Transcription levels of reads per kb were normalized to the maximum value for each predicted open reading frame in parentheses with a representative subunit given for multi–subunit enzymes. Time courses are shown for Cit1 (7,130 reads/kb), aconitase (4,238 reads/kb), isocitrate dehydrogenase (3,495 reads/kb), α–ketoglutarate dehydrogenase (3,094 reads/kb), succinyl–CoA synthetase (5,383 reads/kb), succinate dehydrogenase (2,050 reads/kb), fumarase (2,141 reads/kb), and malate dehydrogenase (6,207 reads/kb).

Figure 5

COG categories for genes differentially expressed with respect to fluoride. COG categories were identified by the IMG–ER annotation pipeline. COG categories represented by genes that are (A) upregulated and (B) downregulated in the presence of fluoride at 48 h. (C) Comparison of COG category representation in the differentially expressed genes with that of the entire genome. The number of open reading frames represented by each COG are given and the percentage of total genes with COG categories are in parenthesis.

Scheme 1. Biological organofluorine pathways.^a

^a(a) The proposed biosynthetic pathway for fluoroacetate and fluorothreonine in S. cattleya. S–adenosylmethionine (1), 5´–fluorodeoxyadenosine (2), 5–fluorodeoxyribose–1–phosphate (3), 5–fluorodeoxyribulose–1–phosphate (4), fluoroacetaldehyde (5), fluoroacetate (6), fluorothreonine (7). Fluorinase (FlA, A), 5´–fluorodeoxyadenosine or methylthioadenosine phosphorylase (FlB, B), methylthioribulose–1–phosphate isomerase (MRI, C), fucolose–1–phosphate aldolase (F1P aldolase, D), fluoroacetaldehyde dehydrogenase (FAldH, E), fluorothreonine transaldolase (FT transaldolase, F). (b) Lethal synthesis of fluorocitrate and inactivation of aconitase. Acetate (8), fluoroacetyl–CoA (9), acetyl–CoA (10), fluorocitrate (11), citrate (12), fluoroaconitate (13), aconitate (14), 4–hydroxy–transaconitate (15), isocitrate (16). Acetate kinase and phosphotransacetylase (AckA and Pta, G), acetyl–CoA synthase (ACS, H), citrate synthase (CS, I), aconitase (J), fluoroacetyl–CoA thioesterase (FlK, K).