Table 2 Estimating global plant microbiome holometabolomes using combinatorics

Table 1 considered endophytes independently, ignoring plant-microbe and microbe-microbe cooperation in NP synthesis. Here, we estimate secondary metabolism that may be more than the sum of its parts through in planta multi-species co-synthesis, syntropy, and synergistic biosynthetic pathways. We first estimate the global number of endophyte communities, then these communities’ additive and synergistic secondary holometabolomes

Estimating the number of distinct endophyte communities on Earth: While the theoretical number of possible combinations of endophyte species within a plant would be 2ⁿ for n endophytes, so that a plant that can host 500 species of endophytes would have 2⁵⁰⁰ possible endophyte communities, real life does not include all possible combinations. Instead, if we calculate the possible unique endophyte sets of size 500 from amongst a set of m unique microbes, we can use the binomial coefficient and take m choose k, with k = 500, then solve for m based on the number of plant species or plant individuals on Earth. Given that m choose k = m!/k!(m-k)!, and assuming a limit of 300,000 combinations (one per plant species) we calculate m is between 502 and 503, meaning that the effective set of unique microbes per plant species would be just 2 or 3 under this endophyte set size of 500 per plant. Note: based on Table 1, we estimated from 113 to 257 fungi per plant species and from 1290 to 32,300 bacteria per plant species, which translates to effectively just over 3 unique fungi per plant species, and no unique bacteria per plant species. If plant individuals rather than plant species are better units for analysis, given the observed variance in endophytes across plant geographic ranges and predicted horizontal exchange of endophytes and we estimate an average of 50,000,000 plant individuals per species, there could be 15 trillion endophyte combinations on Earth, with as many as 6 to 7 unique endophytic fungi or over 3 unique endophytic bacteria per plant.

Estimating endophytic holometabolomes on Earth: Several studies have demonstrated the importance of plant-microbe and microbe-microbe shared metabolic pathways involving intermediate metabolite provision or positive regulatory cues [45]. Plant species diversity is positively associated with bacterial and fungal diversity and metabolism [25, 46], but how much of this is merely additive versus synergistic or cooperative? Results from OSMAC and co-culturing studies that show perhaps 90% of endophyte secondary metabolites depend on in planta conditions [28, 47, 48] pointing to in planta metabolic synergism. Combinatoric models have been helpful for exploring metabolism and biochemical space [49, 50], but these have seldom been applied to understanding synergies between microbes [51, 52]. Biosynthesis of secondary metabolites is particularly amenable to combinatoric synergy because it functions modularly through extending polymer backbones: -CH2-(C=O)- units for polyketides, C5 isoprene units for terpenoids, and non-ribosomomal peptides, that later generate diverse chemicals with the assistance of tailoring enzymes. Recent combinatoric experiments on simple microbiomes also indicate that higher-order interactions in which each species impacts interactions among other species, at least for 2-way and 3-way interactions are widespread in a 5-species microbiome [52]. Here, we will apply a similarly small interaction network for endophytes and consider the holometabolome of a small localized section of plant tissue containing 5 interacting organisms: the plant, two fungal species and two bacterial species, that can interact and cooperate molecularly at close range. In this example, 2ⁿ interactions could occur for n species. If each pair of these 5 species participates in one biosynthetic synergy leading to an additional metabolite, there would be n choose 2 = n!/2!(n-2)! = 10 unique metabolites arising synergistically for this interaction. Within a plant species, if a portion of its endophytes (e.g. 100) participated in these synergies with each of the 10 novel products synthesized once, 100 / 5 = 20 such products would be generated, adding 6 million new secondary metabolites to the global tallies discussed in Table 1, or, at the upper limits, considering individual plants to have distinct communities and synergies, there could be 300 trillion unique in planta synergistic products on Earth. However, this value likely includes extensive redundancy, which is difficult to estimate without further empirical data, or models such as the deep learning models described in Table 3.